G

G4 DNA Gain-of-Function Screens

▶G-Quartet DNA Definition

GABA
Definition
GABA (gamma aminobutyric acid) acts as an inhibi-
tory neurotransmitter in the central nervous system.
GABA-ergic neurotransmission via (inhibitory) inter-
neurons is involved in most brain functions.
▶Addiction, Molecular Biology
Gain-of-function screens are mutational screens based
on the over- or misexpression of genes, aimed at
detecting genes that may show no loss-of-function
effect. Ectopic expression of an endogenous transcrip-
tion unit adjacent to the 5′ end of the randomly
integrated P element is dependent on the expression of
the GAL4 gene (Drosophila melanogaster), and thus
may be spatially and temporally controlled.
▶Drosophila as a Model Organism for Functional
Genomics

GAL4–Expression System
Gag
Definition
Definition The GAL4–expression system is a method for directed
Gag is a retrovirus gene that encodes the retroviral gene expression that can be used to misexpress genes in
internal structural proteins ▶MA, CA, and ▶NC, and specific cell types, or tissues, at different times of
some others. development. This system relies on the generation
▶Retroviruses of transgenic lines that carry “activator” or “effector”
constructs. Activator lines express the yeast transcrip-
tion factor, Gal4, under the control of a desired
promoter, whereas effector lines contain DNA binding
motifs for Gal4–(UAS) linked to the gene of interest.
Gain-of-Function Mutations ▶Drosophila as a Model Organism for Functional
Genomics

Gain of function mutations describes mutations that

result in new (protein) functions in heterozygous
individuals.
▶Huntington’s Disease
▶Loss of Function Mutation
▶Mendelian Forms of Human Hypertension and Gamates
Mechanisms of Disease
▶Mouse Genomics
▶Tumor Suppressor Genes ▶Germ Cells
636 Gamma Rays
Gamma Rays
Definition
Gamma rays are an energetic form of electromagnetic
radiation produced by radioactivity or other nuclear or
subatomic processes such as electron-positron annihi-
lation. Gamma rays are often defined to begin at an
energy of 10 keV, although electromagnetic radiation
from around 10 keV to several hundred keV is also
referred to as hard X-rays. Gamma rays are a form of
ionizing radiation; they are more penetrating than either
alpha or beta radiation (neither of which is electro-
magnetic radiation), but less ionizing.
▶Molecular Imaging
Gammaretroviruses
Definition
Gammaretroviruses refers to the genus of simple
retroviruses that includes murine and feline leukemia
viruses. Many species contain oncogenes and cause
leukemias and sarcomas.
▶Retroviruses
Gammaretroviruses IN (Integrase)
Definition
Gammaretroviruses IN (integrase) is the retrovirus
virion enzyme, and is a product of the pol gene.
▶Retroviruses
Gamma-Secretase (Complex)
The γ-secretase (complex) is a multimeric complex that
is composed of at least four different transmembrane
proteins (presenilin 1 or presenilin 2 (▶PSEN1/
PSEN2); (▶APH–1); nicastrin; PEN–2). The last step
of ▶amyloid generation from amyloid precursor protein
(▶APP) is performed by the γ-secretase complex. The
γ-secretase is also important in other pathways, as for
example in the cleavage of ErbB4, intra cellular domains
of Notch, and similar types of proteins.
▶Alzheimer’s Disease
Gap
Definition
A space introduced into an alignment to compensate for
insertions or deletions in one sequence relative to
another.
▶Protein Databases
Gap Junctions
W. H OWARD E VANS
Medical Biochemistry and Immunology, Cardiff
University School of Medicine, Cardiff, Wales, UK
wmbwhe@cf.ac.uk
Definition
Gap junctions comprise minute regions of the cell’s
plasma (surface) membrane containing arrays of
closely packed membrane channels. These channels
traversing two aligned membranes separated by a
2–3 nm intercellular gap provide a communication
pathway that directly connects cell interiors (Fig. 1).
Communication across gap junctions enables single
cells to co-ordinate, integrate and summate their
metabolic and electrical interactive activities but also
results in some loss of independence. Co-operation and
harmonisation of cellular activities in tissues and
organs is vividly illustrated by the ordered contraction
of heart muscle, made possible because the beating of
individual cells is synchronised and summated to the
organ level by interactions facilitated by gap junctions.
Indeed, each cardiac myocyte can communicate with
about a dozen or more surrounding partner cells. In the
brain, gap junctions enable the synchronisation of the
electrical coupling of neuronal cell networks. In non-
excitable cells, small signalling molecules below 1200
daltons are exchanged across gap junctions. All animal
cells communicate directly with each other across gap
junctions, except striated muscle where the cells have
fused, spermatozoa and non-nucleated erythrocytes and
platelets (1).
Characteristics
Gap junctions are built from one of three biochemically
different families of proteins.
▶Connexins
In vertebrates, gap junctions are constructed from
▶connexin protein units (Fig. 1). Over 20 different
 Gap Junctions 637

Gap Junctions. Figure 1 A gap junction plaque showing diagrammatically the arrangement of paired connexon
hemichannels in the membrane. These dock to generate a channel directly joining two cells. Connexons attach
to the edges of plaques, but they may also function as hemichannels in their own right. In the top box, the
oligomerisation of connexins into hexameric connexons and the various types of gap junctions formed are
illustrated. In the lower box, the topography of a generalised connexin in the lipid bilayer is shown. Bold line
indicates sequences with high homology.

connexins have been found in humans and rodents and
various tissues synthesise different and often over-
lapping types of connexin (Table 1). They constitute a
family of proteins displaying about 40% overall amino
acid sequence identity. A widely adopted method of
naming individual connexins uses the abbreviation Cx
followed by the predicted molecular mass in kD.
Increasingly, a prefix may be added to indicate the
species e.g. h, human; m, mouse; zf, zebra fish, etc.
When the molecular mass of the connexins is similar, a
decimal point is added e.g. mCx 30.2, mCx 30.3 and
mCx 31.1. However, although there is often little size
variation between species, e.g. mCx36 and fish (skate)
Cx35, there is sometimes greater disparity in the
molecular sizes of functionally similar connexins, e.g.
rat Cx46, bovine Cx44 and chicken Cx56 and clearly
this nomenclature is not entirely satisfactory. Mouse
and human connexins have been classified phylogen-
etically as follows: Group 1 or beta: Cx26, Cx30.3,
Cx31, Cx31.1, Cx32; Group 2 or alpha : Cx33, Cx37,
Cx38, Cx40, Cx42, Cx43, Cx50, Cx56; Group3 or
gamma : Cx45, Cx47 and an uncategorised group:
Cx25, Cx29, Cx30.2, Cx36, Cx39, Cx40.1 and Cx58.
Cx43 is by far the most widely distributed connexin
(1). In heart ventricles, myocytes express Cx43
exclusively but myocytes in the atrium and Purkinje
fibres also express Cx40. Endothelial cells lining the
vascular wall express Cx37 in addition to Cx43, which
is also expressed by the smooth muscle cells control-
ling the contraction of arteries. Cx36 is expressed by
neurons in the retina, whereas in the brain astrocytes
express mainly Cx43 and oligodendrocytes Cx32 and
Cx47. Cells comprising the various layers in the skin
express a range of connexins, especially Cx26, Cx30,
Cx31, Cx32, Cx40, Cx43 and Cx45. In liver,
hepatocytes express Cx26 and Cx32, with their relative
abundance varying between species. Lens fibre cells
express Cx46 and Cx50, but epithelial cells enveloping
the lens capsule express Cx43. Up to 196 permutations
of homologous and heterologous connexin based gap
638 Gap Junctions

junction interactions are possible when cells express
two ore more different types of connexins (Fig. 1). The
relative amounts of each connexin in cells also varies
during embryonic and tissue development.
Connexins span the membrane lipid bilayer four times
and in an alpha helical conformation (Fig. 1). The
protein’s amino terminus (a highly conserved sequence of
21 amino acids) and carboxyl terminus (a highly variable
number of amino acids as described below) and a single
intracellular loop face the inside of the cell (Fig. 1). Two
highly conserved amino acid sequences forming a loop
(twenty in the first and forty amino acids in the second)
project in a beta sheet conformation from the cell surface
into the extracellular space; these loops contain three
cysteine residues that are invariable in all connexins
discovered and they are linked to each other by
intramolecular disulphide bonds. These extracellular
loop interactions are crucial for they provide a scaffold
that aligns and extends the transmembrane ▶hemichan-
nel domains across the intercellular gap. The amino acid
sequences in these two loops also set the rules governing
the compatibility of interactions between various con-
nexins. For example, cells with gap junction channels
made from Cx32 and Cx26 communicate but cells
making Cx32 do not communicate with those making
Cx43. The types of connexin synthesised by cells
thus dictate whether homo-or hetero-philic gap
junction channels are generated, with different channel
pore characteristics and molecular selectivities. The
third transmembrane domain and parts of the first
and second transmembrane domains contribute the
wall of the overall channel. The greatest differences in
amino acid sequences between connexins are found in
the intracellular loop projecting into the cytoplasm and
consisting of 30 amino acids in the smaller connexins,
50–55 amino acids in Cx33, Cx37, Cx40, Cx43, Cx46,
Cx50 and Cx57, 80 amino acids in Cx45 and 100 amino
acids in Cx36. The carboxyl terminal tail, often referred
to as the regulatory domain, varies in length from 16
amino acids in Cx26 to 75 in Cx32, 156 in Cx43 and 275
in Cx57. Larger connexins are post-translationally
modified by phosphorylation of several serine and
threonine residues on the carboxyl tail. The functional
consequences of phosphorylation of connexins remain
to be defined as well as the key protein kinases and
phosphatases involved in regulating gap junction
channels. Connexins are not glycosylated, but are
ubiquitinated and acylated.
The genes encoding mouse and human connexins are
located on different chromosomes (Table 1). The
general organisation of connexin genes is similar, and
connexin phylogenetic trees indicate that connexins are
likely to have arisen by gene duplication. Most
connexin genes have a first exon containing 5′
untranslated sequences and a large second exon
containing the complete coding region as well as
untranslated sequences. In contrast, the Cx32 gene
contains two alternative first exons and their expression
is tissue specific. Connexin 36 contains two exons both
with translated and untranslated sequences with the
coding region interrupted by an intron. The Cx45 gene
has three exons, with most transcripts containing only

Gap Junctions. Table 1 Properties of some major mouse/human connexins

Connexin Chromosome mRNA Cells/tissues Disease involvement

type mouse (kb) where present
Cx26 14 2.4 Mammary gland, liver, skin, Deafness, palmoplantar
cochlea hyperkeradosis
Cx30 14 20.23 Skin, cochlea Deafness, ectodermal dysplasia,
hair loss
Cx31 4 1.9, 2.3 Skin, cochlea, uterus Deafness erythrokeratoderma
variabilis
Cx32 X 1.6 Liver, oligodendrocytes Peripheral neuropathy
Cx36 2 2.9 Neurons, retina Visual defects
Cx37 4 1.7 Endothelium ovaries bleeding
Cx40 3 3.5 Heart, endothelium Atrial arrhythmia
Cx43 10 3.0 Diverse Cardiac malformations
Cx45 11 2.2 Heart, brain thalamus, bladder
Cx46 14 2.8 lens cataract
Cx50 3 8.5 lens cataract

exons 2 and 3. Cx40 also has three exons with the third
containing the complete coding sequence. mRNA
encoding connexins is subject to transcriptional control
and tissue specific promoters (acting via multiple
exons) account for hormonal and pharmacological
regulation of expression. This is important, for
example, in the contraction of uterine muscle during
birth when gap junction numbers composed of Cx43
are regulated by oestrogens whereas gap junctions in
the heart are not. Post-transcriptional regulation of
mRNA levels is especially evident with some con-
nexins, for example Cx32 and Cx26 in liver.
How are connexins organised into gap junctions? A
hexagonal arrangement in which six connexins interact
and surround a central pore of 2 nm was suggested by
the regular packing of the presumed channel units in
liver gap junctions stained with heavy metals as well as
by atomic force microscopy. The most up to date
structure is based on a three-dimensional electron
crystallographic analysis of gap junctions prepared
from cultured cells over-expressing recombinant Cx43.
This model (2) shows the alignment of two composite
hexameric connexin hemichannels at a resolution of
0.7 nm in the membrane plane and 2.1 nm in the
vertical plane and it confirms independent biochemical
and physical chemical studies showing the presence of
24 transmembrane alpha helices in each connexon
hemichannel unit. The hemichannel unit has a dumb-
bell shape and is 7 nm wide at the cytoplasmic aspect,
narrowing to 5 nm at the extracellular aspect. The
aqueous channel narrows from 4 nm diameter at the
cytoplasmic entry point to 1.5–2.5 nm depending on
the calcium concentration at the extracellular region
where it becomes continuous with the partner hemi-
channel of a neighbouring cell. The arrangement of the
connexin subunits in the gap junction ensures that
the intercellular channel is completely insulated from
the extracellular ‘gap’.
▶Innexins and ▶Pannexins
Arthropod and vertebrate gap junctions differ in their
overall thickness. Invertebrate gap junctions are
constructed of a biochemically unrelated class of
proteins called innexins. There is no amino acid
sequence homology with connexins, but innexins
adopt a similar topography in the membrane with four
transmembrane domains and cytoplasmically oriented
amino and carboxyl termini. Innexin transcripts
expressed in Xenopus oocytes form functional gap
junctions displaying typical channel gating character-
istics. The Caenorhabditis elegans genome contains as
many as 25 innexin genes, with single cells in this
worm expressing more than one innexin transcript.
Innexins are also present in Drosophila fruit flies
where they feature in the development of the nervous
system and intestine. Innexins have also been identified
Gap Junctions 639
recently in Annelida. The biochemical characterisation
of innexins is awaited.
Pannexins are electrical junctions in vertebrates that are
related to innexins. Pannexin 1 and 2 genes are
abundantly expressed in the central nervous system,
especially the hippocampus, olfactory bulb and cere-
bellum; pannexin 1 is confined to white matter. As with
innexins, expression of pannexin transcripts generates
intercellular channels with similar electrical character-
istics to vertebrate gap junctions constructed of
connexins. Their biochemical characterisation is
awaited.
Assembly and Breakdown of Gap Junctions
Connexins are rapidly degraded; the half-life in heart
and other cells is around two to four hours, a figure G
about 10–20× faster than most membrane proteins.
Connexins are co-translationally inserted directly from
ribosomes into the endoplasmic reticulum, threading
four times into the membrane bilayer to achieve the
typical connexin topography (Fig. 1). Connexins show
a proclivity to oligomerise into hexameric hemichan-
nels that exit the endoplasmic reticulum and enter the
Golgi apparatus. The hemichannels are maintained in a
closed configuration to limit continuity between
cytoplasmic and lumenal environments since ionic
gradients allow cell-signalling responses. The hemi-
channels are then trafficked in membrane vesicles to
the cell’s plasma membrane and attach to the periphery
of pre-existing gap junction plaques, a process that
occurs simultaneously with their docking and align-
ment with hemichannels in the adjacent cell (Fig. 1).
Connexins can be tagged at the carboxyl terminus with
auto-fluorescent proteins such as green fluorescent
protein or short tetracysteine-containing amino acid
motifs to which arsenic-containing chemicals that
fluoresce at different wavelengths will bind. These
approaches have elegantly demonstrated in living cells
how connexin hemichannels are moved to the plasma
membrane and accrete into gap junction plaques and
how gap junction units are internalised (3). Connexins
are transported on 0.5 μm vesicles to the plasma
membrane guided by a microtubular scaffolding and
are removed from the central area of gap junction
plaques as larger vesicles that correspond to annular
gap junctions observed in the electron microscope. Gap
junction plaques are built from up to thousands of
paired hemichannel units that cannot be peeled apart
once formed. Therefore, plaques are internalised into
partner cells as complete units and are ultimately
degraded in phagosomes or lysosomes. In contrast,
incorrectly folded or mutated connexins are transferred
directly from the endoplasmic reticulum for degrada-
tion in proteasomes.
Gap junctions are frequently located next to other
adhesive junctions and it is not surprising that their
640 Gap Junctions
assembly by cells is closely co-ordinated. Connexins
interact via their carboxyl tails with zona occludens 1
and occludin, two proteins associated with tight
▶intercellular junctions, with tubulin, the major
constituent of microtubules and with catenins, a further
class of proteins associated with adhesive junctions.
Specific connexins are also detected in ▶lipid raft
membrane microdomains where they associate with
caveolins.
Regulatory Mechanisms
Gap Junction Channels
Measurements of electrical currents across gap junc-
tions in paired Xenopus oocytes synthesising various
recombinant connexins have shown that opening and
closing (gating) of gap junction channels is determined
mainly by a voltage difference between the paired cells
and features amino acid sequences in the first to second
transmembrane regions. In larger connexins, a second
mechanism is identified involving a “ball and chain”
type interaction between amino acids in the intracel-
lular loop and the carboxyl tail. This chemical gating is
mainly a function of pH with channels closing as pH
drops (4). Gap junctions constructed of different
connexins vary in their gating characteristics. Calcium
ions also regulate gap junctional communication.
Elevation of calcium in cells generally closes gap
junctions. Cell signalling responses such as an increase
in cytoplasmic calcium induced by mechanical aggra-
vation of cells or release of inositol phosphates are
propagated across gap junctions to neighbouring cells
thus generating a calcium wave. The biochemical
nature of the signal transmitted is not known for sure
but one suggestion is that intercellular transmission of
calcium waves involves the passage of inositol tripho-
sphate through the gap junction channel. Calcium
waves are also propagated between cells across
connexin hemichannels. Here, ATP is released across
the hemichannel and then binds in a paracrine fashion
to purinergic receptors on neighbouring cells. In
contrast, progress in establishing the biochemical
nature of molecules/ions transmitted across gap junc-
tions has been slow, probably because its direct nature
has made their interception difficult. All that can be
said is such entities are likely to be 1.2 Kd or less and
with an ionic radius of below 1 nm. Gap junctions
should not be regarded as nonselective pores joining
cells, for the molecular selectivity for a range of
permeants in the context of charge and size is a function
of the connexin makeup of the channel. For example,
gap junction channels distinguish between the passage
of cAMP and cGMP.
Hemichannels
Unopposed connexin hemichannels in non-junctional
regions of the plasma membrane (Fig. 1) were
previously thought of as biogenetic precursors of gap
junctions. However, evidence is building up that they
are regulatable entities in their own right, with
functions influenced for example by calcium levels
inside and outside the cell. These unpaired connexin
hemichannels were first observed in the horizontal cells
of catfish retina and their operation has now been
studied extensively (5). Their presence in mammalian
cells was demonstrated on the basis of passage of small
dyes across the channels or the detection of electrical
currents crossing them. Cells tolerate small numbers
of open connexin hemichannels on the plasma
membrane but the presence of larger numbers is often
a pathological consequence of a metabolic insult such
as in ischaemia, when cells release ATP across the open
hemichannels in cardiac myocytes or glutamate in
astrocytes. Importantly, hemichannels provide a second
mechanism for connexin-dependent intercellular pro-
pagation of calcium waves that complements the
calcium signalling occurring directly across gap
junctions.
Modifications in Disease
Changes in gap junctional communication and espe-
cially mutations in connexin genes have been shown to
correlate with a number of human diseases. Mutations
in Cx43 are extremely rare and diseases in tissues
expressing this connexin mainly involve modifica-
tions in the abundance of functional gap junction
channels and the remodelling of pre-existing gap
junctions as seen in cardiac hypertrophy and infarction
and in endothelial dysfunction in arteries. In contrast,
about 200 Cx32 mutations have been detected in the
X-linked form of ▶Charcot-Marie-Tooth Disease, a
demyelinating syndrome that leads to degeneration of
peripheral nerves. The channels constructed of Cx32
are located mainly in the paranodal loops and Schmidt-
Landermann incisures of myelinating Schwann cells
and provide a direct radial diffusion pathway that is
about 300-fold shorter in distance than a circumfer-
ential route. Many missense, frameshift, deletion and
nonsense mutations lead to a loss of function, many
caused by failure of Cx32 to oligomerise correctly into
hemichannels and to be targeted in a precise manner
to the plasma membrane. Sometimes, gap junctions
are formed by these mutated connexins but their
operation is faulty. Gap junctions constructed form
Cx32 in the liver and pancreatic acinar cells (Table 1)
are unaffected. Surprisingly, myelination by oligoden-
drocytes in the central nervous system is unaffected.
Mutations in Cx26 and Cx30 in the human inner ear are
associated with congenital deafness, a disorder present
in 1 in 1,000 births. Over 50 Cx26 mutations are
known, with one common recessive mutation resulting
in a severely truncated Cx26 protein. Many other site-
specific mutations result in trafficking/▶channel
 Gatekeeper Genes 641

assembly deficiencies. Gap junctions in the inner ear

function in K+ circulation from the interstitial space and GAR Motif
through the cochlear supporting cells, a mechanism
confirmed in mice in which the Cx26 gene was
inactivated. Mutations in Cx26, Cx30 and Cx31.1 are Definition
also associated with disorders of the skin. A GAR motif is a glycine and arginine-rich region of a
Mutations in Cx50 and Cx46 genes are associated with protein in which the arginine residues are often
lens transparency and are prevalent in humans with methylated.
inherited zonular pulverulent cataract. As with Cx43 in ▶Methylation of Proteins
the heart, gap junctional communication is important in
cell migration during organ growth and development.
Knowledge gained by studying genetic mutations in the
laboratory by functional expression in model cell
systems has been extended and complemented in
transgenic mice with single or double gene connexin Gas Chromatography (GC)
“knockouts” and “knockins”. These approaches have G
added important inputs to our understanding of the
physiological, pathological and developmental roles of Definition
individual connexins. The knowledge base will expand Gas chromatography is an analytical method by which
with the application of RNA interference techniques to gaseous compounds are separated based on their
silence connexin genes simultaneously in vivo. Further volatility (boiling point) and interaction with a static
research aims will be to identify functions that can be liquid phase.
attributed to individual connexins or specific combina- ▶Mass Spectrometry: Quantitation
tions of connexins assembled into heteromeric chan-
nels. Higher definition molecular models will help
explain how gap junction channels operate. The
molecular basis of the specificity of transfer of signals
between cells across gap junctions and the way they are
interpreted will continue to be major foci for research. Gastrulation
▶Neuron

References
1. Evans WH, Martin PE (2002) Gap Junctions; structure
and function. Mol Membr Biol 19:121–36
2. Unger VM, Kumar NM, Gilula NB et al (1999) Three
dimensional structure of a recombinant gap junction
membrane channel. Science 238:1176–1180
3. Gaietta G, Deerinck TJ, Adams SR et al (2002) Multicolor
electron microscopy imaging of connexin trafficking.
Science 29:503–507
4. Harris AL (2001) Emerging issues of connexin channels:
biophysics fills the gap. Q Rev Biophys 34:352–472
5. Paul DL, Goodenough DA (2003) Beyond the gap:
functions of unpaired connexon channels. Nat Cell Biol
4:285–294
6. White TW, Paul DL (1999) Genetic diseases and gene
knockouts reveal diverse connexin function. Annu Rev
Physiol 61:283–310
GAPs
▶GTPase-Activating Proteins
Definition
Gastrulation is a morphogenetic process in embryonic
development, during which the three germ layers
ectoderm, endoderm and mesoderm form. Mesoderm is
formed from ectoderm by ▶epithelial-to-mesenchymal
conversion. The primitive ectoderm divides into surface
ectoderm and neuroectoderm.
▶Neural Development
Gatekeeper Genes
Definition
Gatekeeper genes are involved in the control of cell
cycle progression, lifespan of a cell or cell death. They
are often a target for mutations during cancer develop-
ment. The ▶APC gene is the major gatekeeper of the
colon.
▶Cell Cycle - Overview
▶DNA-Repair Mechanisms
▶Tumor Suppressor Genes
642 Gating
Gating
Definition
Gating refers to the conformational changes of ion
channels during the opening and closing of the
permeation pathway.
▶Ion Channels/Excitable Membranes
GBP/FRAT
Definition
GBP (GSK–3 binding protein), and its mammalian
homologue FRAT, bind to the serine-threonine kinase
glycogen synthase kinase 3 (GSK3), and inhibit its
phosphorylation of non-primed GSK–3 substrates. It
has tumor promoting activity in lymphocyte.
▶Wnt/Beta-Catenin Signaling Pathway
GDIs
▶Guanine Nucleotide Dissociation Inhibitors
GEF
▶Guanine Nucleotide Exchange Factor
2D-Gel Electrophoresis
▶Two-Dimensional Gel Electrophoresis
Gelsolin
Definition
Gelsolin and gelsolin-like proteins are ubiquitous F-
actin fragmenting proteins. They perform three major
functions: (1) They sever actin filaments to smaller
fragments; (2) They cap free barbed ends thus
inhibiting elongation; and (3) They nucleate actin
polymerisation by stabilising dimers and trimers. Many
of these proteins are regulated by Ca2+ and the
phosphoinositide PIP2.
▶Actin Cytoskeleton
Gemins
Definition
Gemins are proteins that associate with the ▶SMN
(survival of motoneuron) complex in a stable and
stoichiometric manner. As these proteins and SMN
colocalize in nuclear structures called “gems”, they are
referred to as “Gemins”.
▶Spinal Muscular Atrophy
GenBank
Definition
GenBank, the NIH genetic sequence database, is an
annotated collection of all publicly available DNA
sequences, located at http://www.ncbi.nlm.nig.gov. It is
part of the International Nucleotide Sequence Database
Collaboration, which is comprised of the DNA
DataBank of Japan (DDBJ), the European Molecular
Biology Laboratory (EMBL), and GenBank at NCBI.
▶Protein Databases
Gene
Definition
Gene refers to a segment of DNA coding for a
polypeptide chain of amino acids. A gene includes
untranslated regions before and after the coding region
 Gene Annotation in Plants 643

and intervening (intronic) sequences. Nomenclature:
Human genes are abbreviated using italic capital letters
and numbers without spaces, dashes etc. according to
the approved gene symbols listed in the Human Gene
Nomenclature Database (http://www.gene.ucl.ac.uk/
cgi-bin/nomenclature/searchgenes.pl). Nomenclature
for murine gene symbols underlies the same rules,
but uses lower case letters after the first letter. Example:
PSEN1 = human gene for PS1; Psen1 = murine gene
for PS1.
Gene Annotation in Plants
S. R OMBAUTS , Y. VAN DE P EER , P IERRE R OUZÉ
Department of Plant Systems Biology, University of
Ghent, Ghent, Belgium
pierre.rouze@psb.ugent.be
Synonyms
Just as for gene and genome annotation in any other
organism, gene annotation in plants makes use of
algorithmic approaches based on statistics, artificial
intelligence and machine learning, recently complemen-
ted with homology based approaches depending on the
availability of sequence data, whether related genomes,
complete cDNAs or expressed sequence tags (ESTs).
Definition
All prokaryotic and eukaryotic organisms encode their
genetic information in their genome, built up from
DNA. The process of genome annotation consists of
finding and decoding the information encrypted on the
DNA molecules into known conceptual objects related
to biological entities and functions. In general,
annotation is mostly focused on finding genes, defining
their structure and assigning a function to the product
and the process resulting from the expression of each
gene. But annotation is not restricted to genes, as non-
coding RNAs, transposable elements, promoters and
enhancers also make up the genome and are essential to G
an understanding of the organization and the various
functions encoded by or embedded in genomes.
Characteristics
Gene annotation in plants is in essence not different
from gene annotation in human or mouse, except that
each genome although constituted by the same DNA
has its own style that needs to be captured by the
models used by the different prediction programs.
Anticipating which are the genome specific character-
istics, e.g. codon usage, gene density, length and
composition of introns and intergenic sequences, as
well as conservation of signals such as splice sites
(Table 1) are all important for building adequate
algorithms and for their proper training.

Gene Annotation in Plants. Table 1 Splice site prediction programs

Program Organism Method

GeneSplicer (152) Arabidopsis, human HMM + MDD
NETPLANTGENE (42) (▶http://www.cbs. Arabidopsis NN
dtu.dk/services/NetPGene/)
NETGENE2 (43) (▶http://www.cbs.dtu.dk/ Human, C.elegans, NN + HMM
services/NetGene2/) Arabidopsis

SPLICEVIEW (39) (▶http://l25.itba.mi.cnr. Eukaryotes Score with consensus

it/
webgene/wwwspliceview.html)
NNSPLICE0.9 (44) (▶http://www.fruitfly.
org/seq_tools/splice.html)
SPLICEPREDICTOR (40,153) (▶http://
bioinformatics.iastate.edu/cgi-bin/sp.cgi)
Drosophila, human or other NN
Arabidopsis, maize Logitlinear models: (i) score with
consensus; (ii) local composition

BCM-SPL (▶http://www.softberry.com/ Human, Drosophila, Linear discriminant analysis

berry.phtml; http://genomic.sanger.ac.uk/ C.elegans, yeast, plant
gf/gf.html)

HMM, hidden MM; MDD, maximal dependence decomposition; NN, neural networks
644 Gene Annotation in Plants
The effort undertaken by several laboratories world-
wide to sequence the Arabidopsis thaliana genome
led, by the end of 2000, to the first full catalogue of
genes present in a plant. This work also revealed that
plants have about the same number of protein gene loci
as vertebrate genomes (27,000 for Arabidopsis) but
also that about 5,000 genes remained unknown,
showing no homology to any known sequence in
databases (1). These genes could only be predicted
using ab initio prediction programs and need further
experiment to prove that they truly exist, which has
since been done for a number of them.
Genome annotation can be subdivided in two steps;
structural annotation will provide gene structures
and functional annotation as an as accurate as
possible prediction of the function of each gene. To
acquire structural annotation two main approaches
are combined, intrinsic and extrinsic methods. Intrinsic
(or ab initio) methods are all those methods that decipher
genome content based solely on statistical/lexical
models built by using human-curated data sets of
sequences from the organism under investigation. The
algorithms used for intrinsic approaches can be
subdivided into signal sensors or content sensors. Signal
sensors are algorithms that focus on the retrieval and
identification of functional sites such as e.g. splice
and translation initiation sites, transcription and poly-
adenylation signals and include methods such as
position weight matrices, neural networks and support
vector machines. Content sensors recognize regions
along a sequence that have local characteristics differing
from the surrounding sequence, include mainly methods
such as Markov models and all their variants and are
broadly used to distinguish coding from non-coding
regions.
The purpose of the multiplicity of methods used is to
decompose the problem of gene annotation into key
components and achieve the best results on each
component, which can thereafter be reassembled
towards a whole gene structure (2).
Extrinsic or comparative approaches on the contrary
rely on the availability of sequences from other
genomes and proteins, where regions that show enough
similarity between genomes are believed to have the
same biological meaning, as a result of common
ancestry. The advantage of comparative methods to
predict genes is that it allows the revealing of small and
novel genes without ambiguities and, more impor-
tantly, enables the detection of non-coding features that
can hardly be detected otherwise. One needs never-
theless to be careful as to the choice of organisms used
for comparative methods to achieve good results. Too
closely related genomes might not reveal the informa-
tion hidden in the genomes, as regions larger than
coding sequences will remain conserved, making the
delineation of gene structures impossible. If on the
other hand genomes are too distantly related, only a few
genes will keep significant similarity to be correctly
modeled or even be found. For plants, genome
sequences of one dicot, Arabidopsis, and one monocot,
rice are presently available. Unfortunately they
diverged some 200 million years ago and are therefore
too divergent for comparative annotation. Other
genomes that are currently being sequenced, poplar,
Medicago and Lotus for dicots, maize for monocots
will soon fill this gap.
Besides experimental approaches, functional annotation
can only be achieved through comparative methods
where the knowledge of genes from one organism can
be transposed to the genes and the genome of the
organism concerned. To achieve this, homology
searches and alignment programs are the main algo-
rithms used (Table 2). The quality of the databases
providing the data is of primary importance and is
currently the main limitation. The homology searches
are performed against protein, domain and motif
databases using BLAST or FastA or by using hidden
Markov profiles that show a better sensitivity and
specificity. One point that needs to be stressed is the
importance of consistency in genome annotation across
species. The gene ontology (GO) project is an attempt to
achieve this goal, through a hierarchical description of
the genes in a genome according to the functions of their
products at the various biological levels, from the
molecules to the biological processes and cellular
components with which they are associated. The
classification and standardized terminology of GO
(gene ontology) has been initiated in the annotation of
the D. melanogaster genome and it is hoped that GO
will become a community-curated entity, providing a
central frame and vocabulary for annotation.
Many gene prediction programs are publicly available;
many of them are referred to on the web site maintained
by W. Li (▶http://linkage.rockefeller.edu/wli/gene/
programs.html). Several reviews are available on this
topic too, among which that of Mathé et al. (2) is more
specifically oriented towards plants. The large variety
of gene prediction programs have the drawback that
one does not necessarily know which program to use in
which situation and which performs best depending on
the organism of interest. The issues of specificity, or the
ability to predict only real genes and sensitivity, or the
ability to predict all the genes present in a sequence,
have been addressed by Burset and Guigo (3), Rogic
et al. (4) and in the specific case of plants by Pavy
et al. (5). In those publications it has been made clear
that programs rarely performed well enough to be able
to predict all the genes and that 30–40% of the genes
were likely to be wrongly predicted even by the best
intrinsic methods. In addition, it is clear that the more
extrinsic data (of high quality) become available, the
better the annotation will be.
 Gene Annotation in Plants 645

Gene Annotation in Plants. Table 2 Homology-based gene prediction programs

Program Organism Databank or Alignment Gene

required input reconstr-
uction
AAT (66) (▶http://genome. Primates, cDNA, protein DDS (improved NAP, GAP2
cs.mtu.edu/aat. rodents, other BLASTX), DPS
html) (improved BLASTN)
ALN (62) Protein Tron code, PAM 250
CEM Two genomic BLASTX output, DP
(81) sequence WMM for sites
EbEST (▶http://ares.ifrc. Human, other dbEST BLASTN, 3'-UTR detection,
(73) mcw.edu/EBEST/ EST clustering, Smith assembly of
ebest.html) ± Waterman-based EST-tagged
gapped alignment exons
G
Est2genome EST or cDNA, ModiÆed Smith No
(74) preferably ± Waterman
BLASTN output Needleman-Wunsh
algorithm
GeneSeqer (▶http:// Arabidopsis, dbEST or EST Spliced alignment, Yes
(67,68) bioinformatics. maize, generic database or splice recognition
iastate.edu/ plant proteins with SplicePredictor
cgi-bin/gs.cgi) if missing EST
match
GeneWise (▶http://www. Human One Global alignment DP (dynamite)
(60) sanger.ac.uk/ protein or a HMM translated ORF/
Software/Wise2/ profile protein
genewiseform.
shtml)
GENQUEST (▶http://compbio. dbEST, Smith-Waterman,
ornl.gov/Grail-bin/ SwissProt, Blast, Fasta
EmptyGenquest Prosite,
Form) BLOCKS, GSDB
ICE (64) (▶http://theory. dbEST, OWL Look-up DP
lcs.mit.edu/ice)
INFO (63) Nr 25mer look-up table, No
protein/protein
alignments scored
with PAM 40, PAM
120, PAM 250,
BLO62
ORFgene2 (▶http://l25.itba. Human, mouse, SwissProt BlastP, WAM for Compatibility
(61) mi.cnr.it/ Drosophila, splice sites, graph, DP

webgene/ Aspergillus, identity score on
wwworfgene2. Arabidopsis, frequencies of
html) Caenorhabditis dipeptides
PredictGenes (▶http://cbrg.inf. Invertebrates, SwissProt PAM 250 DP
ethz.ch/Server/ vertebrates,
subsec- prokaryotes,
tion3_1_8.html) plants
646 Gene Annotation in Plants

Gene Annotation in Plants. Table 2 Homology-based gene prediction programs (Continued)

Program Organism Databank or Alignment Gene

required input reconstr-
uction
PRO- (▶http://www. Vertebrates One homologous Protein/protein DP
CRUSTES hto.usc.edu/ protein alignments scored
(59) software/ with PAM 120
procrustes/
wwwserv.html)
Pro-Gen (83) (▶http://www. Two genomic DP
anchorgen.com/ sequences
pro_gen/ Alignment of
pro_gen.html) translated
sequences scored
with PAM 120
ROSETTA (▶http:// Human, mouse Two genomic GLASS (global DP
(80) crossspecies. sequences alignment system),
lcs.mit.edu/) PAM 20, Genscan
method for splice
sites
SGP-1 (82) (▶http://soft.ice. Vertebrates, Two genomic Local alignment DP
mpg.de/sgp-1) angiosperms sequences or a
pairwise local
alignment output
SIM4 (69) All eukaryotes cDNA/genomic HSP from Blast No
SLAM (85) (▶http://baboon. Human, mouse Two genomic Generalized pair DP
math.berkeley. sequences HMM
edu/
syntenic/
slam.html)
Spidey (70) (▶http://www. Vertebrates, One genomic Two Blasts: high
ncbi.nlm.nih.gov/ Drosophila, sequence/set of stringency and low
IEB/Research/ C. elegans, mRNAs stringency
Ostell/Spidey/ plant
index.html)
SYNCOD (72) (▶http://l25.itba. Human, mouse, BLASTN output Silent/replacement No
mi.cnr.it/
Drosophila, ratio,Monte Carlo
webgene/ Arabidopsis, simulations
wwwsyncod. Aspergillus,
html) Caenorhabditis
TAP (75) (▶http://sapiens. Human, mouse, dbEST WU-BLASTN, SIM4 Yes
wustl.edu/
zkan/ Drosophila
TAP/)
Utopia (84) All eukaryotes Two genomic Local alignment Yes
sequences

The above described programs are different from Clinical Relevance

annotation platforms, which do not attempt to make
predictions themselves, but present the results from
different prediction programs graphically and thus have
to be seen as complementary tools that facilitate human
driven annotation.
The use of plants for medical purposes dates back
thousands of years and was part of the magic exerted by
medicine man, sorcerers and druids. Even now many
pharmaceutical compounds that are commonly used are
or were once extracted from plants. Genetic engineering
 Gene Annotation in Plants 647

Gene Annotation in Plants. Table 3 Ab initio gene prediction programs (possibly with homology integration)

Program Organism Gene elements Gene model Homology

DAGGER (91) Site scores Directed acyclic
graphs
EuGène (31) (▶http://www.inra. Arabidopsis Three-periodic DP EST/cDNA,
fr/bia/T/EuGene) IMM for exons, one protein
IMM for introns,
one for intergenic
regions, one for
UTR. NetGene2/
SplicePredictor for
splice sites
GeneId3 (89) (▶http://www1. Vertebrates, plants Rule-based DP EST
imim.es/geneid. method; WAM,
html) discriminant G
analysis.
GENEFINDER (▶http://genomic. Human, mouse, Linear DP Protein
(28): FGENE, sanger.ac.uk/gf/gf. Drosophila, discriminant
FEX html;;▶http://www. Caenorhabditis analysis
softberry.com/ elegans, yeast,
berry.phtml) dicots, monocots,
Schizosaccharomy-
ces pombe,
Neurospora crassa
GENEFINDER Log likelihood ratio DP
(Green) score matrix on
MM
GeneGenerator Maize Logitlinear models DP
(92) for splice sites,
start; 3rd to 5th
order MM for
exons and introns
GeneMark (29) (▶http://opal. Prokaryotes, 5th order MM No
biology.gatech. eukaryotes (homogeneous for
edu/GeneMark/ introns, three-
genemark24.cgi) periodic for exons)
GeneMark. (▶http://opal. Human, mouse, 5th order MM GHMM, DP Under
hmm (35) biology.gatech. Drosophila, Gallus (homogeneous for development
edu/GeneMark/ gallus, Arabidopsis, introns, three-
eukhmm.cgi) rice, maize, periodic for exons)
Chlamydomonas
reinhardtii,
C. elegans,
Hordeum vulgare,
Triticum aestivum
GeneModeler (▶ftp://ftp.tigr.org/ Eukaryotes Nucleotide and Rule-based
(57) pub/software/gm/) dinucleotide method
composition,
consensus for
splice sites
GeneParser (▶http://beagle. Vertebrates NN DP EST
(27) colorado.edu/

eesnyder/
GeneParser.html)
648 Gene Annotation in Plants

Gene Annotation in Plants. Table 3 Ab initio gene prediction programs (possibly with homology integration)
(Continued)
Program Organism Gene elements Gene model Homology
Genie (44,96) (▶http://www. Drosophila, human, NN GHMM, DP Protein
fruitøy.org/ other
seq_tools/genie.
html)
GenLang (154) (▶http://www.cbil. Vertebrates, Chart parsing, DP
upenn.edu/ Drosophila, dicots
genlang/ Grammar rules,
genlang_home. WAM, hextuple
html) frequencies
GenomeScan Vertebrates Genscan method, GHMM, DP Protein
BLASTP or
BLASTX
GENSCAN (30) (▶http://genes.mit. Vertebrates, WAM for acceptor; GHMM, DP Protein,
edu/GENSCAN. Arabidopsis, maize MDD for donor; 5th GenomeScan
html) order MM (99)
(homogeneous for
introns, three-
periodic for exons)
GENVIEW2 (▶http://l25.itba. Human, mouse, Linear DP
(25) mi.cnr.it/ Diptera combination,

webgene/ dicodon statistic
wwwgene.html)
GlimmerM (▶salzberg@cs. Small eukaryotes, Three-periodic DP
(33,34) jhu.edu) Arabidopsis, rice IMM for exons
(order 0-8), IMM for
introns, 2nd order
MM for splice sites
GRAIL/GAP3/ (▶http://compbio. Human, mouse, NN DP EST, cDNA
GrailEXP ornl.gov/public/ Arabidopsis,
(90,155) tools/) Drosophila
GRPL (97) Human, Drosophila, Reference point GHMM, DP Protein
Arabidopsis logistic for splice
sites, 5th order MM
(homogeneous for
introns, three-
periodic for exons)
HMMgene (98) (▶http://www.cbs. Vertebrates, Three-periodic 4th CHMM
dtu.dk/services/ C. elegans order MM for
HMMgene/) exons, 3rd order
MM for introns
MORGAN (48) (▶http://www.cs. Vertebrates Decision tree DP
jhu.edu/labs/ system
compbio/morgan.
html)
MZEF (26) (▶http://argon. Human, mouse, Quadratic No
cshl.org/ Arabidopsis, fission discriminant
geneænder/) yeast analysis
 Gene Annotation in Plants 649

Gene Annotation in Plants. Table 3 Ab initio gene prediction programs (possibly with homology integration)
(Continued)
Program Organism Gene elements Gene model Homology
SORFIND (24) Matrix method for No
start and splice
sites, hexamer
usage (Fourier
measure)
Twinscan (100) Mouse, human Genscan method; GHMM Genomic
5th order MM for sequence
UTR and inter-
genic, WAM for
acceptor sites
VEIL (47) (▶http://www.cs. Vertebrates HMM DP
jhu.edu/labs/ G
compbio/veil.html)
Xpound (156) (▶http://bioweb. Human Three-periodic 1st HMM
pasteur.fr/seqanal/ order MM for
interfaces/ exons, 1st order
xpound-simple. MM for introns and
html) intergenic

of plants to produce biopharmaceuticals is much more
recent. Identification and mining of genes involved in
the metabolism of such compounds in medicinal plants
and in model systems is then an important issue. Gene
annotation is the basic step for it, and its high quality can
speed up and better focus the research on genes of
potential pharmaceutical importance, especially the
ones involved in secondary metabolism.
Besides being a source of pharmaceuticals, plants are
also seen as a convenient and inexpensive way to
produce proteins and other molecules of medicinal
interest, a practice often referred as “pharming”. Most
genes can indeed be expressed in a wide range of
organisms. Therefore expression systems need to be
tested for efficiency, cost and the biological activity of
the products as the demand for high production levels
at low cost is important to make modern medicine
available for an ever expanding world population.
Modified mammalian cells are in that respect valuable
as far as the biological activity is concerned but their
use is limited because of expensive culturing and
difficult scaling up. The advantage of microbial
organisms is that as well as the easier modification of
the organisms, larger quantities can be manufactured
using industrial bioreactors. Their disadvantage is that
proteins do not become correctly glycosylated for
usage in humans and that some proteins lack the proper
folding and disulfide bridges. Plants on the other hand,
have many potential advantages for the production of
recombinant proteins and the engineering of pharma-
ceuticals. First, growing plants is more economical than
industrial facilities with bioreactors. Second, starting
from plant material is already documented. Third,
purification is not required when the therapeutic product
can be administrated as food. Fourth, plants can be
directed to target proteins into organs and intracellular
compartments that confer better and more stable
conservation (e.g. seeds). Fifth, the production levels
that can be reached using modified plants approaches
industrial scales. Last, the risk of human health threats
due to contamination is reduced to a minimum.
The first recombinant therapeutic proteins, successfully
produced in tobacco plants were different forms and
parts of immunoglobulin (Ig), making plants virtually
unlimited sources of inexpensive monoclonal antibo-
dies. Ig produced in plants can effectively prevent
infectious diseases and cancers in the mouse model or
be used for in vivo tumor imaging. None are available
commercially yet.
Glycosylation of proteins though remains a potential
problem, as N-glycans in plants are structurally more
diverse with a majority of oligosaccharides having
β-(1,2)-xylose and α-(1,3)-fucose linked to the Man3-
GlcNAc2 core. These are not found in mammalian
N-glycans, while plant engineered proteins are lacking
the sialic acid that represents 10% of the mouse sugar
650 Gene Chip Technology
content. These differences appear to have no influence
on the binding affinities of Ig in vitro, however, there is
some concern about the immunogenicity and allergeni-
city of plant engineered proteins. To circumvent this
problem, proteins have been made edible, suppressing
the problem since plant materials are ubiquitous in the
human diet, the rationale being that plant engineered
proteins can trigger a mucosal immunity. A high
dosage is needed to maximize the chances of the
protein reaching the gut intact. Therefore high expres-
sion levels in the plant tissues must be achieved. If
annotation proper is not giving an answer to this issue,
taking into account the features selected for making
efficient, ab initio prediction software can be used as a
guide. This tells us what makes a gene most adapted to
its genome style and in turn most expressed. The latest
approach to achieving high expressions levels was to
express the proteins in the chloroplast. Expressing
proteins in chloroplasts is an alternative for high
expression with some advantages such as the absence
of silencing of the engineered gene and the envir-
onmentally friendliness of the process, as spreading of
chloroplast-targeted genes to other plants becomes very
unlikely (6, 7).
References
1. The Arabidopsis Genome Initiative (2000) Analysis of the
genome sequence of the flowering plant Arabidopsis
thaliana. Nature 408:796–815
2. Mathé C, Sagot M-F, Shiex T et al (2002) Current methods
of gene prediction, their strengths and weaknesses. Nucl
Acids Res 19:4103–4117
3. Burset M, Guigo R (1996) Evaluation of gene structure
prediction programs. Genomics 34:353–367
4. Rogic S, Mackworth A, Ouellette F (2001) Evaluation of
genefinding programs on mammalian sequences. Genome
Res 11:817–832
5. Pavy N, Rombauts S, Déhais P et al (1999) Evaluation of
gene prediction software using a genomic data set:
application to Arabidopsis thaliana sequences. Bioinfor-
matics 15:887–899
6. Daniell H, Streatfield SJ, Wycoff K (2001) Medical
molecular farming: production of antibodies, biopharma-
ceuticals and edible vaccines in plants. Trends Plant Sci
6:219–226
7. Price B (2003) Conference on Plant-Made Pharmaceu-
ticals. IDrugs 6:442–445 http://www.cpmp2003.org/
Gene Chip Technology
▶Biochip Technology
▶DNA Chip Technology
▶Gene Chip Technology
▶Microarray Technology
Gene Chip Technology and Its
Application to Molecular Medicine
H EIKE Z IMDAHL , N ORBERT H ÜBNER
Max Delbrück Center for Molecular Medicine (MDC),
Berlin, Germany
nhuebner@mdc-berlin.de
Definition
DNA ▶microarrays, or DNA chips consist of thou-
sands of individual DNA sequences arrayed at a high
density on a single matrix, usually glass slides or quartz
wafers, but sometimes on nylon substrates. Probes with
known identity are used to determine complementary
binding, thus allowing the analysis of ▶gene expres-
sion, DNA sequence variation or protein levels in a
parallel format.
Description
The analysis of gene expression levels in a certain
tissue, cell type or stage of development provides
essential information for any attempt to understand
biological processes and to isolate differently regulated
genes associated with a disease phenotype. Gene
expression profiling may allow linkage of specific
genes to disease susceptibility and drug response and
the identification of genes and components of complex
molecular pathways.
Various methods are available for detecting and
quantitating gene expression levels, e.g. Northern blot,
RNase protection assay, differential display, represen-
tational difference analysis (RDA) and serial analysis
of gene expression (SAGE). For the high throughput
analysis of gene expression on a global level, the
microarray has emerged.
Microarrays usually contain thousands of arrayed
▶cDNAs or ▶oligonucleotides.
After completion of the human genome sequencing, the
estimated number of human genes is
30,000, but
there are more ▶mRNA species that arise e.g. by
▶alternative splicing. Most investigators use in their
experiments arrays comprising a substantial subset of
the entire transcriptome. Gene expression analysis
arrays are commercially available for a number of
organisms, e.g. H. sapiens, M. musculus, R. norvegi-
cus, Arabidopsis, C. elegans and Drosophila.
Expression profiling using microarrays represents the
most efficient way of measuring gene expression –
alterations in transcript levels in tissues or entire
genomes can be simultaneously assayed (1-3). In
addition to the capacity for parallel analysis of
thousands of genes with a minimal amount of sample,
 Gene Chip Technology and Its Application to Molecular Medicine 651

Gene Chip Technology and Its Application to Molecular Medicine. Figure 1 Schematic representation of the
experimental strategy of cDNA and oligonucleotide arrays (modified according to Schulze A, Downward J, Nat Cell
Biol 2001; 3:E190-E195(36). cDNA arrays: For the array preparation, inserts from cDNA clones (libraries) are
amplified and PCR products are spotted onto glass slides or nylon membranes at specific positions using arraying
robots. Target preparation: RNA from the two tissues or cell populations under comparison is used to synthesize
cDNA in the presence of either radioactively labeled nucleotides or nucleotides labeled with two different fluorescent
dyes, Cy3 and Cy5, during cDNA synthesis. Samples labeled with two different fluorescent dyes are mixed and are
hybridized to the array, whereas samples labeled radioactively (or with one fluorescent dye) are hybridized to
separate arrays. Signal intensity ratios are obtained by comparing either two different signals (Cy5/Cy3) on one array
or by comparing signals of genes represented on two arrays (array 1/array 2). High-density oligonucleotide arrays:
For the array preparation sequences of 16–20 short oligonucleotides (typically 25mers) are chosen from the mRNA
reference sequence of each gene. Light-directed, in situ oligonucleotide synthesis is used to generate high-density
probe arrays, usually containing over 300,000 individual elements. Target preparation: polyA+ RNA is prepared from
different tissues and used to generate double-stranded cDNA carrying a transcriptional start site for T7 DNA
polymerase. During in vitro transcription, biotin-labeled nucleotides are incorporated into synthesized cRNA
molecules. Each cRNA sample hybridizes separately to the array. Target binding is detected by staining with a
fluorescent dye coupled to streptavidin. Signal intensities on different arrays are used to calculate relative mRNA
abundance for genes represented on the array.

other advantages include the high sensitivity, the
economy of size (miniaturization) and the use of non-
toxic chemicals.
Despite the enormous potential of the technology a
number of issues attenuate the power of microarrays,
e.g. the control for biological and environmental factors
and fluctuations, the validation of data, the need for
computational tools to analyze the vast amount of data
and to enable comparisons between arrays and finally
the high costs of commercial microarrays.
Conceptually different approaches to the develop-
ment of microarray technology have resulted in the
generation of two different array formats, oligonucleo-
tide and cDNA (‘targets’) arrays (Fig. 1). The ‘target’
652 Gene Chip Technology and Its Application to Molecular Medicine
cDNAs (or oligonucleotides) are immobilized on nylon
membranes or glass slides. cDNA arrays are generated
by arraying PCR products of ▶cDNA libraries or clone
collections usually onto glass or nylon substrates.
cDNA arrays offer flexibility in the choice of arrayed
elements and lower costs, particularly for the prepara-
tion of smaller, customized arrays for specific inves-
tigations of a small number of genes. In addition,
arraying of unsequenced clones from cDNA libraries
can be useful for gene discovery.
The advantage of the in situ synthesized, high-density
oligonucleotide arrays (Affymetrix, www.affymetrix.
com) is the high reproducibility of in situ synthesis on
oligonucleotide chips, allowing an accurate compar-
ison of signals generated by samples hybridized to
separate arrays.
Microarray Fabrication
The concept of being able to characterize large numbers
of clones by ▶hybridization analyses of high-density
arrayed cDNA libraries was established more than a
decade ago (1).
In general, arrays are described as macroarrays or
microarrays, the difference being the size of the sample
spots and size of the array. Macroarrays are usually
printed on nylon membranes, contain sample spot sizes
of about 250 microns or larger and can easily be imaged
by existing gel and blot scanners. The sample spot sizes
in microarrays are typically less than 200 microns in
diameter and probes are attached onto glass-like
substrates. Depending on the arrayed material, the most
commonly used array platforms are cDNA arrays and
oligonucleotide arrays (▶http://www.gene-chips.com/).
For the generation of a cDNA microarray (4), cDNA
clones representing as many unique transcripts as
possible are either selected within ▶EST data (▶http://
www.ncbi.nlm.nih.gov/ UniGene and ▶http://www.
tigr.org/tdb/tgi/hgi) or tissue-specific cDNA libraries
are constructed or ordered (www.rzpd.de). The cDNA
clone inserts are PCR amplified from plasmid DNA or
amplified from bacterial cultures. In high-throughput
applications the amplification of clones in cultures
stored in 384-well plates is more cost efficient and less
labor intensive than amplification from plasmid DNA.
PCR products can be purified to remove unincorpo-
rated nucleotides and primers, e.g. by filtration using
silica systems. Amplified PCR products are spotted,
usually in denaturing or high-salt buffer, onto glass
slides or nylon membranes using robotic systems.
Spots are typically 100–300 μm in size and are spaced
about the same distance apart. Using this technique,
arrays consisting of more than 30,000 cDNAs can be
fitted onto the surface of a conventional microscope
slide.
Commercially available cDNA arrays consisting of
thousands of distinct sequence-verified genes are
provided by companies, like Clontech, Agilent, Incyte
Genomics, Invitrogen (Research Genetics) or non-
profit, public, limited institutions like the German
Resource Center (▶www.rzpd.de).
For oligonucleotide arrays, short oligonucleotides from
20–25 mers (Affymetrix) up to 60mers (Agilent
Technologies) are usually synthesized in situ, either
by photolithography onto silicon wafers (high-density
oligonucleotide arrays from Affymetrix) or spotted by
ink-jet technology (e.g. Agilent Technologies). Alter-
natively, presynthesized oligonucleotides can be
spotted onto glass slides or glass-like matrices.
Oligonucleotide arrays have certain advantages over
cDNA arrays, namely high reproducibility and the facts
that the sequence information (usually EST sequences)
alone is sufficient to generate the DNA to be arrayed.
Furthermore the oligonucleotide arrays can be designed
to allow both ▶SNP and alternative splicing analysis
and do not require amplification and purification of
cDNA fragments (5).
Since short oligonucleotides may result in less specific
hybridization and reduced sensitivity, the arraying of
presynthesized longer oligonucleotides (50–100 mers)
has recently been developed to counteract these
disadvantages. However, the high costs of commer-
cially available, in situ-synthesized oligonucleotide
arrays and their accessories (spotters, scanners, analysis
software) as well as the time-consuming design of
oligonucleotide sets may limit their use for academic
laboratories.
Affymetrix has pioneered the oligonucleotide array
technology and generated a number of different
commercially available arrays for various organisms
(▶www.affymetrix.com).
The Microarray Experiment
Careful experimental design of the microarray will
ensure the maximal potential gain in efficiency and is
particularly important if the resulting experiment is to
be maximally informative, given the effort and the
resources.
There are many protocols and different types of
systems available; the basic procedure for a large-scale
measurement of gene expression involves the prepara-
tion of total or mRNA from the biological sample(s)
under investigation (e.g. ‘candidate’ organ) and the
hybridization of copied ‘labeled’ RNA to the array
(Fig. 1).
In most cases, the extracted mRNA is converted to
cDNA (▶reverse transcription), labeled and hybridized
to the DNA elements on the array surface of the array.
In some cases (e.g. hybridization of Affymetrix chips)
the cDNA is labeled during in vitro transcription and
▶cRNA is hybridized. To ensure a high reproducibility,
fluctuations in sample preparation and hybridization
need to be reduced to a minimum. Major sources of
 Gene Chip Technology and Its Application to Molecular Medicine
random fluctuations to be expected are in probe, target
and array preparation, e.g. in mRNA preparation,
reverse transcription, labeling, target volume, hybridi-
zation parameters, overshining effects, non-specific
background, variations in pin geometry during spotting
of cDNA, slide inhomogeneities and image analysis
(6). Replicates of each experiment should be used in
order to reduce variability and to differentiate between
experimental variation and real expression differences.
Suitable internal controls ensure quality control
measurements for samples and array. After the
hybridization process, intensity signals from the
hybridized RNA samples are detected by phospho-
imaging or fluorescence scanning and independent
images are generated.
Analysis and Data Management
Microarray experiments generate large and complex
data sets, e.g. lists of spot intensities and intensity
ratios. Basically, the data obtained from microarray
experiments provide information on the relative
expression of genes corresponding to the mRNA
sample of interest. Computational and statistical tools
are required to analyze the large amount of data in order
to address biological questions.
Once images of hybridized microarrays are processed,
arrayed spots are identified, relative signal intensities
for each spot are measured and background intensity is
subtracted. Signal intensities are usually normalized to
compensate for experimental variability and to ‘bal-
ance’ the signals from the two samples being compared
(7). All normalization techniques assume that all or a
subset of spots (e.g. genes) on the array have an
average expression ratio equal to one. The normal-
ization factor is then used to adjust the data (signal
intensities) from the two samples and to ensure that that
the total quantity of RNA hybridized to the array is the
same. Finally mean spot or transcript intensities are
calculated and ratios of intensities are used to account
for relative expression differences. In a simple pairwise
comparison of gene expression between two samples,
the results can be shown in plots of the intensities or the
log of the intensity ratios. Scatter plots are widely used
to make the observed differential expression visible.
A variety of software tools utilizing different mathe-
matical algorithms to perform microarray image
analysis are available; a detailed discussion of the
analytical tools is beyond the scope of this article (see
reviews 8, 9).
The first information obtained after data analysis and
extraction of gene expression analysis is identification
of those genes with significant differential expression
in two samples or in a time series after a given
treatment. To address the full potential of genome-scale
experiments a ▶cluster analysis is performed to
analyze the entire repertoire of transcripts. Basically,
653
cluster analysis uses a standard statistical algorithm to
arrange and organize genes according similar patterns
of gene expression (10).
The data management is of particular importance for
further downstream analyses. Databases are an im-
portant resource for storing and retrieving the vast
amount of data generated in a microarray experiment.
A number of gene expression databases have been
generated and are accessible to the public (e.g. Gene
Expression Omnibus at the National Center for
Biotechnology Information, www.ncbi.nlm.nih.gov/
geo and ArrayExpress at the European Bioinformatics
Institute, EMBL-EBI www.ebi.ac.uk/ arrayexpress/).
Due to the experimental variation inherent in micro-
array experiments, the validation of the results by
alternative techniques, such as quantitative real-time G
PCR or Northern blotting is advisable.
The most challenging part is “making sense” of the
complex data retrieved, including to distinguish, whether
the gene’s expression change is part of the etiology of the
disease or part of the pathology of the disease. The first
task is the identification of the relevant gene(s), the
prediction of protein function and the identification of the
genetic variation that modulates gene expression,
including the number of loci involved, the effect of each
single locus and the interaction between loci.
Clinical Applications
Microarrays certainly have multiple applications many
of which will develop and evolve over time. Although
the first application of microarrays was in monitoring
gene expression, the strategy of using arrayed biomo-
lecules to examine a biological sample is generally
applicable, e.g. for mutation screening, ▶polymorph-
ism analysis, mapping and other applications. An
increasing number of human diseases result from
alterations in DNA sequence and/or altered gene
expression patterns. Therefore, information about up-
and down-regulation of multiple genes is important for
identification of disease genes, understanding of gene
function and for potential therapeutic and/or diagnostic
applications. The first clinical application may be the
use of microarrays for the molecular classification of
cancer (see disease diagnosis).
Although gene expression analysis is a powerful
approach to identify characteristics of disease states
or signaling pathways, it should be noted that gene
expression levels often represent complex, quantitative
phenotypes, influenced by environmental and genetic
factors and the regulation of mRNA levels is only one
aspect of biological control. Protein levels are also
controlled at several post-transcriptional steps and
protein activity is controlled by post-translational
modification. A complete picture may be obtained by
studying the global level of cellular proteins by
proteomics (e.g. protein microarrays).
654 Gene Chip Technology and Its Application to Molecular Medicine
Disease Diagnosis
The real promise of the examination of gene expression
using microarrays is to identify genes, which are
consistently up- or down-regulated and play significant
roles in the development and progression of disease.
For example, the over-expression of certain genes is
correlated with a certain type of cancer. By monitoring
expression a new generic approach for cancer classi-
fication has been established and a comprehensive and
commonly accessible catalog of gene expression
profiles will make an accurate multiclass cancer
classification feasible. Improvement in precise, objec-
tive and systematic tumor classification at the mole-
cular level will advance cancer treatment (3, 11).
Pharmacogenomics
The information about gene expression and sequence
variation will also have an impact on many aspects of
the drug discovery process and on drug efficacy (12).
Use for prognostic markers or to identify therapeutic
targets has great potential.
Microarrays are used at specified steps in the process of
drug development:
. Target discovery, to identify genes or pathways with
altered expression in diseased human tissues or in
animal models of disease.
. Target validation, to determine that a gene product is
causative of disease symptoms or that activation of
the target protein ameliorates disease symptoms. An
agonist/activator or an inhibitor, which may be
therapeutic, could be identified using microarrays.
. Compound optimization, to screen a series of
therapeutic drug candidates to find the compounds
that are most specific for the target protein and those
that cause unintended effects.
. Drug metabolism, to predict whether a drug
candidate will cause drug–drug interactions.
. Drug efficacy, to identify the individual mode of
action or adverse effects of a given drug.
. Microarrays should therefore prove useful in under-
standing the mechanistic basis of action of many
drugs.
Toxicological research: Toxicogenomics
Toxicogenomics is concerned with the identification of
potential human and environmental toxicants and with
finding correlations between toxic responses to tox-
icants and changes in the genetic profiles of the objects
exposed to such toxicants through the use of genomics
resources. DNA microarrays allow the identification of
highly sensitive and informative markers for toxicity,
by monitoring the expression levels of thousands of
genes simultaneously.
Therapeutic Consequences
It has been suggested that microarrays will be routinely
used not only in the selection, assessment and quality
control of the best drugs for pharmaceutical develop-
ment and for disease diagnosis, but also for monitoring
disease status and the outcomes of therapeutic inter-
ventions (11-15). Microarrays may be of great benefit
with respect to personalized medicine, i.e. to select the
most likely effective drug, to individualize dosing and
to minimize the safety risk for cost effective health care
management. Microarrays may also be useful to
identify bacterial strains that are resistant to known
antibiotics to avoid a lack of response in certain
patients.
One important aspect is to find a correlation between
therapeutic responses to drugs and the genetic profiles
of patients to address questions, such as why do some
drugs work better in some patients than in others and
why some drugs may even be highly toxic to certain
patients?
Additionally to gene expression profiling, microarrays
are especially suited for high-throughput genotyping to
examine sequence variation, i.e. to screen SNPs (single
nucleotide polymorphisms). An individual’s genotype
or genetic profile may allow 1. determination of disease
association and assessment of disease risk and 2.
determination of dosage and type of drug. This will
enable the selection of the most appropriate and
efficient drug and reduce chances of an adverse drug
reaction (16, 17).
To realize the potential of microarrays, it will be a
challenge to develop a cooperative framework that
meets the requirements of basic research and clinical
medicine.
▶Microarray Data Analysis
References
1. Lennon GG, Lehrach H (1991) Hybridization analyses of
arrayed cDNA libraries. Trends Genet 7:314
2. Schena M, Shalon D, Heller R et al (1996), Parallel
human genome analysis: microarray-based expression
monitoring of 1000 genes. Proc Natl Acad Sci USA
93:10614
3. De Risi J, Penland L, Brown PO et al (1996) of a cDNA
microarray to analyse gene expression patterns in human
cancer. Nat Genet 14:457
4. Schena M, Shalon D, Davis RW et al (1995) Quantitative
monitoring of gene expression patterns with a comple-
mentary DNA microarray. Science 270:467
5. Lockhart DJ, Dong H, Byrne MC et al (1996) Expression
monitoring by hybridization to high-density oligonucleo-
tide arrays. Nat Biotechnol 14:1675
6. Lee ML, Kuo FC, Whitmore GA et al (2000) Importance
of replication in microarray gene expression studies:
statistical methods and evidence from repetitive cDNA
hybridizations. Proc Natl Acad Sci USA 97:9834

7. Schadt EE, Li C, Ellis B et al (2001) Feature extraction
and normalization algorithms for high-density oligonu-
cleotide gene expression array data. J Cell Biochem 37:120
8. Butte A (2002) The use and analysis of microarray data.
Nat Rev Drug Discov 1:951
9. Quackenbush J (2001) Computational analysis of
microarray data. Nat Rev Genet 2:418
10. Eisen MB, Spellman PT, Brown PO (1998) Cluster
analysis and display of genome-wide expression pat-
terns. Proc Natl Acad Sci USA 95:14863
11. Ramaswamy S, Ross KN, Lander ES et al (2003) A
molecular signature of metastasis in primary solid
tumors. Nat Genet 33:49
12. Gerhold DL, Jensen RV, Gullans SR (2002) Better
therapeutics through microarrays. Nat Genet 32:547
13. Golub TR, Slonim DK, Tamayo P et al (1999) Molecular
classification of cancer: class discovery and class prediction
by gene expression monitoring. Science 286:531
14. Jain KK (2000) Applications of biochip and microarray
systems in pharmacogenomics. Pharmacogenomics 1:289
15. Marton MJ, De Risi JL, Bennett HA et al (1998) Drug
target validation and identification of secondary drug
target effects using DNA microarrays. Nat Med 4:1293
16. Shi MM (2002) Technologies for individual genotyping:
detection of genetic polymorphisms in drug targets and
disease genes. Am J Pharmacogenomics 2:197
17. Guzey C, Spigset O (2002) Genotyping of drug targets: a
method to predict adverse drug reactions? Drug Saf
25:553
Gene Cluster
Definition
Gene cluster refers to a set of co-regulated genes,
presumably belonging to the same functional class.
▶RNAi Interference in Mammalian Cells
Gene Conversion
Definition
Gene conversion refers to a non-reciprocal, limited
transfer of sequence information from one chromo-
some to another, or from one chromosome region to
another region, of the same chromosome. The donating
sequence is copied to the receiving chromosome, which
is thus “converted”. The donating sequence remains
unchanged.
▶Chromosomal Instability Syndromes
▶Spinal Muscular Atrophy
Gene Duplications 655
Gene Dosage Analysis
Definition
Gene dosage analysis is aimed at quantifying the copy
number of a gene in an individual.
▶Spinal Muscular Atrophy
Gene Duplications
S EBASTIAN M. S HIMELD
Department of Zoology, University of Oxford, G
Oxford, UK
sebastian.shimeld@zoo.ox.ac.uk
Synonyms
segment duplication, chromosome duplication,
▶trisomy and polyploidy. Sister genes separated by
duplication are referred to as paralogues.
Definition
The term gene duplication describes a situation where a
single ancestral gene has been copied such that two (or
more) copies of that gene now exist in a single genome.
Gene duplications are saltatory events in genome
evolution in that the change occurs in one generation,
with the parents having one copy while the offspring
have two copies. Therefore, as with other genetic
changes, gene duplications typically first exist in one
individual. Whether they spread to become fixed in a
population or are purged will primarily depend upon
the effect they have on the organism’s ▶fitness. At a
practical level, gene duplications that have become
fixed within a taxon are recognised retrospectively by
the presence of homologous genes within one genome.
These are best known in the context of gene families,
groups of genes of related sequence inferred to have
originated by repeated gene duplication from a single
ancestral gene.
Characteristics
The Origin of Duplicated Genes
The prevalence of gene families in eukaryotic and
prokaryotic genomes suggests gene duplication has
played a prominent role in evolution. How do gene
duplications occur? Probably the most common event
is tandem duplication, as evidenced by the prevalence
of homologous genes situated in tandem in most
genomes. Tandem duplication probably occurs most
frequently due to mis-recombination at ▶meiosis,
656 Gene Duplications
although other factors might also be important.
Repeated tandem duplication without purging of
duplicated genes results in ▶gene clusters, that is a
group of related genes situated next to each other in the
genome. Such gene clusters can be evolutionarily
ancient (for instance the ▶Hox gene cluster), but are
more commonly of relatively recent origin, for example
the cluster of zinc finger genes on chromosome 19q12.
Genes can also duplicate in other ways. Single genes
can be duplicated elsewhere in the genome by several
potential mechanisms, including via piggybacking on
adjacent active retrotransposons or insertion of a
reverse transcribed mRNA into the genome by a viral
reverse transcriptase. Several mechanisms allow multi-
ple genes to be duplicated simultaneously. Sections of
DNA containing multiple genes can duplicate either in
tandem or elsewhere in the genome (for example by
transposition). Evidence suggests that recently dupli-
cated segments form an estimated 5% of the human
genome, suggesting this is a frequent and ongoing
process (1). These are also known as segmental or
block duplications. Duplication of an entire chromo-
some with all its incumbent genes can also occur.
Although this is strongly selected against in humans
and typically unviable (an exception is trisomy of
chromosome 21), some other lineages appear to be able
to tolerate such duplications more easily and aberrant
chromosome numbers are especially common in
flowering plants. Finally, there is evidence that whole
genome duplication has occurred in several lineages,
including in yeast, plants and salmonid fish (2). There
is also evidence that genome duplication occurred early
in vertebrate evolution and therefore that all vertebrates
including humans are ancestrally polyploid. Duplica-
tion of an entire genome includes duplication of all its
incumbent genes.
The Fate of Duplicated Genes
When examining the genomes of living organisms, we
infer the presence of previous gene duplications by the
existence of genes with homologous sequences. By their
nature, these represent gene duplications that have
become fixed within a population such that all members
of a species (or higher taxonomic division) possess both
copies. The route from the initial gene duplication to
▶fixation or loss is affected by several factors. Some
gene duplications confer an obvious ▶selective advan-
tage and spread rapidly through a population, for
example the tandem amplification of esterase genes in
mosquitoes can confer a degree of resistance to specific
pesticides. Conversely, fixation of a duplicated gene
within a population is by no means certain. Many gene
products are required at a specific dosage and duplica-
tion disrupts this balance. This is likely to reduce the
fitness of the individual carrying the gene duplication,
leading to it being rapidly purged from the population.
For many genes, however, it is likely that duplication has
little or no effect on fitness and is effectively neutral. A
common way to view the fate of duplicated genes in this
context was as a race between the accumulation of
mutations that eventually silence one gene (turning it
into a ▶pseudogene which would accumulate more
mutations over evolutionary time and eventually
become unrecognisable) and the acquisition of diver-
gent functions by the two duplicates and hence the
necessity for an organism to maintain both. Empirical
data does not support this model, however, suggesting a
high rate of retention of duplicate genes (3).
More recently, a modified form of this model has been
proposed. Many genes, particularly in multicellular
organisms, are multifunctional, either at the biochem-
ical level or in terms of regulated expression in different
organs or cell types. This creates the possibility of
duplicated genes diverging by subfunctionalisation,
which implies that duplicate genes diverge by main-
taining separate functions that were all initially
maintained by the single gene ancestor. An organism
must maintain both copies in its genome or lose one of
the functions, providing a selective reason for gene
duplicates to be retained in a population. Genes whose
expression in multiple tissues is regulated by separate
▶enhancers may be particularly prone to subfunctio-
nalisation, as mutations in different enhancers of two
duplicate genes would necessitate the retention of both,
even if the encoded amino acid sequences were
identical. This provides a potential explanation for the
apparently high retention rate of duplicate genes, at
least in multicellular organisms (4).
A corollary of duplicate gene divergence is redundancy
and compensation. Mutational analysis of families of
homologous genes often reveals a degree of redun-
dancy, such that the phenotype of a ▶double mutant is
more severe than the sum of the phenotypes of each
single mutant. This implies the genes are partially
redundant and that one gene can in part compensate for
the lack of the other, due to co-expression in the same
tissue and a similar biochemical function.
Evolutionary Implications of Gene Duplication
Gene duplication has the potential to provide a lineage
with ‘new’ genetic material, in that one copy of the
gene is, in principle, free to evolve new functions,
while the other maintains existing functions. This
concept has led to the suggestion that gene duplication
and entire genome duplication may play an important
role in evolutionary innovation. However direct
experimental support for this suggestion is lacking
and some evidence suggests that, contrary to the above,
both duplicate copies experience purifying ▶selection
following duplication (5). Nevertheless, it is common-
place to find gene family members that have undoubt-
edly evolved by duplication playing different roles in

the development, biochemistry or physiology of an
organism. This implies that some degree of evolu-
tionary innovation frequently follows gene duplica-
tion. A second possible evolutionary consequence
of gene duplication is the disruption of successful
interbreeding between populations in which different
genes have duplicated. This suggests gene duplication
might be a powerful force behind the evolution of
reproductively isolated populations, and therefore of
new species (6).
Clinical Relevance
The phenotypic effect of chromosomal aberrations
involving duplication of segments of DNA or the
possession of extra chromosomes is due to the
imbalance of gene products deriving from the dupli-
cated genes. ▶Down syndrome (trisomy of chromo-
some 21) is probably the best-known case involving an
entire chromosome, as, unlike most trisomies in
humans, embryos carrying an extra chromosome 21
are viable. There are numerous other syndromes
involving the duplication of specific chromosomal
regions (7). Incomplete gene duplications may also
result in the fusion of two genes at the boundary between
the original and duplicated DNA. This can result in
novel gene products with deleterious properties.
References
1. Eichler EE (2001) Recent duplication, domain accretion
and the dynamic mutation of the human genome. Trends
Genet 17:661–669
2. Skrabanek L, Wolfe KH (1998) Eukaryotic genome
duplication – where’s the evidence? Curr Opin Genet
Dev 8:694–700
3. Hughes MK, Hughes AL (1993) Evolution of duplicate
genes in a tetraploid animal, Xenopus laevis. Mol Biol
Evol 10:1360–1369
4. Lynch M, Force A (2000) The probability of duplicate
gene preservation by subfunctionalisation. Genetics
154:459–473
5. Wagner A (2002) Selection and gene duplication: a view
from the genome. Genome Biol 3:1012.1–1012.3
6. Lynch M, Conery JS (2000) The evolutionary fate and
consequences of duplicate genes. Science 290:1151–1155
7. Inoue K, Lupski JR (2002) Molecular mechanisms
for genomic disorders. Annu Rev Genom Hum Genet
3:199–242
Gene Expression
Definition
Gene expression describes the process by which a
gene’s coded information is converted into the
Gene Expression Data Analysis: Supervised Analysis 657
structures present and operating in the cell. Expressed
genes include those that are transcribed into mRNA and
then translated into protein, and those that are
transcribed into RNA but not translated into protein
(e.g. transfer and ribosomal RNAs).
▶Microarrays in Colorectal Cancer
Gene Expression Data Analysis:
Classification
G
Definition
For classification, algorithms such as weighted-voting,
k-nearest-neighbor classifiers, support vector machines
and artificial neural networks can be applied to the set
of genes selected using supervised analysis of gene
expression to build models capable of predicting the
class of a particular sample. To test the robustness of
classification, these methods are often coupled with a
leave-one-out cross-validation analysis, in which one
of the samples from the original ‘training’ set is
withheld and a class prediction is made on the withheld
sample. For complete validation, gene lists should be
tested on a second ‘test’ set of samples that were not
used to derive the discriminatory gene list.
▶DNA Chips
▶Gene Chip Technology and Its Application in
Molecular Medicine
▶Microarrays in Colorectal Cancer
Gene Expression Data Analysis:
Supervised Analysis
Definition
Using this method, one searches for genes whose
expression patterns correlate with an external para-
meter. The most commonly used ‘supervising’ para-
meters are clinical features such as survival, presence of
metastases and response to therapy. Many statistical
metrics have been used successfully in ‘supervised’
analyses, including the standard t-test, permutation-
based tests, and signal-to-noise ratios.
▶Microarrays in Colorectal Cancer
658 Gene Expression Data Analysis: Unsupervised Analysis
Gene Expression Data Analysis:
Unsupervised Analysis
Definition
No external feature is used to guide the analysis process
of gene expression. The data are used to search for
patterns without any a priori expectation concerning
the number or type of groups that are present. The most
common ‘unsupervised’ analysis method is hierarchi-
cal cluster analysis, based on similarity metrics.
▶Microarrays in Colorectal Cancer
Gene Expression Data Matrix
Definition
Gene expression data matrix refers to a table where
each row represents a gene, each column represents a
particular sample, or a particular experimental condi-
tion, and each position contains a number or a set of
numbers characterising the expression level of the
particular gene under the particular experimental
condition.
▶Microarray Data Analysis
Gene Expression Profile
▶Expression profile
Gene Gun
Definition
A gene gun is a tool for in vivo transformation of cells
or organisms (e.g. gene delivery; DNA vaccination).
The gun is loaded with DNA- or RNA-coated gold
particles that are injected into cells or tissues using a
helium pressure pulse.
▶DNA-based Vaccination
Gene Mapping
▶Genetic Epidemiology
Gene Ontology
Definition
Gene Ontology is a project to produce a controlled
vocabulary of terms relating to molecular function,
biological process, or cellular components, developed
by the Gene Ontology Consortium. Such controlled
vocabulary allows consistent use of terminology when
describing the roles of genes and protein in cells.
▶Protein Databases
Gene Silencing
Definition
Gene silencing refers to repression of genes by
the formation of a specialized chromatin structure
(heterochromatin). Silenced genomic regions carry
specific histone modification patterns (hypoacetyla-
tion, H3 K9 methylation in some organisms) and are
bound by heterochromatic proteins.
▶Chromatin Acetylation
Gene Silencing by Double-Stranded
RNA
▶RNA Interference in Mammalian Cells
Gene Targeting
Definition
Gene-targeting describes the production of a modified
allele of a gene by the process of homologous
recombination between a modified exogenous DNA
 Genetic Algorithm 659

molecule with its endogenous counterpart (target). In

transgenic technology, it is used to introduce a targeted Gene-Environment Interaction
mutation into the genome of mouse embryonic stem
cells (▶ES-cells). When injected into blastocysts,
mutant ES-cells contribute to all tissues of the embryo, Definition
including germ cells. Once the mutant gene has entered Gene-environment interaction describes an interplay
the germ line, mouse strains heterozygous and homo- between genetic variation and environmental triggers
zygous for the mutated gene can be bred and analysed. (e.g. smoke exposure, infections), which influences
▶Jun/Fos susceptibility to a certain disease.
▶Morpholino Oligonucleotides, Functional Genomics ▶Atopy Genetics
by Gene ‘Knock-down’
▶Mouse Genomics
▶Transgenic and Knockout Animals

Gene-Gene Interaction
G

Definition
Gene Therapy Gene-gene interaction describes an interplay between
variations in two different genes that influence(s)
susceptibility.
▶Clinical Gene Transfer ▶Atopy Genetics

General Transcription Factors

Gene Trapping
Definition
General transcription factors describes a set of common
Definition transcription factors that, together with RNA polymer-
Gene trapping comprises of a strategy by which the ase, are necessary and sufficient to direct accurate
insertion of a targeting vector into a gene leads to transcription initiation from a core promoter in vitro.
reporter gene activation. The inserted vector sequence ▶Core Promoters
acts as a tag that facilitates rapid cloning of the trapped
gene.
▶Large-Scale ENU Mutagenesis in Mice
▶Medaka as a Model Organism for Functional
Genomics
▶Mutagenesis Approaches in the Zebrafish Genetic Algorithm

Definition
Genetic algorithm is a statistical/mathematical term/
method that is aimed at finding the optimal solution to
a question. The process is the same as natural selection:
Gene-Based Therapies (1) selection of the strongest individuals/solutions,
(2) production of new organisms/new solutions,
by mixing the previously selected elements, and
Definition (3) mutations, which are accidental changes in the
Gene-based therapies involve the transfer of genetic organisms/solutions. The process is reiterated until no
material into a host with the hope of ameliorating or more improvement can be done. The last solution is
curing a disease. taken as the final one.
▶Limb Girdle Muscular Dystrophies ▶EST Mining for Expression Analysis
660 Genetic Background
Genetic Background
R OBERT G ERLAI
Psychology Department, University of Toronto at
Mississauga, Mississauga, Canada
robert_gerlai@yahoo.com
Synonyms
Host genotype; host genome
Definition
Genetic background represents all the genes in the
▶genome. The effects of “background” genes are often
considered with regard to their ability to influence or
modify the effects of ▶mutations artificially generated
by the experimenter in model organisms such as the
mouse. Mutations may be induced or introduced using
targeted mutagenesis (▶Targeted Gene Disruption), for
example by ▶homologous recombination in ▶em-
bryonic stem cells in the mouse or by using random
mutagenesis with chemical mutagens including ethyl
nitroso urea ▶ENU. These mutations interact with the
genetic background and will express their effects at the
phenotypical level in a manner modulated and
modified by the genetic background, a phenomenon
called ▶epistasis. The present essay will focus on the
genetic background from this perspective. It will
explain the complications associated with epistasis
and with genetic linkage using examples of ▶knock out
(or ▶null mutant) mice developed for the analysis of
molecular mechanisms of mammalian brain function
and behavior. The issues raised in this essay, however,
are general and will be valid for several other genetic
manipulation approaches and for all fields of biology.
Characteristics
▶Gene targeting allows to create null mutations in
mice and to analyze how the mutant organism responds
to the lack of the product of a ▶single gene. This has
facilitated the molecular dissection of such complex
traits as mammalian brain function and behavior.
However, numerous problems have been pointed out
(2, 4, 5, 6) with regard to the interpretation of the
phenotypical changes observed in null mutant mice.
Briefly, the most controversial issues stem from the fact
that scientists overlooked the influence of the genetic
background. These issues can be divided into two main
categories, both of which have general importance in
genomics. The first is a cluster of problems associated
with compensatory mechanisms. This problem is rather
difficult as there is no general solution to avoid it. The
second problem is associated with genetic linkage (the
so called flanking region problem). Practical solutions
to this problem exist and will be presented. While the
examples will be drawn mostly from the field of
behavioral neuroscience, the points illustrated are valid
for any biological trait.
The Complicating Effect of Compensatory Mechanisms
With gene targeting one can knock out a gene in vivo
and create a mutant organism that lacks the gene
product. The promise of gene targeting has been to
reveal the in vivo function of the gene of interest.
However, the functional relevance of gene targeting has
been questioned [reviewed in (3)] because the mutation
may lead to an avalanche of compensatory processes
(e.g. up- or down-regulation of other genes) and
resulting secondary phenotypical changes. Compensa-
tion may be due to ▶genetic redundancy. Genetic
redundancy in this context means that putative “helper”
genes take over the function of the targeted one, e.g.
become up-regulated and compensate for the absence
of the product of the targeted gene. Although labor
intensive, proper analysis of compensatory changes
may allow one to reveal how biochemical pathways
interact. For example, Chen et al. (1) showed that
although a null mutation in protein kinase C γ subtype
(PKCγ) in mice resulted in an apparently normal long-
term depression (LTD) in the cerebellum of the
mutants, LTD could be blocked by a PKC inhibitor
only in control mice but not in the null mutants,
suggesting that LTD was mediated, at least partly, by
non-PKC dependent processes in the null mutant mice.
“Compensatory” processes, however, can also induce
phenotypical changes. For example, assume gene α
serves hypothetical function ‘A’. Also assume that
targeted gene α is compensated for by gene β in the
genetic background; gene β becomes up-regulated
in response to the absence of α gene product. The
excess of gene β product is able to compensate for the
lack of gene α product and no change is observed in
function ‘A’ at the phenotypical level. However, over-
expression of gene β may have some pleiotropic
effects, i.e. may affect functions other than ‘A’,
similarly to the way over-expression of genes alters
the phenotype of transgenic mice. These functional
alterations when observed by the investigator will be
assigned to gene α. Although they are indeed due to the
introduced mutation of gene α, they need not reveal
the function of this gene per se because they are related
to this gene only indirectly. This example is not
hypothetical. Empirical evidence for similar situations
can be found in the literature (2).
Teasing out the direct and indirect effects of the
mutation is not trivial and dissection of the molecular
mechanisms underlying complex phenotypical traits
will require meticulous studies in which numerous
factors need to be controlled. Lathe (6) discusses

several confounding factors. Instead of reiterating his
arguments, the reader’s attention is now drawn to what
is proposed to be the main issue, the necessity of a
“systemic approach” view in gene targeting (5) (also
see ▶Phenomics).
The Need for a Systemic View
The principal problem in genomics is a systemic one
that concerns biological organization and the functional
units of this organization. From a geneticist’s viewpoint
the units of biological organization are the genes and
their function is to encode particular proteins. How-
ever, when it comes to the question of phenotypical
effects, genes may not be the units and the definition
of their function may be complicated. Clusters of genes
in the genetic background defined by higher organiza-
tional level phenomena, including developmental,
physiological or even behavioral, may represent the
functionally relevant unit. Disruption of a single gene
may alter a biochemical cascade within the functional
gene cluster. Expression levels of the genes belonging
to a functional cluster may change in concert.
Investigation of such changes may reveal the biological
organization of the organism. The boundaries of
putative gene clusters may not be sharp. Some genes
may belong more, others less, to a specific functional
gene group. This also implies that the gene group
organization may not be orthogonal, i.e. some genes
may belong to more than one functional group.
Functional groups may be hierarchically organized. A
smaller number of genes may define subgroups that
may make up groups that in turn may be organized into
super-groups, etc. Disrupting single genes will perturb
the organism and will force it to respond in a way
inherent in its biological organization. The phenotypi-
cal changes one observes are the reflection of this
organization. Instead of looking for the function of
single genes, it is proposed that investigators should
take a systemic organizational view into consideration,
an approach conceptually similar to metabolic control
analysis employed in biochemistry.
In summary, the effects of genetic manipulation must
be investigated at all practically feasible levels of
biological organization, including gene expression
patterns, protein-protein interactions and a broad
spectrum of phenotypical traits that may be affected
by the direct and indirect effects of the mutation.
Polymorphism in the Genetic Background May
Make the Results of Gene Targeting Studies
Difficult to Interpret
Assume that knock out of gene α leads to differential
expression of alleles b vs. B of gene β, and a regulatory
change of gene β leads to different phenotypical effects
depending on which allele (b or B) is present in the α
null mutant mouse. Consequently, ▶polymorphism in
Genetic Background 661
the genetic background will not allow one to conclude
with certainty that a particular phenotypical change
observed in the null mutant was due to the null
mutation or to the genetic background. This issue is
especially problematic if the genetic background of the
null mutant animals is different from that of their wild
type control counterparts, which is a typical problem in
a large number of gene targeting studies.
Null Mutant Mice of Gene Targeting Studies
Are Often the F2 Offspring of Two Mouse Strains
The genetics of the breeding strategy usually employed
to generate null mutant mice is explained in Fig. 1. The
figure also depicts why the hybrid genetic background
leads to complications.
G
The Flanking Allele Problem
The F2 population is a segregating population in which
mice have recombinant genotypes derived from the two
parental mouse strains (Fig. 1). The difficulties arising
from this are threefold. First, the recombination pattern,
i.e. which locus contains strain 129 and which B6
alleles and whether in a homozygous or heterozygous
form, may be different between littermates. Thus not
even wild type littermates of their mutant counterparts
represent an appropriate control, since their alleles
could be different from those of the mutants not only at
the locus of the gene of interest but also at other loci.
This may lead to false positive results. Second, due to
the genetic variation resulting from the hybrid
segregating background, detecting significant effects
of the mutated gene may be difficult, which leads to
false negative results. These two problems can be
alleviated by measuring larger numbers of animals, i.e.
by increasing the power of statistical comparisons and
decreasing the possibility of sampling error. Increasing
the sample size, however, will not solve the third
problem, which is associated with genetic linkage. If
the targeted mutagenesis is conducted in ES cells from
strain 129, the chromosome with the targeted locus will
carry alleles of genes of 129-type. The probability of
genetic recombination is generally inversely related to
the distance between the loci of the genes (linkage).
Thus, the 129-type alleles of the genes whose loci are
close to the locus of the mutated gene will remain
together with the mutated allele of the gene of interest
(Fig. 1). In other words, any time the mutation is
detected in a mouse, e.g. by ▶Southern blotting or
PCR, that particular animal will also carry the linked
129-type genes with high probability. Conversely, a
non-mutant control, animal will not carry these
129-type alleles and will have B6 alleles with high
probability if the 129-ES cell chimera was crossed to
B6. In effect, the mutation can be seen as a ▶marker for
the 129-type genes linked to the locus of the targeted
gene. Consequently, any phenotypical differences
662 Genetic Background
Genetic Background. Figure 1 The genetic back-
ground of mice generated by gene targeting. Most gene
targeting is carried out in cultured embryonic stem (ES)
cells derived from one of the substrains of mouse strain
‘129’. The 129-type ES cells carrying the targeted
mutation are introduced into a blastocyst and the
surviving chimeric embryos develop to term, are raised
to adulthood and are mated to “wild type”, i.e. non
mutated, mice. ES cells originating from mouse strain
129 carry one chromosome (white) with the disrupted
allele (double lines) of the targeted gene. If these ES
cells populate the germ-line in the chimeric mice, the
mutation will be transmitted when the chimera is mated.
A cross between a germline transmitting chimera and a
C57BL/6 (B6) mouse (black chromosomes (a) will
produce an F1 population (b) in which 50 % of the
animals will have one copy of the mutant allele
(heterozygous mutants) and 50% of them will have no
mutant allele (wild type animals) at the targeted locus.
Using Southern blotting or PCR (polymerase chain
reaction) one can detect the presence of the mutant
allele and identify the heterozygous mutant animals. If
these animals are mated with each other, according to
Mendel’s law, homozygous mutant (two mutant alleles),
heterozygous mutant (one mutant and one wild type
allele) and wild type (two wild type alleles) animals will
be obtained. It is also important to remember, however,
observed between mutant and control littermates of
the hybrid genetic origin may be due either to the
introduced null mutation or to the background genes
linked to the targeted locus. Thus, one may find false
positive results [for experimental examples see (3)].
Solutions to the Flanking Allele Problem
In order to decrease the probability of contribution of
variable background genes, one could backcross the
mutant hybrid animals for several generations to the
strain of choice, e.g. to B6, and create a ▶congenic
strain that carries the mutation on the desired genetic
background. However, complete elimination of 129-
type genes that surround the locus of the gene of
interest is not practically possible. For example, even
with 12 generations of backcrossing (approximately 2
years of breeding) to B6, the length of the 129-type
chromosome segment introduced to the B6 genome
how genes at loci other than the targeted one will be
inherited. Crossover events during the meiotic process of
gametogenesis will “shuffle” the alleles of these back-
ground genes and will create recombinant chromosomes
(c), which will characterize the genotype of the sperm and
the egg of the F1 mice. The genotype of an F2 individual,
therefore, will be represented by a pair of such
recombinant chromosomes. For example, a homozy-
gous null mutant mouse may have chromosomes a and
b, a and c, or b and c; a heterozygous mouse may have
one of the recombinant chromosomes with the null
mutation (a, b, or c) and another without the null mutation
(d, e, or f); whereas a wild type control mouse may have
chromosomes d and e, d and f, or e and f. Panel C shows
that the null mutant allele of the targeted gene will be
surrounded by 129-type genes, however, the wild type
allele of the gene will be surrounded by B6 type genes.
This linkage disequilibrium is simply due to the fact that
the null mutant allele came from a strain 129 genetic
background. In an animal produced from mating such F2
mice (F3 or the following generations), the null mutant
allele could be surrounded by B6 genes only if, during the
meiotic processes of gametogenesis, crossovers oc-
curred precisely flanking both sides of the targeted gene,
events whose combined probability is infinitesimally
small. (d) shows the probabilistic distribution of 129
(white) vs. B6 (black) alleles in an F2 segregating
population. The depicted chromosomes thus represent
the genotype “average” of the F2 population. Note that in
mice carrying the null mutation (chromosome ‘a’), the
probability of finding 129 alleles on the mutant chromo-
some increases the closer a given locus is to the locus of
the targeted gene. However, in mice carrying the wild
type allele of the targeted gene (chromosome ‘b’), the
probability of finding 129 alleles decreases the closer a
given locus is to the locus of the targeted gene. Also note
that as the distance increases from the locus of the
targeted gene, the probability of the presence of 129 vs.
B6 allele approaches 50-50%. (Modified from (3))

would be, on average, about 16 centiMorgans (cM)
representing about 1% of the genome, i.e. about 3–400
genes. But if no alternatives are available, backcrossing
is recommended because it stabilizes the genetic
background of the mutant line by reducing the variation
in recombination patterns across generations. Other,
more complicated breeding strategies have also been
proposed [reviewed in (3)]. They represent better
solutions than simple backcrossing but they do not
completely eliminate the flanking allele problem.
Additional suggestions have also been made [for
review see (3)]; rescue experiments may rule out the
potential effects of linked 129-type genes and genera-
tion of “knock in” mice may be a control for the null
mutant gene “knock out” animals. Furthermore,
inducible knock out techniques, e.g. tetracycline
transactivator systems, are recommended because
comparison of pre- and post-induction phenotypes
represents appropriate within subject control. Finally,
generating null mutant mice with a pure genetic
background is known to be the perfect solution for
the problems associated with the genetic background,
but up to date, this has not been preferred because most
ES cells are derived from 129 type mice and these
animals are not good breeders.
Clinical Relevance
Numerous genetic models of human diseases have been
and will be created. However, the genetic manipulation
is expected to lead to complex phenotypical changes
that are influenced by a large number of genes in the
genetic background as well as by environmental
factors. Understanding compensatory mechanisms
and systemic responses to the induced mutation and
eliminating or addressing the confounding effects of
background genes are important steps forward that will
facilitate the modeling and ultimately the understand-
ing of human diseases.
References
1. Chen C, Kano M, Abeliovich A et al (1995) Impaired
motor coordination correlates with persistent multiple
climbing fiber innervation in PKC gamma mutant mice.
Cell 83:1233–1242
2. Crusio WE (1996) Gene-targeting studies: new methods,
old problems. Trends Neurosc 19:186–187
3. Gerlai R (2001) Gene targeting: Technical confounds and
potential solutions for the behavioral neuroscientist.
Behav Brain Res 125:13–21
4. Gerlai R (1996) Gene targeting studies of mammalian
behavior: Is it the mutation or the background genotype?
Trends Neurosc 19:177–181
5. Gerlai R (1996) Gene targeting in Neuroscience: The
systemic approach. Trends Neurosci 19:188–189
6. Lathe R (1996) Mice, gene targeting and behaviour: more
than just genetic background. Trends Neurosci 19:183–186
Genetic Code 663
Genetic Code
J EAN L EHMANN
Theoretical Biophysics, Department of Physics,
Royal Institute of Technology, Stockholm, Sweden
jean@theophys.kth.se
Definition
The genetic code is the set of correspondences between
the codons of mRNAs and the amino acids of the
proteins produced on the ribosomes through the
▶translation of these mRNAs. This set also includes
“stop” codons without an amino-acid assignment, G
which specify the termination of translation.
Codons are the units of information in mRNA. They
are made up of three consecutive ▶nucleotides, each of
which can be one of the four bases U, C, G or A. Sixty-
four different codons result from all the combinations.
Twenty amino acids are coded through the phenomen-
on of translation. A twenty-first, selenocysteine, is
coded in some organisms in particular situations (1, 2)
and a still very rare twenty-second (pyrrolysine) has
recently been discovered (2).
The set of correspondences between the codons and the
amino acids is highly conserved in all organisms. The
most common one is called the canonical genetic code,
previously also called the universal genetic code before
deviations were found in different ▶taxa. It is usually
depicted in a table with three entries (Fig. 1).
Since the sequences of mRNAs can be different from
those of the genes for proteins from which they are
transcribed, the coding rules are generally only valid
for the mRNAs prior to translation. These differences
arise from modifications on mRNAs through ▶matura-
tion processes occurring before translation (observed
however almost only in Eukaryotes).
Characteristics
Main Molecular Processes Involved in Decoding
The coding rules are the outcome of extremely
sophisticated molecular processes. Basically, the transla-
tion system converts genetic information contained in
genes into functional proteins. The information is always
processed in the form of an mRNA. In Eukaryotes, the
latter can be the outcome of a complex maturation process
from its primary transcript(s), which can involve several
genes. In Eubacteria and Archebacteria, it is generally
translated during or immediately after its ▶transcription
from a gene without any rearrangement. It may also come
from the genome of an invasive entity (virus).
A start codon upstream of the mRNA signals the place
of the beginning of translation. This codon is almost
always AUG, but is also sometimes GUG (very rarely
664 Genetic Code
Genetic Code. Figure 1 The canonical genetic code
table. The codonic positions are arranged from left to
right of the table, corresponding to the reading direction
of the mRNAs (5′–3′). Each of the sixty-four possible
codons code for a particular amino acid or the stop
function, which binds a release factor RF1 or RF2 at the
end of translation. The amino acids are abbreviated to
their three-letter notation.
UUG or CUG). Any of these codons can bind
the initiator tRNA coding a modified methionine,
N-formylmethionine. The start codon also positions
the sequence into the correct ▶reading frame. The
process generally ends at a stop codon (UAA, UAG
or UGA in the canonical genetic code), which binds
a release factor RF1 (UAA, UAG) or RF2 (UAA,
UGA).
The path leading a free amino acid to incorporation into
a coded protein involves three main steps (Fig. 2):
1. ▶Activation of the amino acid by the corresponding
aminoacyl-tRNA synthetase (aaRS) through hydro-
lysis of a molecule of ATP.
2. The aaRS subsequently binds the amino acid to a
cognate tRNA. Prior to translation, a complex made
up of an ▶elongation factor Tu (EF-Tu) and GTP
binds the amino acid at the 3′ end of the tRNA,
giving it a much higher affinity for the ribosome.
3. During translation, occurring on the ribosomes, the
successive codons of a particular mRNA are tested
by the incoming tRNAs through anticodon-codon
associations. A proofreading mechanism is pre-
ceded by hydrolysis of the GTP of the complex at
the 3′ end of the tRNA. Anticodon-codon comple-
mentarity is the decisive factor leading to the
formation of the peptide bond between the last
amino acid of the nascent protein and the amino acid
carried by the incoming tRNA.
Accuracy of the Coding Rules
The reliability of the coding rules thus depends on the
accuracy of two major processes of molecular recogni-
tion:
a) Binding of both an amino acid and a tRNA by an
aaRS (steps 1–2).
b) Binding of the anticodon of a tRNA by a codon of
an mRNA through base pairing (step 3).
The first process (a) constitutes a textbook case of
RNA-protein recognition (3) and has been referred to
as the second genetic code; as soon as a particular aaRS
has bound an amino acid to a cognate tRNA, the code is
virtually already established since the anticodon-codon
associations occurring in (b) follow relatively strict
base-pairing rules.
Each of the (generally) twenty different aaRSs thus
recognizes both a tRNA and an amino acid. The
recognition elements on the tRNAs are mainly situated
in the acceptor stem and anticodon domains, which
constitute the two major spatial regions of that molecule.
Both positive and negative elements are used by the
aaRSs to identify their associated tRNAs, and differ-
entiation between two tRNAs can sometimes depend on
the identity of only a few bases (3). Since more than one
tRNA molecule is often necessary for the translation of
the whole set of codons belonging to a particular amino
acid, in several cases an aaRS must be capable of
recognizing different tRNAs, which implies that the
anticodon is sometimes not used as a discriminatory
element (e.g. for ser-, leu- and ala-accepting tRNAs).
The aaRSs are among the most studied enzymes, and
their structural analyses have given important clues on
the evolution of the genetic system (4).
The second process (b) involves an initial selection
and a proofreading mechanism that are used by
the ribosomes to discriminate between correct and
incorrect anticodon-codon associations (5). These
coupled mechanisms, separated by GTP hydrolysis,
ensure a very low error frequency which in E. coli has
been estimated as being between 6 × 10–4 and 5 × 10–3.
The initial selection step occurs when an incoming
tRNA charged with its cognate amino acid (combined
with EF-Tu and GTP) binds the ribosome and tests the
 Genetic Code 665

Genetic Code. Figure 2 The main molecular processes involved in the assemblage of free amino acids into proteins
according to the rules of the genetic code. Three sets of molecules participate in the translation of the mRNAs: the
encoded amino acids (20 different kinds), the aminoacyl-tRNA synthetases (aaRSs) (at least one for each encoded
amino acid, not all shown here) and the tRNAs (at least 22 but often more than 30 different kinds, not all shown here).
The example shows the events leading to the translation of a CUU codon of an mRNA. First, a leuRS binds a leucine
and subsequently catalyses its activation through the hydrolysis of a molecule of ATP, resulting in an aminoacyl-
adenylate (leu
AMP) and ppi [reaction (1)]. As soon as the leuRS recognizes a cognate tRNA (in this case, a tRNA
with an anticodon GAG), the aminoacylation reaction occurs and the tRNA is thus charged with the amino acid [reaction
(2)]. The AMP molecule is then released. After the [EF-Tu + GTP] complex has bound the amino acid at the 3′ end of
the tRNA (not shown here), the latter can participate in translation, occurring on a ribosome. In accordance with the
wobble rules (Table 1), it can successfully bind the CUU codon, thereby enabling the formation of the peptide bond
between the incoming leucine and the nascent protein [reaction (3)]. The previous tRNA (with the anticodon AAG) then
leaves the ribosome. Note that the codons read from 5′ to 3′ (thus from right to left in the figure) and the anticodons from
3′ to 5′. The arrow above the mRNA shows the starting place of translation, while the cutting of the strand into codons is
indicated by small dots. For clarity, details on structures and events are omitted at the level of the ribosome.

codon at the point of being translated. At this stage,
most of the non-cognate tRNAs are rejected. However,
in addition to cognate tRNAs, some near-cognate
tRNAs (whose anticodons can form partial base-pairs)
may also cross this first stage, associated with the
hydrolysis of the GTP. This hydrolysis results in a
▶conformational change of EF-Tu, which subse-
quently leaves the 3′ end of the tRNA. The amino acid
is then free to move to the ▶peptidyl-transferase center
of the ribosome where peptide bond formation can take
place. Before that event however, almost all remaining
near-cognate tRNAs are rejected (proofreading) (5).
Caveat
The standard rules of the genetic code can be altered
during translation events that have been referred to as
“recoding” (1). These alterations are specific to
individual mRNAs, and involve three possible kinds
of event, ▶programmed frame shifting, ▶translational
bypassing and ▶redefinition of codons. Albeit not
666 Genetic Code

common, it is thought that any of these events may

occur in all living organisms (1). They serve notably as
control mechanisms of gene expression.

Order in the Genetic Code Table

Degeneracy
Since most of the amino acids are coded by more than
one codon, the genetic code is degenerate (Fig. 3). The
degeneracy is connected with the base in third position
of the codons, which often has only a small influence
on the nature of the encoded amino acid. A particular
codon generally belongs to one of two major
categories, the four-fold and the two-fold degenerate
families. In two cases, the latter family is further split
into individual codons, each with a specific assign-
ment. The presence of a degeneracy symmetry over the
entire table, appearing when the succession [U, C, G,
A] (or its inverse) is used to write the table, shows that
the splitting into four-fold and two-fold degenerate
families is conditioned by the nature of the bases in the
first and second positions of the codons (Fig. 3).
The origin of the degeneracy stems from the existence
of wobble rules in the base-pairing of the third position
of the anticodons that can be specific to the type of
family (four-fold or two-fold degenerate), and which
are controlled by the ribosome (Table 1). Thus, in the
case of mitochondrial and chloroplastic systems where
wobble rules are the most extensive, a tRNA with U in
the third position of the anticodon can translate any of Genetic Code. Figure 3 Degeneracy in the canonical
the four codons of the corresponding four-fold genetic code table. Two families of degeneracy are
degenerate family, while a tRNA with U (G) in the mainly present in the table: the four-fold degenerate
third position of the anticodon can translate any of the families (gray rectangles) and the two-fold degenerate
two codons with G or A (U or C) in the third position of families (squares). In two cases (AUR and UGR), the
the anticodon of the corresponding two-fold degenerate latter type of degeneracy is further split into individual
family. As a result, only 22 different tRNAs are codons. The succession [U, C, G, A] used to write the
necessary in some systems (in which also the AGR table highlights a degeneracy symmetry over entire the
family is reassigned to stop; see next subsection) in table (dashed line), highlighting the fact that simple rules
order to read all the codons for amino acids. The based on the nature of the bases in the first and second
positions of the codon account for the distribution of the
wobble rules are, however, generally less extensive and
two main families in the table:
in most systems more than 30 kinds of tRNAs are used
for the translation of all the codons. Moreover, the
Four-fold degenerate Two-fold degenerate
tRNAs often have modified bases in the third position
families families
of the anticodon (e.g. I, Q), each with particular
matching properties (Table 1). 1st pos. 2nd pos. 1st pos. 2nd pos.
An important consequence of this wobble phenomen- (W) (Y,S) (W) (Y,W), (R,S), (R,W)
on, enabling a limited number of different tRNAs to
cope with the whole set of codons, is an improvement (S) (Y,W), (Y,S), (S) (R,W)
(R,S)
in the efficiency of translation.
Moreover, some amino acids are present in more than
one codon family. Thus, leu, ser and arg are coded by a where the following categories are used to differentiate
total of six codons in two codon families in the the bases: S (strong matching) = G or C; W (weak
canonical genetic code. matching) = A or U; R (purine) = A or G; Y (pyrimidine) =
C or U
Reassignments
Although the general organization of the genetic code
is conserved over all living organisms, many variants
 Genetic Code 667

Genetic Code. Table 1 Wobble rules in the genetic code

3rd base 3rd base codon (3′)

anticodon (5′)
Standard Limitation Possible extension of Limitation known
matching(s) known so far matchings so far
Standard A U C,G,U(A) ACN and CGN
bases families in
Mycoplasma ssp,
yeast mt.,
nematode mt.
C G
G C,U
U A,G A,C,G,U All 4-fold degenerate
families G
in mt. and ch.
Modified I A,C,U 4-fold degenerate families (except GGN)
bases
Cm A(G) UUR family in E. coli
5
f C A,G AUR family in nematode, bovine, squid mt. and Drosophila mt.
L A AUA in eubacteria and plant mt.
Q C,U 2-fold degenerate families in eubacteria and eucaryotes
7
m G A,C,G,U AGN family in echinoderm mt. and squid mt.
5
xo U A,G,U UCN, GUN, GCN and ACN families in eubacteria
xm5Um, Um, A,G 2-fold degenerate families in mt., eubacteria and eucaryotes
xm5U
xm5s2U A(G) 2-fold degenerate families in eubacteria and eucaryotes
G in anticodon A,C,U AAU, AAC, AAA in echinoderm mt.
(UψG)

Main source: ref (6). N = U, C, G or A; R = A or G; Y = C or U. Mt. = mitochondria; ch. = chloroplasts

have been discovered in different taxa. In addition, the
organelles (mitochondria and chloroplasts) have their
own translation systems with specific variants of the
canonical genetic code (Fig. 4).
An examination of all the variants discovered so far
indicates that some codons are more prone to
reassignment than others (6). The stop codons
constitute a typical example; the two-fold degenerate
family UAR is frequently reassigned to gln in the
nuclear system, while UGA almost always codes for trp
in mitochondria. This particular versatility may be
explained by the rarity of these codons, which implies
that their reassignments might not cause catastrophic
disturbance. Another interesting change concerns the
AUA codon, which often codes for met in the
mitochondria. As a result, the degeneracy symmetry
pointed out in Fig. 3 is entirely valid for the
mitochondrial systems that also have UGA coding for
trp. These changes imply that the wobble rules can be
altered within the codon families concerned.
Furthermore, it has been observed that all codons that
have been reassigned in the nuclear systems have also
been subject to reassignment in the mitochondria, while
no codon has been reassigned in the nuclear systems
only (6).
Some codon families seem to be excluded from any
reassignment procedure. This is especially the case for
all codons with G in first position, corresponding to the
amino acids val, ala, gly, asp and glu, as compared with
the codons with U, C or A in this same position, for
which at least four changes have been reported (Fig. 4).
Moreover, all these amino acids are only present in this
part of the table in the canonical genetic code. This
particularity shows that the genetic code has a core.
668 Genetic Code
Genetic Code. Figure 4 Reassignments and para-
meters of order in the canonical genetic code table. The
numbers refer to reassignments that have been reported
so far [main source: ref (6)]: 1 thr, 2 ser, 3 met and
unassigned, 4 stop, 5 trp and cys, 6 unassigned, 7 ser, gly,
stop and unassigned, 8 ser, gly, stop and unassigned
(7 and 8 changes are independent), 9 leu, ala and gln,
10
tyr, gln and glu, 11 asn. Unassigned means that the
codon(s) disappeared in the entire genome. Changes
that have been reported in mitochondria only are
indicated by an asterisk (*). The gray area within the
table points out all codon families with G in first position,
for which any reassignment has been reported so far.
The corresponding amino acids generally constitute
more than 97% of the amount of all amino acids
produced in experiments thought to reproduce the
conditions of the early Earth (the results however differ
slightly depending on the hypothesized conditions). The
hydrophobicity parameter is shown on the top of the table.
It is connected with the 2nd anticodonic position (over-
drawn above the codonic one), the succession
[A, G, C, U] ranking the bases in decreasing order on the
hydrophobicity scale. The hydrophobicity correlation
between the base at this position and the amino acids
result in most of the hydrophobic amino acids being on the
left of the table, while most of the hydrophilic ones are on
the right.
The Core of the Genetic Code
As revealed by statistical analyses of coding sequences,
all codons of the genetic code do not occur with equal
probability. Some codons occur very rarely while others
are frequent. Many factors can affect their distribution,
but a general trend connected with the organization of
the genetic code, which is independent of the organism
or the genes under consideration can be highlighted.
The most general pattern occurring in coding se-
quences is GNN, meaning that the codons with G in
first position are overrepresented in comparison with
those with U, C or A at this same position (N stands for
any nucleotide).
These codons code for the amino acids val, ala, gly, asp
and glu, which display the simplest side chains of all
the amino acids of the genetic code. This can explain
their relative abundance in the proteins of which they
constitute the scaffold.
One can, furthermore, connect this abundance to the
observed stability of their assignment (see previous
section). Changing the signification of any of the
corresponding codons may cause malfunctions in some
proteins, resulting in disturbances in biochemical pro-
cesses (it has, however, been experimentally shown that
an organism such as E. coli can survive treatment of this
kind).
Interestingly enough, this set of amino acids generally
constitute more than 97% of the total quantity of all amino
acids found in the so-called prebiotic synthesis experi-
ments (Fig. 4). It thus seems likely that the primitive code
was limited to the use of these amino acids, which were
only assigned to the codons with G in first position for
physico-chemical reason. Later, more codons and more
amino acids were added, eventually constituting the
genetic code as we know it in present-day organisms.
A Hydrophobicity Parameter in the Table
Correlation studies on physico-chemical properties of
the constituents of the genetic code have revealed that
the ▶hydrophobicity of the base in the second position
of the anticodon is positively correlated with that of the
encoded amino acid. It has also been established that
the succession [A, G, C, U] ranks the bases from the
most hydrophobic (A) to the most hydrophilic (U).
Thus, Fig. 4 naturally arranges the amino acids into the
genetic code table with regard to hydrophobicity; the
most hydrophobic amino acids are mainly on the left of
the table while the hydrophilic ones are on the right.
This order in the genetic code table is noteworthy since
the hydrophobicity of the amino acids has a major
influence on the folding of the proteins of which they
are the constituents. Thus, an analysis of coding
sequences based on this correlation can provide
information on the structure of the corresponding
encoded proteins (7).
Clinical Relevance
Since the aaRSs are responsible for the reliability of the
coding rules, genetic diseases affecting these proteins
can create important disorders in the organism.

Autoantibodies to five aaRRs have been described, and
each is associated with a syndrome of inflammatory
myopathy with interstitial lung disease and arthritis (8).
At the level of the ribosome, antibiotics such as
paromomycin and streptomycin can significantly affect
the fidelity of translation. Paromomycin stabilizes the
tRNAs irrespective of whether the codon-anticodon
pair is cognate or near-cognate and hence increases
amino acid misincorporation (5).
▶Nucleotide Biosynthesis
▶Translational Control in Eukaryotes
References
1. Baranov PV, Gesteland RF, Atkins JF (2002) Recoding:
translational bifurcation in gene expression. Gene
286:187–201
2. Ibba M, Söll D (2002) Genetic code: introducing
pyrrolysine. Curr Biol 12:R464–R466
3. Beuning PJ, Musier-Forsyth K (1999) Transfer RNA
recognition by aminoacyl-tRNA synthetases. Biopoly-
mers (Nucleic Acids Sciences) 52:1–28
4. Ribas de Pouplana L, Schimmel P (2001) Aminoacyl-
tRNA synthetases: potential markers of genetic code
development. Trends Bioch Sci 26:591–596
5. Rodnina MV, Wintermeier W (2001) Ribosome fidelity:
tRNA discrimination, proofreading and induced fit.
Trends Bioch Sci 26:124–130
6. Knight RD, Freeland SJ, Landweber LF (2001) Rewriting
the keyboard: evolvability of the genetic code. Nat Rev
Gen 2:49–58
7. Chiusano ML, Alvarez-Valin F, Di Giulio M et al (2000)
Second codon positions of genes and the secondary
structure of proteins. Relationships and implications for
the origin of the genetic code. Gene 261:63–69
8. Hirakata M, Suwa A, Nagai S et al (1999) Anti-KS:
identification of autoantibodies to asparaginly-transfer
RNA synthetase associated with interstitial lung disease.
J Immun 162:2315–2320
Genetic Counselling
Definition
The following definition of genetic counselling was
adopted by the American Society of Human Genetics:
“Genetic counselling is a communication process
which deals with the human problems associated with
the occurrence or the risk of an occurrence of a genetic
disorder in the family. This process involves an attempt
by one or more appropriately trained persons to help
the individual or family to (1) comprehend the medical
facts including the diagnosis probable course of the
disorder and the available management; (2) appreciate
the way heredity contributes to the disorder and the risk
of recurrence in specified relatives; (3) understand the
Genetic Epidemiology 669
alternatives for dealing with the risk of occurrence; (4)
choose the course of action which seems to them
appropriate in view of their risk their family goals and
their ethical and religious standards to act in accordance
with that decision; and (5) to make the best possible
adjustment to the disorder in an affected family
member and/or the risk of recurrence of that disorder.”
▶Fragile X Syndrome
▶Peutz-Jeghers Syndrome
Genetic Distance
G
Definition
Genetic distance describes the linear distance between
genes on a genetic map, measured in Morgan (=100
centi-Morgan); a genetic distance of 1 centi-Morgan
between two loci entails that they are expected to be
involved, on average, in one recombination per 100
meioses.
▶Genetic Epidemiology
Genetic Epidemiology
M ICHAEL K RAWCZAK
Institute for Medical Informatics und Statistics,
Christian-Albrechts-University Kiel, Kiel, Germany
krawczak@medinfo.uni-kiel.de
Synonyms
Linkage analysis; positional cloning; gene mapping
Definition
Genetic epidemiology is the scientific discipline
concerned with the causes and distribution of human
diseases in groups of relatives and with the inherited
predisposition to disease in populations. In many ways,
genetic epidemiology is an interdisciplinary science
that has more aspects of population and evolutionary
genetics to it than of classical epidemiology. Only
recently has the discipline changed its focus to target
diseases that are characterized by the small-to-moder-
ate relative risks that are usually the objective of
conventional epidemiological research. So far how-
ever, genetic epidemiology has been most successful in
elucidating the genetic basis of so-called “monogenic”
disorders, including cystic fibrosis, Huntington disease
and Duchenne muscular dystrophy (Table 1). The
670 Genetic Epidemiology

Genetic Epidemiology. Table 1 A list of human disease genes characterized through genetic epidemiological
research employing a positional cloning approach

Year Gene Disease OMIM

1986 CYBB chronic granulomatous disease 306400
1986 DMD Duchenne muscular dystrophy 310200
1986 RB1 retinoblastoma 180200
1989 CFTR cystic fibrosis 219700
1990 CHM choroideremia 303100
1990 NF1 neurofibromatosis type 1 162200
1990 SRY sex reversal 306100
1990 WT1 Wilms tumour 194070
1991 APC familial adenomatous polyposis 175100
1991 FMR1 fragile X syndrome 309550
1991 KAL1 Kallmann syndrome 308700
1991 PAX6 aniridia 106210
1992 DMPK myotonic dystrophy 160900
1992 NDP Norrie disease 310600
1992 OCRL Lowe syndrome 309000
1993 ABCD1 adrenoleukodystrophy 300100
1993 ATP7A Menkes syndrome 309400
1993 ATP7B Wilson disease 277900
1993 BTK X-linked agammaglobulinemia 300300
1993 FMR2 fragile X syndrome 309548
1993 GK hyperglycerolemia 307030
1993 HD Huntington disease 143100
1993 NF2 neurofibromatosis type 2 101000
1993 PAFAH1B1 Miller-Dieker syndrome 247200
1993 SCA1 spinocerebellar ataxia type 1 164400
1993 TSC2 tuberous sclerosis 191092
1993 VHL von Hippel Lindau syndrome 193300
1994 BRCA1 hereditary breast and ovarian cancer 113705
1994 DAX1 X-linked adrenal hypoplasia congenita 300200
1994 DRPLA dentatorubral-pallidoluysian atrophy 125370
1994 EMD Emery-Dreifuss muscular dystrophy 310300
1994 FGD1 Aarskog-Scott syndrome 305400
1994 FGFR3 achondroplasia 100800
1994 MJD Machado-Joseph disease 109150
1994 PKD1 polycystic kidney disease type 1 173900
1994 SLC26A2 diastrophic dysplasia 222600
 Genetic Epidemiology 671

Genetic Epidemiology. Table 1 A list of human disease genes characterized through genetic epidemiological
research employing a positional cloning approach (Continued)

Year Gene Disease OMIM

1994 WAS Wiskott-Aldrich syndrome 301000
1994 XK McLeod syndrome 314850
1995 ARSE X-linked recessive chondrodysplasia punctata 302950
1995 ATM ataxia telangiectasia 208900
1995 BLM Bloom syndrome 210900
1995 BRCA2 hereditary breast cancer 114480
1995 CAPN3 limb-girdle muscular dystrophy type 2A 253600
1995 CLN3 Batten disease 204200
1995 EXT1 multiple exostoses 133700 G
1995 OA1 ocular albinism 300500
1995 PHEX hypophosphatemia rickets 307800
1995 POU3F4 X-linked mixed deafness 304400
1995 PSEN1 early onset familial Alzheimer disease 104311
1995 PSEN2 early onset familial Alzheimer disease 600759
1995 SGCB limb-girdle muscular dystrophy type 2E 604286
1995 SGCG limb-girdle muscular dystrophy type 2C 253700
1995 SMN1 spinal muscular atrophy type 1 253300
1996 CHS1 Chediak-Higashi syndrome 214500
1996 CSTB progressive myoclonus epilepsy 254800
1996 ED1 ectodermal dysplasia type 1 305100
1996 EXT2 multiple exostoses type 2 133701
1996 FANCA Fanconi anaemia 227650
1996 FRDA Friedreich ataxia 229300
1996 GPC3 Simpson-Golabi-Behmel syndrome type 1 312870
1996 HFE haemochromatosis 235200
1996 HPS Hermansky-Pudlak syndrome 203300
1996 KCNQ1 long QT syndrome type 1 192500
1996 MTM1 X-linked myotubular myopathy type 1 310400
1996 PITX2 Rieger syndrome 180500
1996 PKD2 polycystic kidney disease type 2 173910
1996 PTCH Gorlin syndrome 109400
1996 RECQL2 Werner syndrome 277700
1996 RPGR X-linked retinitis pigmentosa 300389
1996 SGCD limb-girdle muscular dystrophy type 2F 601287
1996 TAZ Barth syndrome 302060
1996 TCF1 maturity-onset diabetes of the young type 3 600496
672 Genetic Epidemiology

Genetic Epidemiology. Table 1 A list of human disease genes characterized through genetic epidemiological
research employing a positional cloning approach (Continued)

Year Gene Disease OMIM

1996 TCOF1 Treacher-Collins syndrome 154500
1997 ABCA4 Stargardt disease type 1 248200
1997 AIRE autoimmune polyglandular syndrome type 1 240300
1997 DIAPH1 non-syndromic autosomal dominant deafness type 1 124900
1997 DYT1 early-onset torsion dystonia 128100
1997 JAG1 Alagille syndrome 118450
1997 MEFV familial Mediterranean fever 249100
1997 MEN1 multiple endocrine neoplasia type 1 131100
1997 MID1 Opitz syndrome 300000
1997 MYOC juvenile primary open angle glaucoma 137750
1997 NPC1 Niemann-Pick disease 257220
1997 RS1 retinoschisis 312700
1997 SCA7 spinocerebellar ataxia type 7 164500
1997 SLC26A4 Pendred syndrome 274600
1997 TBX5 Holt-Oram syndrome 142900
1997 TSC1 tuberous sclerosis 191100
1997 UBE3A Angelman syndrome 105830
1997 ZIC3 situs inversus 306955
1998 CACNA1F X-linked congenital night blindness type 2 300071
1998 CLN5 neuronal ceroid lipofuscinosis type 5 256731
1998 CTNS cystinosis 219800
1998 DCX X-linked lissencephaly 300067
1998 DFNA5 non-syndromic autosomal dominant deafness type 5 600994
1998 DYSF limb-girdle muscular dystrophy type 2B 253601
1998 EPM2A progressive myoclonus epilepsy (Lafora) 254780
1998 FCMD Fukuyama type congenital muscular dystrophy 253800
1998 KCNQ2 benign familial neonatal convulsions type 1 121200
1998 MYO15A non-syndromic sensorineural recessive deafness type 3 600316
1998 NBS1 Nijmegen breakage syndrome 251260
1998 NPHS1 nephrotic syndrome type 1 256300
1998 PABPN1 oculopharyngeal muscular dystrophy 164300
1998 PARK2 juvenile Parkinson disease 600116
1998 RP2 X-linked retinitis pigmentosa 312600
1998 SH2D1A X-linked lymphoproliferative disease 308240
1998 STK11 Peutz-Jeghers syndrome 175200
1998 USH2A Usher syndrome type 2A 276901
 Genetic Epidemiology 673

Genetic Epidemiology. Table 1 A list of human disease genes characterized through genetic epidemiological
research employing a positional cloning approach (Continued)

Year Gene Disease OMIM

1998 VMD2 Best disease 153700
1998 WFS1 Wolfram syndrome 222300
1999 ABCA1 Tangier disease 205400
1999 ATP2A2 Darier disease 124200
1999 CCM1 cerebral cavernous malformations type 1 116860
1999 CLN8 progressive epilepsy with mental retardation 600143
1999 EFEMP1 malattia leventinese 126600
1999 LMNA Emery-Dreifuss muscular dystrophy 181350
1999 PRG4 camptodactyly-arthropathy-coxa vara-pericarditis syndrome 208250 G
1999 SLC17A5 Salla disease 604369
1999 SLC19A2 thiamine-responsive megaloblastic anaemia 249270
1999 SLC25A13 adult-onset citrullinemia type 2 603471
1999 SPG4 autosomal dominant hereditary spastic paraplegia type 4 182601
1999 WISP3 progressive pseudorheumatoid dysplasia 208230
2000 AIPL1 Leber congenital amaurosis type 4 604393
2000 ATP2C1 Hailey-Hailey disease 169600
2000 FGF23 autosomal dominant rickets 193100
2000 MCOLN1 mucolipidosis type 4 252650
2000 MKKS McKusick-Kaufman syndrome 236700
2000 MTMR2 Charcot-Marie-Tooth disease type 4B 601382
2000 MYH9 May-Hegglin anomaly 155100
2000 NPHS2 idiopathic steroid-resistant nephrosis 600995
2000 PRKAR1A Carney complex 160980
2000 PVRL1 cleft lip/palate-ectodermal dysplasia syndrome 225000
2000 RELN autosomal recessive lissencephaly 257320
2000 SPINK5 Netherton syndrome 256500
2000 USH1C Usher syndrome 1C 276904
2001 BBS2 Bardet-Biedl syndrome 209900
2001 BBS4 Bardet-Biedl syndrome 209900
2001 BSND Bartter syndrome 602522
2001 CDH23 Usher syndrome 1D 601067
2001 ELAC2 familial prostate cancer 176807
2001 FANCD2 Fanconi anaemia 227646
2001 PRPC8 retinitis pigmentosa 13 600059
2001 SOST sclerosteosis 269500
2001 USH3A Usher syndrome 3 276902
674 Genetic Epidemiology

Genetic Epidemiology. Table 1 A list of human disease genes characterized through genetic epidemiological
research employing a positional cloning approach (Continued)

Year Gene Disease OMIM

2002 BBS1 Bardet-Biedl syndrome 209900
2002 GDAP1 Charcot-Marie-Tooth disease type 4A 214400
2002 RNASEL hereditary prostate cancer type 1 601518
2002 SMARCAL1 Schimke immuno-osseous dysplasia 242900
2002 TMC1 non-syndromic autosomal dominant deafness type 36 606705
2002 TRPM6 hypomagnesemia with secondary hypocalcemia 602014

OMIM: Entry number in “Mendelian Inheritance in Man Online” at ▶http://www.ncbi.nlm.nih.gov/omim/

approach taken to detect the genetic variants respon-
sible for these diseases is generally known as
“positional cloning”. Instead of relying upon prior
knowledge about the disease-causing biochemical
defect(s), positional cloning utilizes the segregation
pattern of genetic markers (e.g. SNPs, microsatellites,
RFLPs) in affected families to localize the genes
involved in a given phenotypic trait (“linkage analy-
sis”). The more often the trait and a particular marker
allele are co-inherited by family members, the stronger
the evidence that a gene in the vicinity of the marker
influences the trait, i.e. that marker and disease gene are
linked.
Characteristics
Gene Mapping and Meiotic ▶Recombination
Formally, linkage analysis involves the assessment of
the recombination fraction θ between two genetic loci
like, in the context of genetic epidemiology, a marker
and an unknown disease gene. Parameter θ equals the
rate at which children receive from a given parent either
the grand-maternal allele at one locus and the grand-
paternal allele at the other or vice versa. Assuming
Mendelian inheritance, θ = ½ for a pair of genes located
on different chromosomes. For two loci residing on the
same chromosome, meiotic recombination is only
possible via “▶crossing-over”, signifying the breakage
and re-union of homologous, non-sister chromatids
during the metaphase of meiotic division I (Fig. 1).
Indeed, it can be shown mathematically that θ equals
exactly half the probability of at least one crossing-over
occurring between the two loci in question, provided
that some critical assumptions about the randomness of
crossing-overs are correct.
In any case, one corollary of the above is that θ
represents an increasing function of the physical
distance between two loci and therefore provides a
key parameter for gene mapping. Unfortunately, θ is
not an additive measure of distance since it can never
exceed ½. In order to facilitate gene mapping, θ
therefore has to be transformed into a linear “▶genetic
distance” d(θ) by some ▶mapping function (the most
widely used being d(θ) = ½ln(1–2θ), proposed by
British geneticist J.B.S. Haldane in 1919). Genetic
distance is measured in units of Morgan (M) in order
to honour T.H. Morgan, the American Nobel
prize-winning biologist who first discovered the role
of chromosomes in heredity. One centi-Morgan (cM)
roughly corresponds to one expected recombination per
100 meioses.
Parametric Linkage Analysis
Linkage analysis has two major objectives, namely
(i) to clarify with some statistical confidence whether
θ = ½ or θ < ½, and (ii) to estimate θ in the latter case.
Both goals are easily achieved in laboratory animals
where controlled breeding can be performed such that,
after a few generations, recombinants and non-
recombinants can simply be counted. In humans as
well as in animals with longer generation times
however, linkage analysis has to fall back upon family
data. How such data can be used to draw statistical
inferences about linkage depends upon their complex-
ity, i.e. on how much prior knowledge is available
about the genetic, environmental and stochastic nature
of the phenotypes of interest.
For the simple genotype-phenotype relationships
encountered with most monogenic disorders, family
data can be analysed by explicitly modelling the co-
inheritance of the disease and marker in a family, based
upon the underlying genotype frequencies and ▶pene-
trances (“parametric linkage analysis”). In such cases,
the likelihood L of a given recombination fraction θ0
between disease gene and marker is a computable
function of the phenotypic data D observed in the
family. This leads to the definition of zðy0 Þ ¼ log10
fLðy ¼ y0 jDÞ=Lðy ¼ 1=2jDÞg.
Quantity z, termed the “▶lod score” and introduced by
N.E. Morton in 1955, is used as a sequential statistic to
 Genetic Epidemiology 675

Genetic Epidemiology. Figure 1 Process of crossing-over during germ cell development. The two nearly
duplicated chromosomes align during the late metaphase of meiotic division I, where an overlap and breakage
of their constituent non-sister chromatids may occur (red: maternal chromosome, blue: paternal chromosome).
Re-annealing and re-synthesis by the cellular repair mechanisms leaves two chromatids with genetic material
flanking the site of crossing-over that is not of the same parental origin (i.e. the resulting chromosomes would
represent recombinants with respect to the two loci shown).

test whether θ < ½, and to quantify the evidence in . relatively minor effects exerted by individual
favour or against θ0. When z(θ0) > 3, then linkage variants and
between disease gene and marker locus is regarded as . an important modifying role of environmental
being proven and θ is estimated by that recombination factors.
fraction that yields the highest lod score. This
Since no prior information is usually available as to
procedure is exemplified in Fig. 2 for a large family
how genetic variation at a given locus modifies the risk
affected by an autosomal dominant disorder. The
for a complex disease (i.e. the genetic model of the
results of a linkage analysis are usually presented in
disease is unknown), gene mapping for complex
the form of lod score tables or graphs (Fig. 2b) where
diseases has to adopt robust, albeit less powerful,
ceteris paribus, studies of independent (i.e. unrelated)
“non-parametric” or “model-free” linkage analysis
families can be aggregated by summation of the family-
such as, for example, the study of pairs of relatives.
wise lod scores. If a disease gene is to be integrated into
The idea underlying this approach, which is both
a pre-existing map of linked markers, then this is most
simple and intuitively appealing, goes back to a 1935
efficiently performed by parametric multi-locus linkage
paper by British medical geneticist L.S. Penrose. The
analysis, which has been shown to be up to twice as
number of sib-pairs, out of a total of n independent
accurate as the pair-wise approach, measured in terms
pairs, who share k parental alleles of an autosomal
of the variance of the ensuing recombination fraction
locus identical by descent (“ibd”) follows a multi-
estimates.
nomial distribution with parameters zk, k = 0, 1, or 2
(Fig. 3). Under the null hypothesis of no etiological
Non-Parametric Linkage Analysis of Complex Diseases
connection between marker and disease, the inheritance
Increasingly, the major challenge to genetic epidemiol-
of a marker can be assumed to follow Mendelian rules
ogy is being posed by so-called “complex” diseases,
and to be independent of the disease status of the
which comprise conditions such as diabetes, heart
siblings. This implies that, irrespective of whether the
disease, cancer and psychiatric illness. When compared
sibs are concordant or discordant, z0 = ¼, z1 = ½ and
to monogenic disorders, this category of disease is
z2 = ¼. Any test for a deviation from these proportions
characterized by
represents a test for linkage between the marker
. a substantially higher population frequency, and a putative disease gene. In its original form, the
. the involvement of multiple genes, most probably affected sib-pair test required that the ibd status at each
interacting with one another, marker be determined unequivocally for all sib-pairs.
676 Genetic Epidemiology

Genetic Epidemiology. Figure 2 Linkage analysis of a family affected by an autosomal dominant disease.
(a) shows the pedigree with patients marked by black symbols. Genotypes observed for a biallelic marker locus
with alleles “A” and “B” are displayed alongside each individual. (b) is a graphical display of the lod scores as
calculated from the family data, assuming full penetrance and lack of de novo mutation. The lod score at θ0=0.00
equals minus infinity owing to a recombination that has occurred during the paternal meiosis leading to an
affected girl in the most recent generation (marked by *).

However, a number of derivatives of the test have since Quantitative Phenotypes

been developed which incorporate posterior distribu-
tions on k (as inferred from other relatives), utilize
relatives other than sibs or are based upon identity-by-
state (“ibs”) rather than ibd allele sharing. In general,
these methods are less powerful than the original test,
but can extract mapping information that would
otherwise not be used.
Non-parametric methods have also been devised for the
genetic analysis of quantitative phenotypes which, in
many instances, may be more powerful than the
consideration of dichotomized disease outcomes (“af-
fected” vs “non-affected”) derived from them. The idea
underlying the concept of quantitative trait mapping is
nevertheless the same as for qualitative characters in

Genetic Epidemiology. Figure 3 Level k of autosomal identical-by-descent (ibd) allele sharing between two sibs.
For each value of k (i.e. k=0,1, or 2), shared alleles are marked in red for the second sib.
Genetic Epidemiology. Table 2 Haplotype
frequencies of two biallelic loci
Marker 2 Marker 1 Total
allele 1A allele 1B
allele 2A a b a+b
allele 2B c d c+d
Total a+c b+d 1 mapping resolution that can be achieved using (family-
Linkage disequlibrium (LD) is usually measured by D=ad-bc.
However, since D depends upon the marginal sums (i.e. the
allele frequencies), LD is often quantified by D'=D/Dmax instead.
Here, Dmax denotes the maximum absolute value of D that is
possible for the same allele frequencies. If D>0, then Dmax=min
{(a+c)(c+d),(a+b)(b+d)}; if D<0, then Dmax=min{(a+b)(a+c),
(b+d)(c+d)}.
that the observed level of marker ibd sharing between
relatives is compared to their phenotypic similarity. If a
marker is linked to a gene that influences the trait, then
sibs with similar phenotypes, for example, will tend to
share more than ½ of their marker alleles ibd whereas
dissimilar sibs will not. A formal test for this effect was
proposed by American statisticians J.K. Haseman and
R.C. Elston in 1972. For each sib-pair, the squared
difference Y of their phenotypic values is calculated,
and the number π of their ibd marker alleles determined
(or estimated). A linear regression analysis of Y on π
then reveals (i) whether the two variables are correlated
and (ii) whether a significant relationship, if found,
is a biologically plausible indication of linkage. The
Haseman-Elston approach has since been expanded
and refined, for example by considering the mean-
corrected product of the sib-specific phenotypes
instead of Y, so as to increase informativity about
linkage. An alternative method aims at decomposing
the variance of the phenotype into components that are
due to genes which are either linked to the marker, or
not (“variance component analysis”). For a marker
Genetic Epidemiology 677
linked to a gene of strong effect, the phenotypic G
covariance among relatives should be positively related
to their degree of marker ibd sharing, and this
relationship translates into larger estimates of the
corresponding variance components.
▶Linkage Disequilibrium
Intervals of 1 Mb are generally regarded as the limit of
based) linkage analysis, and more precise mapping of
disease genes can only be expected from population-
based association studies, exploiting linkage disequili-
brium (LD). The most general definition of LD is the
condition that the alleles of linked loci do not occur
statistically independent on the chromosomes observed
in a population. In the simplest case of two biallelic
loci, haplotype frequencies can be arranged in a
two-by-two table with cell probabilities a,b,c and d
(Table 2), and LD is meaningfully quantified by the
cross product D = ad − bc. When a new allele first
arises in a population by either mutation or migration, it
occurs as a single copy that resides on a certain
haplotype background, together with certain alleles of
other loci. Only in later generations will the allele
become more frequent through either selection or
genetic drift or both. In any case, chromosomes
carrying the new allele will recombine with chromo-
somes carrying other haplotypes so that the strong
original LD will erode with time. This loss of LD will
be slower for closely linked loci and, under some
simplifying assumptions about mutation rates, migra-
tion and mating patterns, D can indeed be shown to
decrease by a factor of 1 − θ in each generation.
Therefore, strong LD is an indication of close linkage
and assessment of LD between a marker and putative
disease gene can be regarded as linkage analysis in a
super-pedigree tying all analysed individuals together.
In principle, any kind of genetic marker can be
employed in disease association studies provided that
(i) the marker mutation rate is low and (ii) the density of
markers chosen for analysis is high enough to ensure
678 Genetic Epidemiology

Genetic Epidemiology. Figure 4 Transmission disequilibrium test (TDT) of disease-association for biallelic marker
genes. The sampling units of the TDT are nuclear families comprising both parents and an affected child (“trios”).
Only transmissions from heterozygous parents to their children are evaluated (cells x and y in the table shown). The
TDT statistic equals McNemar’s (x-y)2/(x+y), which follows a χ2 distribution with 1 degree of freedom under the null
hypothesis of no association.

sufficiently strong LD with disease gene(s). Empirical
data and theoretical considerations suggest that a
sensible marker density should be no lower than
approximately 1 in 50,000 nucleotides. Ideally, asso-
ciation markers should be chosen from within genes
that represent biologically plausible candidates for an
involvement in the disease of interest. The chance of
detecting association would then be increased further if
marker alleles were themselves of functional signifi-
cance by altering, for example, the protein product or a
regulatory sequence.
Family-based Association Studies
The simplest form of a population-based association
study is that invoking a case-control design. As in
classical epidemiology, relative risks or odds ratio of
particular marker genotypes or haplotypes can be
estimated from the respective frequencies in patients
and unrelated healthy controls. However, concerns
have arisen over the potentially confounding effects of
ethnic, social or geographical population stratification
that would generate systematic differences between the
genetic characteristics of the two samples, unrelated to
disease. To solve this problem, family-based associa-
tion designs have been proposed of which the
transmission disequilibrium test (TDT) is the most
widely used. A TDT is basically a McNemar test for
preferential transmission of particular marker alleles
from heterozygous parents to their affected offspring
(Fig. 4). Any deviation of the transmission to non-
transmission ratio from the expected 1:1 is indicative
of both linkage between marker and disease gene
in the presence of LD and of LD in the presence of
linkage.

Since chromosomes of close relatives act as internal
controls in the TDT and similar tests, it has become
almost paradigmatic for the genetic epidemiology of
complex disease that family-based association studies
are superior to case-control designs. However, family-
based designs also have disadvantages that might not
always be fully outweighed by their apparent robust-
ness. For example, gene-environment interactions
cannot be analysed in family-based studies since no
genuine controls are available for comparison to
patients. Furthermore, parental genotypes are required
for the TDT and these may be difficult to obtain for
late-onset disorders. The use of other family members
as surrogates to try to reconstruct parental genotypes
with some certainty has been suggested in such
instances. However, such methods are usually costly,
inaccurate and inefficient. On the other hand, possible
confounding of data by population stratification can
be avoided in case-control studies through careful
matching by ethnic and geographic origin. Further-
more, if a sufficiently large set of genetic markers is
available that is not itself tested for association, then
these markers can be used to estimate the level of
population stratification and to correct the employed
test statistic accordingly. Finally, although population
stratification may represent a theoretical possibility,
empirical evidence for its practical importance as a
confounding factor in genetic epidemiology is still
lacking.
Clinical Relevance
The reasons for the apparent lack of success that plagues
genetic studies of complex disease are manifold and the
most critical issue is probably the reduction in power
caused by genetic heterogeneity. Genetic heterogeneity
can occur at two levels, either within genes (“allelic
heterogeneity”) or between genes (“locus heterogene-
ity”). In order to increase the power of genetic studies of
complex diseases, the major goal in their planning and
performance is thus to control for genetic heterogeneity
at all levels. First and foremost, this requires a careful
choice of the population under study. Ideally for a
disease association to be detectable, all copies of the
predisposing allele should be ibd in patients. The best
populations to analyse for LD are therefore those that
have been small and isolated for most of their history or
that have undergone recent expansion from a small
number of founders. Not surprisingly, genetic epide-
miology has been particularly successful in Finland and
some inward breeding communities in the USA (e.g.
Amish, Hutterites, Ashkenazim). In addition to popula-
tion genetic issues, an appropriate definition of
phenotypes, a breakdown by sub-phenotypes and the
use of sensible covariates to define etiologically homo-
geneous sub-populations can help to reduce genetic
heterogeneity further. Finally, genetic epidemiology
Genetic Heterogeneity 679
is a constantly evolving scientific discipline so that
improved power may also arise from the development
and application of new analytical tools that take genetic
and etiological heterogeneity into account (e.g. multi-
locus statistics, time series analysis).
Complex human diseases are typically common and
have a substantial economic impact upon national health
systems. The resulting public interest renders disorders
such as cancer, heart disease and diabetes particularly
attractive for genetic research and a large number of
studies into these diseases are often being performed in
parallel. On the other hand, for the reasons mentioned
above, the prior probability of successfully mapping and
characterizing genes for complex diseases is compara-
tively low and usually unknown. Even with high
significance levels imposed, most positive gene map- G
ping results may therefore be wrong. This implies that
genetic epidemiological research will almost inevitably
continue to be driven towards the generation of false
positive results. Claims as to the elucidation of a disease
predisposition should therefore always be received with
some caution and judged as preliminary until confirmed
by controlled replication, meta-analysis or independent
laboratory experiments.
References
1. Elston RC (1998) Linkage and association. Genet
Epidemiol 15:565–576
2. Schork NJ, Cardon LR, Xu X (1998) The future of genetic
epidemiology. Trends Genet 14:266–272
3. Risch NJ (2000) Searching for genetic determinants in the
new millennium. Nature 405:847–856
4. Terwilliger JD, Göring HHH (2000) Gene mapping in the
20th and 21st centuries: statistical methods, data analysis,
and experimental design. Hum Biol 72:63–132
5. Sham P (2001) Shifting paradigms in gene-mapping
methodology for complex traits. Pharmacogenomics
2:195–202
Genetic Hearing Disorder
Definition
Genetic hearing disorders mainly comprise of non-X
linked genetic hearing loss, which is only observed in
homozygous individuals having inherited the mutated
gene from each parent.
▶Microvilli
Genetic Heterogeneity
▶Heterogeneity/Heterogenous
680 Genetic Immunization
Genetic Immunization
Definition
Genetic immunization is a technique to induce specific
immune responses by injecting antigen-encoding
expression plasmid DNA.
▶DNA-based Vaccination
Genetic Interactions
Definition
Genetic interactions describe interactions between two
or more mutations that result in a phenotype.
▶Cell Polarity
Genetic Map
Definition
Genetic map (also known as a linkage map) is a map of
a genome, which shows the relative positions (order
and distances) of the genes and/or markers on the
chromosomes. The map is based on pairwise coin-
heritance (linkage) of markers. Genetic maps are
generally composites of data from many experiments.
▶Chromosome 21, Disorders
▶YAC and PAC Maps
Genetic Modification
Definition
Genetic modification describes the introduction of a
new nucleic acid into a cell, organism or micro-
organism. In the context of clinical gene transfer, the
term is used in conjunction with the transfer of a nucleic
acid. This can be achieved by either introducing an
expression construct or by modification of cellular
nucleic acid e.g. by revision of a point mutation. Nucleic
acids used (e.g. for triple helix formation or RNA with
ribozyme function that is not part of a transgene) are not
ment to lead to genetic modification as far as the term is
used in the context of clinical gene transfer.
▶Clinical Gene Transfer
Genetic Polymorphism
Definition
Genetic polymorphism is the presence of multiple
inherited forms of a gene with at least an allele
frequency of 1% within the population.
▶Pharmacogenomics
Genetic Predisposition
to Multiple Sclerosis
A LASTAIR C OMPSTON
University of Cambridge Neurology Unit,
Cambridge, UK
alastair.compston@medschl.cam.ac.uk
Definition
▶Multiple sclerosis is a typical complex trait. There is
an increased familial recurrence risk and evidence from
▶linkage and ▶association studies for functional
▶polymorphisms that increase disease susceptibility
but without Mendelian patterns of inheritance. There is
epidemiological evidence for the role of environmental
factors in determining the distribution of the disease.
Characteristics
Familial Multiple Sclerosis
Multiple sclerosis has a familial recurrence rate of
approximately 15%. Overall, the reduction in risk
changes from 3% (relative risk 9) in first-degree
relatives to 1% (relative risk 3.4) and 1% (relative risk
2.9) in second and third degree relatives, respectively,
compared with a population lifetime rate of 0.3%. At
one extreme, the risk of multiple sclerosis is 35% for
the monozygotic twin partner of an affected proband, or
the children of parents who are both affected (▶con-
jugal pairs), compared to 0.3% for individuals related
by adoption to a proband. Familial clustering is
therefore genetically determined.
Cellular and Molecular Recognition
The Analysis of Complex Traits
Two methods—linkage and association—underpin the
analysis of complex traits. Linkage has low statistical
power but operates within families across relatively
large genetic distances. Association has high statistical
power but is only informative within the boundaries of

▶linkage disequilibrium present in the population
under study. In founder populations, polymorphisms
that increase susceptibility to disease are necessarily
located within a large group of linked genes. This block
is subject to recombination during subsequent meioses
and is gradually whittled down until there is no residual
linkage disequilibrium. It seems that genetic factors
determine susceptibility and, to some extent, also shape
the clinical course. The choice of markers has been
driven either by a priori guesses on the nature of
susceptibility (▶candidate genes) or systematic screen-
ing of the genome. Candidates selected because they
map within linked regions combine both strategies.
Markers are analyzed individually (single point
analysis) or corrected for information available from
their neighbours (multipoint analysis). The cumulative
probability that a given marker or region of interest is
linked or associated with multiple sclerosis can be
formally tested by ▶meta-analysis of available studies.
The lesson learned thus far is that no one gene makes a
major contribution to susceptibility although collec-
tively they determine a relative risk (for siblings) of
around 20.
Candidate Genes in Multiple Sclerosis
Much effort has gone into the assessment of candidate
susceptibility genes chosen on the basis of prevailing
ideas concerning the pathogenesis of multiple sclerosis.
Population studies comparing unrelated cases and
controls show an association between the class II
▶major histocompatibility complex alleles DR15 and
DQ6 and their underlying genotypes (DRB1*1501,
DRB5*0101 and DQA1*0102, DQB2*0602). This is
seen in almost all populations (Caucasian, Oriental,
Arab, Hispanic, Finnish, Russian and Jewish) although
the strength of the association differs. Even those ethnic
groups in which the frequency of multiple sclerosis is
low, or the phenotype distinct from that usually
observed in northern Europe, are now acknowledged
to be primarily DR15 associated with one exception. In
Sardinians, the association is with DR4 (DRB1*0405-
DQA*0301-DQB1*0302). In some other Mediterranean
populations (Canaries and Turkey), the association is
with DR2 (DBR1*1501, DQA1*0102, DQB1*0602)
and DR4 (DRB1*04, DQA1*03, DQB1*0302). Most
investigators assume that—based on the genetics
and obvious candidature through its role in restricting
the immune response—DR (or DQ) is itself the
susceptibility gene encoded at 6p21.
Outside the major histocompatibility complex, many
candidates have been screened. The case made on the
basis of ideas concerning the pathogenesis of multiple
sclerosis is much strengthened by prior knowledge that
the candidate gene also maps to a region already
implicated by linkage studies (positional candidates).
The range of candidates now studied includes adhesion
Genetic Predisposition to Multiple Sclerosis 681
molecules, immune receptors, accessory molecules,
cytokines, chemokines and their receptors or antagonists,
structural genes of oligodendrocytes or myelin, and
molecules regulating cell death and survival. In a small
and regionally restricted population of Finns, multiple
sclerosis is associated and linked to the gene for myelin
basic protein, encoded on chromosome 18. The effect can
be traced to a subset of families with common ancestry
and does not hold up in the larger cohort. It is the nature of
screening so many potential effects that a proportion will
appear to be associated or linked but by chance. Equally,
it would be difficult to show unambiguously that one or
more genes exerting a small biological effect is making a
contribution to susceptibility in a single study. That said,
some plausible associations or linkages have been
provisionally reported, although no one of these has yet G
stood up to repeated replication. These mainly involve
factors that appear to increase susceptibility to multiple
sclerosis, but there is provisional evidence for primary
effects on resistance and effects on severity or clinical
features of the disease.
DR15 is associated with younger age at diagnosis and
female gender but does not distinguish features relating
to disease course, outcome, specific clinical features or
paraclinical investigations. This suggests that DR15
exerts its effect on susceptibility rather than modifying
the course of multiple sclerosis. Loci apparently
associated with disease protection are FAS-670, IL-
12p40, FcR and MCP-3. Genes that may influence the
course or phenotype of multiple sclerosis include
CTLA4, IL-1Ra/IL-1B, IL-2, CCR5, oestrogen recep-
tor, CNTF and Apo-E and mutations of mitochondrial
DNA.
Linkage Genome Screens
The dividend from attempting to fast-track the solution
to susceptibility in multiple sclerosis by the candidate
gene approach has been small but the problem is also
not solved by the nine whole genome linkage analyses
using variable numbers of families from the United
States, Canada, United Kingdom, Finland, Sardinia,
Italy, Turkey, Scandinavia and Australia. These screens
have involved between 21–225 families each typed for
257–443 microsatellite markers chosen to provide an
average spacing of around 10 centiMorgans. Although
several new genomic regions of interest were revealed,
many are false positives. These whole genome screens
have been used to explore regions of interest in more
detail hoping to consolidate their status based on
mapping but without picking out positional candidates.
Linkage on chromosome 17q is supported by addi-
tional positional screens from Denmark, Canada, and
Finland. There is collateral support for the involvement
of chromosomes 5p, 7p and 12q based on direct
evidence and synteny with genes determining sus-
ceptibility to experimental forms of demyelination.
682 Genetic Predisposition to Multiple Sclerosis
Meta-analysis has been deployed in the expectation
that this will reduce the evidence for false positive
peaks and strengthen the candidature of those which are
genuine providing the best guide to shared regions of
interest as the map is serially up-dated. This was last
completed in 2005.
Whole Genome Association Screening
Until recently, whole genome linkage disequilibrium
mapping was considered impractical and dependent on
chance co-localisation of susceptibility genes and
markers applied randomly and at low density. This
situation changed with the increased availability of
widely distributed microsatellite markers and is set to
increase further with the identification and mapping of
▶single nucleotide polymorphisms. A first pass at
screening the genome for association was completed in
2003 based on a 0.5 cM map of microsatellite markers
and using DNA pools derived from cases with multiple
sclerosis and unrelated controls. Individual results
provided provisional evidence for associations based
on linkage disequilibrium outside the major histo-
compatibility complex on 6p21. The number of
micro-satellite markers used necessarily made this a
low-density screen, especially since the number of
informative markers was less than the full set of 6000
used in this Genetic Analysis of Multiple sclerosis in
EuropeanS (GAMES). With considerable variation
depending on the stochastic nature of linkage dis-
equilibrium on individual chromosomes in European
populations, it may only have covered 10% of the
genome in detail and another 20% in part, leaving
much yet to be explored. Perhaps its main value lies in
the exclusion of many microsatellite markers lying in
blocks of linkage disequilibrium of varying size rather
than in the provisional positive associations. New
screens based on single nucleotide markers present on
individual chips, and at a much higher density are now
in progress.
Future Strategies for Identifying Susceptibility Genes
Once regions of interest are mapped, the next aim is to
move from whole genome screening to the identifica-
tion of functional polymorphisms which condition one
component or another of the disease process and
determine variations in the clinical course and features.
How to reach that position is less clear and several
parallel strategies have been suggested. One is to add
incrementally to the number of available famil``õõies
until thresholds for linkage are reached for the
identification of secure loci using statistical criteria
for genome wide significances. An alternative is to
accept that the combination of linkage and association
now available is sufficient to concentrate the search for
positional candidates within regions of interest. Each
provisional site already offers several interesting
possibilities, although the number of genes encoding
components of the nervous, immune and signalling
systems is such as to make practically any region
suggestive with respect to sensible candidates. Rapid
progress is being made in characterising the whole
genome for the size, distribution and diversity of blocks
containing a restricted number of haplotypes. If the
preliminary evidence holds up, it will be possible to tag
the common variants within each block in populations
(such as Europeans) retaining significant linkage
disequilibrium and screen individuals for the suscept-
ibility haplotypes with relative economy.
Clinical Relevance
▶Concordance within families can be used to gauge
the influence of genetic factors in determining the
clinical phenotype of multiple sclerosis. Time to reach
the later stages of disability does not differ between
familial and sporadic cases. Conjugal pairs show no
evidence for clinical concordance, clustering at year of
onset or distortion of the expected pattern of age at
onset in the second affected spouse. The most recent
assessment of concordance in co-affected siblings and
parent-child pairs supports a role for genetic factors in
determining age at onset and progression either from
onset or after a phase of relapsing remitting disease, but
not the initial presentation or disability. Concordant
parent-child pairs show no distortion in the random
distribution of male-female pairings and neither sex nor
line of inheritance influence disability, age at onset or
course. In this situation, disability is highest in the male
offspring of affected fathers, who more commonly
follow a primary progressive course.
The risk of ▶autoimmunity is increased in the relatives
of probands with multiple sclerosis. Three surveys,
together involving around 4000 relatives of 1000
probands, have shown recurrence of multiple sclerosis
in 15% with another autoimmune disease (Graves’
disease, rheumatoid arthritis and diabetes) in about 5%
of pedigrees. Several other disorders have been
considered more frequent than expected in patients
with multiple sclerosis. None of these is entirely
secure but the there may be co-morbidity between
neurofibromatosis 1 and primary progressive multiple
sclerosis.
A minority of patients who meet clinical criteria for the
diagnosis of multiple sclerosis and in whom there are
associated magnetic resonance imaging abnormalities
and cerebrospinal fluid oligoclonal bands have an
illness in which there is disproportionate involvement
of the anterior visual pathway. These are commonly
women with male relatives already known to be
affected by ▶Leber’s hereditary optic neuropathy and
they have pathological mutations of mitochondrial

DNA. The clinical features of demyelinating disease
seen in Orientals and Africans are distinct and provide
another example of clinical heterogeneity. In Japan,
multiple sclerosis shows either a Western phenotype, in
which a number of sites are involved, or an optico-
spinal pattern in which the clinical picture is dominated
by involvement of visual and spinal cord pathways
with a specifically different genetic background
(HLA-DP*1501 rather than DRB1*1501 seen with
the Western phenotype). However, recent reports of
multiple sclerosis in Japanese highlight the pre-
viously under-reported extent of the so-called Western
phenotype. Demyelinating disease is considered ex-
tremely rare in Africans, but a number of cases are
described and the phenotype is typically a severe illness
dominated by one or more episodes usually affecting
the anterior visual pathway and spinal cord—again
combining the anatomical features of ▶Devic’s disease
with the clinical course of moderately severe relapsing
remitting multiple sclerosis. ▶Phenocopies may con-
fuse the analysis of complex traits where diagnosis
depends on pattern recognition of symptoms, signs and
laboratory investigations in the absence of a test for the
disease. Reassuringly, one large cohort screened for
other diseases was shown not to be contaminated by
cases of ▶CADASIL, ▶spinocerebellar degeneration,
or ▶adrenoleukodystrophy.
Conclusions
Six main categories of susceptibility genes can be
predicted: genes which determine susceptibility to the
process of inflammation across a range of disorders –
the autoimmune genes; those which determine the
specificity of that process for the development of
multiple sclerosis – the ubiquitous genes; those which
are relevant for the pathogenesis in isolated populations
– the domestic genes; those which determine particular
phenotypes – the pleiotropic genes; those which
determine variations in the clinical course – the
modifying genes and those which cluster to provide
specifically different (heterogeneous) contributions to
the pathogenesis – the epistatic genes. A major part
of future studies will be to resolve the question of
disease heterogeneity in multiple sclerosis. When
eventually in place, the potential of this genetic
knowledge for improved understanding of the patho-
genesis of multiple sclerosis and designing novel
treatments is considerable. Resolving the issues of
complexity and heterogeneity in multiple sclerosis and
other complex traits has practical dividends. Without
knowledge linking aetiology to pathogenesis and
phenotype, putative new treatments will continue to
be screened in cohorts who may or may not have an
appropriate pathological substrate for that particular
intervention.
Genetic Screen 683
References
1. Ebers GC, Bulman DE, Sadovnick AD et al (1986) A
population based study of multiple sclerosis in twins.
N Engl J Med 315:1638–1642
2. Gabriel SB, Schaffner SF, Nguyen H et al (2002) The
structure of haplotype blocks in the human genome.
Science 296:2225–2229
3. Harding AE, Sweeney MG, Brockington M et al (1992)
Occurrence of a multiple sclerosis-like illness in women
who have a Leber’s hereditary optic neuropathy mito-
chondrial DNA mutation. Brain 115:989–989
4. Risch NJ (2000) Searching for genetic determinants in the
new millennium. Nature 405:847–856
5. Sawcer S, Maranian M, Setakis E et al (2002) A whole
genome screen for linkage disequilibrium in multiple
sclerosis confirms disease associations with regions
previously linked to susceptibility. Brain 125:1337–1347
6. The Transatlantic Multiple Sclerosis Genetics Coopera- G
tive (2001) A meta-analysis of genome screens in multiple
sclerosis. Mult Scler 7:3–11
Genetic Redundancy
Definition
Genetic redundancy refers to the presence of genes in
multiple forms in the DNA of eukaryotes. Two or more
genes are capable of executing the same tasks, thus
eliminating one gene’s function. It does not alter the
development of function.
▶Drosophila Model of Cardiac Disease
▶Muscle Development
Genetic Screen
Definition
Genetic screening is analysing a group of individuals to
identify those who are at high risk of having or passing-
on a specific genetic disorder. In experimental genetics,
the term describes an approach to identifying genes and
gene functions by random gene knock out and
identifying genes causing a specific phenotype; or vice
versa, identifying phenotypes caused by specific gene
knock out.
▶Genetic Screening in Populations
▶Mutagenesis Approaches in the Zebrafish
684 Genetic Screening in Populations
Genetic Screening in Populations
V ILMUNDUR G UDNASON
Icelandic Heart Association Heart Preventive Clinic
and Research Institute, Kopavogur, Iceland
v.gudnason@hjarta.is
Definition
“Genetic screening may be defined as any kind of test
performed for the systematic early detection or
exclusion of a genetic disease, the genetic predisposi-
tion or resistance to a disease or to determine whether a
person carries a gene variant which may produce
disease in offspring. Screening may be concerned with
the general population or with specific sub-populations
defined on some basis other than their health” (from the
European Society of Human Genetics, ▶http://www.
eshg.org/).
Characteristics
Population screening has been applied in the newly
born for a long time for medical conditions known to be
preventable by medical intervention, such as phenyl-
ketonuria. In more recent times considerably more
knowledge on underlying genetic defects for various
diseases has become available. What conditions to
screen for, medically, biochemically or genetically has
been a matter of discussion for decades. Most common
diseases have complex multifactorial and probably
▶polygenic underlying causes. For that reason, genetic
screening for complex diseases in populations is not
really feasible for the time being. However, a number
of genetic variations have been associated with the risk
of developing disease and may be applied as specific
tests in the evaluation of risk in groups of patients
identified by a given disease. Despite that, only
▶monogenic diseases or disorders fit the criteria set
for appropriate population screening including genetic
screening.
Wilson and Jungner (1) put forth ten criteria, which
should be considered before population wide screening
should be set up. These criteria are that:
1. the condition being screened for should be an
important health problem.
2. the natural history should be well understood
3. there should be a detectable early stage
4. treatment at an early stage should be of more
benefit than at a later stage
5. there should be a suitable test for identifying
people at the early stage
6. the test should be acceptable
7. intervals for repeating the test should be determined
8. there should be adequate health service provision
for the extra clinical workload resulting from the
screening
9. the risk of screening, both physical and psycholo-
gical should be less than the benefits
10. the costs should be balanced against the benefits
There are not many diseases or disorders that fulfil
these criteria completely and far from all monogenic
diseases do so. ▶Familial hypercholesterolemia (FH)
is one condition, which does fit exactly. FH is a
monogenic disorder that is due to a defect in the low
density lipoprotein (LDL) receptor gene with a lifelong
elevation of blood cholesterol (2). It has a prevalence of
1 in 500 in most populations and a high risk of
premature coronary artery disease and can be described
as an important health problem. The natural history of
FH is well understood and the disorder can be detected
early in life. Treatment is available and there is strong
evidence that early treatment is beneficial. The diag-
nosis of FH, whether performed by clinical or genetic
testing does not require more complex intervention than
venipuncture. Once the diagnosis is made, there is no
need for further diagnosis. Considerable experience in
screening for FH has accumulated in many populations
and FH can be looked at as a paradigm for screening for
other monogenic disorders or diseases.
Cellular and Molecular Regulation
Familial hypercholesterolemia is caused by a mutation
in the ▶LDL receptor gene. The ▶LDL receptor takes
up cholesterol from the bloodstream by binding LDL
cholesterol particles, mainly in the liver and thus reduces
blood levels of cholesterol. Individuals who lack LDL
receptor or have a reduced number of functional
receptors demonstrate considerably increased levels of
blood cholesterol. FH is an autosomal dominant
disorder with a gene dosage effect. This means that
heterozygotes for a mutation in the LDL receptor gene
frequently have double the amount of blood cholesterol
compared to individuals from the general population
and homozygotes or ▶compound heterozygotes have
several fold increase in blood cholesterol.
A variety of different mutations in the LDL receptor
gene have been found to cause FH. These can be
accessed on the FH web site www.ucl.ac.uk/fh/. The
sheer number of mutations makes the identification of
FH complicated. Most mutations are confined to a
single or a few families, but others are more wide-
spread. However, there are a number of mutations that
have been found to be population specific allowing for
population based screening for a given set of mutations.
There are two main approaches for identifying
previously unidentified individuals with FH; first,
examination of first-degree relatives for a given index
case and secondly, genealogical tracing in defined
 Genetic Screening in Populations 685

populations to a common ancestor for a known records or genealogical information is electronically

mutation in the LDL receptor gene.
First Degree Relative Screening
The conventional approach to identify new FH patients
is to ask FH index cases for informed consent to contact
their first-degree relatives. This is the method recom-
mended by the Med Ped project (Make Early Diagnosis
– Prevent Early Deaths in Medical Pedigrees) (▶http://
www.medped.org/). The main advantage of such an
approach is the high probability of diagnosing family
members carrying the LDL-receptor mutation when
contacting close relatives. The probability is 50% for
first-degree relatives of the index case (parent, sibling and
child) and then declines by about half for each generation.
In the Netherlands, experience from FH screening using
similar first degree relative approaches, revealed 2039
individuals identified as ▶heterozygous FH of 5442
relatives tested (3) The main disadvantages are that the
doctor needs to rely on consent from the patient to contact
his/her relatives and that pedigrees based on information
from the patients are seldom complete for more than one
or two generations back. Therefore the search for new
patients can reach a blind end.
Genealogy Tracing for Identification of
Affected Individuals
The genealogy tracing approach is applicable where
there is a common mutation in a specific population in
an isolated area where genealogical information is
available either from church records or other similar
available such as in the example from Iceland described
below (4).
A common mutation in the LDL receptor gene (I4T
+2C) has been identified and found to be responsible for
up to 60% of FH in Iceland. For the genetic screening
only probands with this mutation were included. These
probands were genealogically traced to common
ancestors by The Icelandic Genetic Council’s family
tracing office. The family tracing was performed
through a partly computerized database derived from
censuses (first carried out in Iceland 1703), church
records and birth and marriage certificates. Once a
common ancestor had been identified, a list of all
descendants was produced. The oldest individual alive
in each family lineage was identified as key individual G
and contacted for cholesterol measurements and for
genetic testing (Fig. 1) after obtaining informed consent.
If positive for the common mutation, his or her offspring
were recruited for testing. Relatives of key individuals
negative for the common mutation were not recruited.
Fourteen probands positive for the common mutation
were genealogically traced to four family clusters, one
cluster with four probands, one with three probands and
two with two probands each. The ancestors for the
clusters were born in the late 18th century and early
19th century and were traced back for 3 and 4
generations. Three of the probands could not be linked
to any other proband. The tracing revealed 2201 live
individuals in the four family clusters and of these, 364
(17%) key individuals were identified (Fig. 1). Three

Genetic Screening in Populations. Figure 1 The pedigree shows how two individuals with FH (probands) are
traced to a common ancestor (upwards tracing arrows). The oldest individual alive in each family lineage was
identified as key individual and contacted for genetic testing (downward tracing arrows). Offspring of the key
individuals positive for the mutation were recruited for testing. If positive, their offspring were also called in and so on.
Relatives of key individuals negative for the mutation were not recruited. + means a deceased individual and black
filling an affected individual.
686 Genetic X-Linked Disease
hundred and six key individuals (84%) responded.
Thirty five (11%) of the 306 key individuals who
responded were positive for the common mutation or
nearly one in every 9 key individuals tested. This yield
is a fifty-six-fold enrichment from the 1 in 500 yield of
screening the general population. No homozygotes
were detected. Of the 35 positive key individuals, seven
had not been diagnosed before.
This demonstrates that screening extended families is a
feasible approach for achieving the goal of finding
individuals with FH previously unknown to have this
treatable condition. This approach may well be
practical in other populations where genealogical
information is available.
Clinical Relevance
Familial hypercholesterolemia is a condition with
considerably elevated risk of developing coronary
artery disease, which may eventually lead to a heart
attack. A report from The World Health Organization
(WHO) shows the mean age of onset for coronary heart
disease in untreated individuals to be 45–48 years in
males and 55–58 in females (5) This enormously raised
risk of a heart attack that is potentially preventable calls
for an active search for and identification of affected
individuals at an early age and an aggressive treatment
of all known risk factors for coronary heart disease. The
WHO conference in Paris 1997 (5) urged an early
diagnosis and treatment of individuals with FH. The
main challenge is to prevent premature atherosclerosis
in individuals with FH.
In recent years a new class of drugs for the treatment of
hypercholesterolemia has become available. These are
HMG CoA reductase inhibitors called statins, which
directly affect intracellular production of cholesterol
and hence lead to an increase in the number of LDL
receptor molecules on the cell surface of the liver. This
in turn leads to enhanced uptake of cholesterol rich
particles from the circulation with a corresponding
reduction in the level of blood cholesterol. It has been
demonstrated that cholesterol-lowering drugs are
effective in reducing coronary stenosis assessed by
coronary angiography in patients with FH (6) and there
is evidence for improved survival of patients with FH
in recent years especially after the introduction of statin
therapy (7).
The prognosis for primary prevention of coronary artery
disease in heterozygous FH patients is excellent and for
that reason it is a major important challenge to identify
undiagnosed or inadequately treated individuals.
The above example of genetic screening fulfils all the
criteria for screening in populations set by Wilson and
Jungner in 1968 (1). There are a number of monogenic
diseases that may benefit from the experience obtained
from the effort of identifying new FH patients by
systematic search.
References
1. Wilson J, Jungner YG (1968) Principles and practice of
mass screening for disease. Public Health Papers 34,
Report 65 (iv) WHO, Geneva
2. Goldstein JL, Hobbs HH, Brown MS (1995) Familial
Hypercholesterolemia. In: Scriver CT, Beaudet AL, Sly
WS (eds) et al The Metabolic and Molecular Bases of
Inherited Diseases, McGraw-Hill, New York, pp 1981–2030
3. Eckenhausen MA, Defesche JC, Sijbrands EJ et al (2001)
Review of first 5 years of screening for familial
hypercholesterolaemia in the Netherlands. Lancet
357:165–168
4. Thorsson B, Sigurdsson G, Gudnason V (2003) Systema-
tic Family Screening for Familial Hypercholesterolemia in
Iceland. Arterioscler Thromb Vasc Biol 23:335–333
5. Report of a WHO Consultation (1997) Familial Hyperch-
olesterolaemia (FH). WHO Human Genetics Programme,
Geneva
6. Kane JP, Malloy MJ, Ports TA et al (1990) Regression of
coronary atherosclerosis during treatment of familial
hypercholesterolemia with combined drug regimens.
JAMA 264:3007–3012
7. Scientific Steering Committee on behalf of the Simon
Broome Register Group (1999) Mortality in treated
heterozygous familial hypercholesterolaemia: implications
for clinical management. Atherosclerosis 142:105–112
Genetic X-Linked Disease
Definition
A genetic x-linked disease is determined by mutation of
a gene located on the X chromosome.
▶Microvilli
Genetically Engineered Animals
▶Transgenic and Knock-out Animals
Genome
Definition
The term genome refers to the complete set of genetic
information contained in an organism or a cell, which
includes both the chromosomes within the nucleus and
in mitochondria.
 Genome-Wide Analysis 687

▶Biochemical Engineering of Glycoproteins

▶Chromosome 21 Disorders Genome Scan
▶COPD and Asthma Genetics
▶Functional Genomics, the Systematic Analysis of
Gene Function of all Genes and Gene Products in Definition
Parallel Genome scan refers to a genetic research method in
▶Protein Microarrays as Tool for Protein Profiling and which the entire DNA of an organism is searched
Identification of Novel Protein-Protein Interactions systematically for locations on the chromosomes that
are inherited in the same pattern as a specific trait. This
method is usually applied to collections of families that
show multifactored inheritance of specific traits, such as
type 1 diabetes.
▶Diabetes Mellitus, Genetics
▶Manic Depression
Genome Analysis in Plants
G
▶Plant Genomics
Genome Screen

Definition
Genome screen describes the testing of a population
Genome Engineering group to identify a subset of individuals at high risk for
having or transmitting a specific genetic disorder, by
using several hundred markers selected from the whole
genome to identify chromosomal regions that are co-
▶Cre/loxP Strategies
inherited (linked) with a specific disease.
▶Atopy Genetics
▶COPD and Asthma Genetics
▶Genetic Screening in Populations

Genome Functionalization by Arrayed

cDNA Transduction Genome Walking

▶Automated High Throughput Functional Character-

ization of Human Proteins
Definition
Genome walking is a local physical mapping technique
for obtaining unknown DNA regions on either side of
chromosomal regions of known nucleotide sequences.
▶YAC and PAC Maps

Genome Instability
Genome-Wide Analysis
Definition
Genome instability describes processes in cells that
accumulate mutations with high frequency. These Definition
mutations include point mutations, insertions, deletions A genome-wide analysis is the systematic investigation
and translocations. of all regions of the genome to determine those
▶Chromosomal Instability Syndromes polymorphisms more often associated with a disease.
▶DNA-Repair Mechanisms ▶Common Diseases, Genetics
688 Genomic Analysis of Single Disseminated Cancer Cells
Genomic Analysis of Single
Disseminated Cancer Cells
C HRISTOPH A. K LEIN
Institute of Immunology, Ludwig Maximilian
University, Munich, Germany
christoph.klein@med.uni-muenchen.de
Definition
The term “disseminated cancer cells” refers to cells that
originate from a malignant primary tumour and are
found at ectopic sites as single cells or small cell
clusters. More than 80% of human malignant tumours
stem from epithelial tissues (such as mammary glands,
lung or gastro-intestinal organs). The major cause of
death for patients that suffer from these types of cancer
(carcinomas) is metastasis, i.e. emergence of tumour
colonies that are found in organs distant from the
primary site of tumour growth. Today many cancer
patients are diagnosed before metastatic disease is
detected by clinical imaging techniques (such as X-ray
or CT scans) and will be submitted to surgery of their
primary tumour. A substantial percentage of these
patients, however, will eventually succumb to metas-
tasis months, years of even decades after initial
diagnosis of their primary cancer, indicating that
tumour cells left the primary tumour before the surgeon
removed it, and that some of these cells have the
potential to found a metastasis. Therefore, great effort
was undertaken to detect the precursor cells of later
arising metastasis at the time of surgery in order to
develop means for prevention of later arising metas-
tasis. For cancers of epithelial origin, detection of
disseminated cancer cells is often based on histogenetic
markers that enable discrimination from the surround-
ing cells, i.e. identification of epithelial cells in purely
mesenchymal organs. Clinically accessible mesenchy-
mal organs are blood, bone marrow or lymph nodes.
Thus the applied markers (e.g. epithelial cytokeratins)
do not detect tumour cells directly but epithelial cells
that are usually not found in mesenchymal organs of
donors without epithelial malignancy. Clinical studies
revealed that the finding of cytokeratin positive cells
(with most studies performed on bone marrow or
lymph node) at the time of surgery puts carcinoma
patients at high risk for the development of metastasis
later on. Depending on tumour type and clinical stage,
approximately every third carcinoma patient without
manifest metastases harbours disseminated cancer cells
in bone marrow or macroscopically tumour-free lymph
nodes. However, absolute and relative tumour cell
frequencies are extremely low, being about one or two
cancer cells per two million bone marrow cells. Thus,
disseminated cancer cells belong to the rarest cells in
the human body and their molecular-genomic char-
acterization requires special techniques.
Characteristics
(Whole) genome analysis of single disseminated
cancer cells
The first studies that characterized cytokeratin-positive
cells intended to confirm their malignant nature. It was
shown by interphase fluorescence in-situ hybridisation
(▶FISH) that some cytokeratin-positive cells harbour
chromosomal abnormalities. Then, protein expression
of a variety of cancer-associated molecules was tested
on cytokeratin-positive cells. Although sporadic
insights into the biology of disseminated cancer cells
could be obtained, double staining (or labelling in the
case of FISH) was very cumbersome because of the
extreme rarity of the investigated cells.
A single diploid cell contains about 6 pg genomic
DNA. Multiple molecular-genetic analyses of such
minute amounts therefore require amplification. To this
end several methods have been developed with the
most frequently used being the ▶DOP-PCR (degen-
erate oligonucleotide-primed ▶PCR) and the PEP
(primer extension, preamplification) method or deriva-
tives thereof. These methods use mixtures of degen-
erate or random primers to amplify the whole genome,
which leads to the problem that it is impossible to
control for equal binding of the primers to complex
sequences for unbiased amplification. However un-
biased amplification is mandatory for the application of
whole genome screening techniques such as compara-
tive genomic hybridization that measures numerical
aberrations in tumour cell genomes. To circumvent this
problem an adaptor-linker approach was developed.
Here, the genome is cut into small fragments (about
100–2000 bp) by a frequently cutting restriction
enzyme. Then, adaptors are ligated to both ends of
the fragments and the fragmented genome is
subsequently amplified using a single primer that
binds to the adaptor and thereby amplifies all fragments
alike.
The approach is increasingly applied to disseminated
cancer cells of various types of tumours and the results
have changed the prevailing view on metastatic
progression. Of the many interesting findings perhaps
the most important are that dissemination occurs often
earlier than previously thought. The cancer cells often
display less or different genomic aberration than their
matched primary tumours. Thus metastases and
primary tumours seem to develop independently to a
large degree.

Gene expression analysis of single disseminated
cancer cells
With the completion of the human genome project and
the introduction of technologies such as DNA micro-
arrays and laser microdissection, many fields in
biology and medicine await the application of compre-
hensive gene expression analyses of specific cell types
isolated from defined tissues. For the amplification of
single cell mRNA the first protocols were introduced in
the late eighties and early nineties of the last century. So
far the protocols are based on either of two principal
approaches, linear amplification by T7 RNA polymer-
ase or PCR-based amplification.
As a general rule, PCR-based methods are easier to
handle and less time consuming, while there are
concerns about the quantitative reliability of measure-
ments obtained after exponential amplification. The
linear amplification achieved by T7 RNA polymerase,
also referred to as the Eberwine protocol, has the
advantage that a potentially occurring failure to amplify
a given transcript will not be exponentially transmitted.
Here, mRNA is transcribed by a primer containing the
promotor of the T7 RNA polymerase. After ▶cDNA
synthesis, in-vitro transcription is performed and the
procedure is repeated once or twice. The effect of the
few cycles is thought to change only marginally – if at all
– the original template ratios. On the other hand, several
groups have observed that the relative abundance of
transcripts is also preserved by PCR-based methods –
provided that the correct conditions are applied.
Our preferred method belongs to the approaches using
PCR. The protocol uses a single primer that binds to
two binding sites artificially introduced to all mRNA
sequences. First, a poly-C flanking region is incorpo-
rated during cDNA synthesis and after reverse
transcription a poly G-tail is added. Four aspects seem
to be particularly important. Firstly, single cell mRNA
is bound to a solid phase enabling the change of buffers
and thereby always optimal conditions for each
enzymatic reaction. Secondly, random primers for
cDNA synthesis reduce the length of primary transcript
and allow for subsequent amplification within the
optimal range for PCR. Thirdly, a poly-G tail provides
a much better primer binding site than a poly A or poly
T tail. Fourthly, introducing a poly-C flank on one side
of the template and a poly-G tail on the other makes all
sequences equally G/C-rich at their primer-binding site.
Adequate conditions for a single poly-C PCR primer,
i.e. high annealing temperature and the addition of
denaturing agents such as formamide enable highly
specific and unbiased amplification of such sequences.
With this amplification method in hand, gene expres-
sion profiling of single cells has become possible and
first interesting results have been obtained. For
example, we found that the minor histocompatibility
Genomic Analysis of Single Disseminated Cancer Cells 689
antigen HA-1 is aberrantly expressed on single
disseminated cancer cells. This finding makes it
reasonable to apply allogeneic bone marrow transplan-
tation as immunotherapy in a HA-1 mismatch situation.
We recently adopted the protocol for high-density
oligonucleotide microarrays. Thus, screening of all
expressed human genes may reveal new target
structures on single disseminated cancer cells, the
precursor cells of lethal metastasis.
Clinical Relevance
Clinically manifest metastatic disease can rarely be cured.
One likely reason is that the cancer cells have
genomically and phenotypically progressed so far that
they are highly resistant against current ways to induce
apoptosis by any type of treatment, another that the G
tumour burden is just too large for complete tumour cell
eradication at tolerable drug doses. Therefore, systemic
therapies are added to loco-regional treatment (e.g.
surgery or irradiation therapy) before metastasis becomes
manifest. Such therapies target the relatively few tumour
cells that spread throughout the body, have therefore been
called “adjuvant” and are currently in the centre of clinical
efforts. The underlying rational is to destroy the tumour
seed timely when the tumour load is low and the cells still
vulnerable. However, adjuvant chemotherapies, which
are currently the best characterised and most effective,
have not fulfilled the hopes so far. Although several
therapy regimens improve significantly the overall- and
the disease-free survival of the patients, the absolute
benefit is rather low being in the range of few percent and
improving survival time for the individual patient by few
months. It is increasingly recognized that one reason for
the failure of adjuvant therapies is the almost complete
lack of knowledge about the target cells – the
disseminated cancer cells. Disseminated cancer cells are
genomically and phenotypically often very different from
their matched primary tumours. Therefore, therapies that
are based on mechanisms active in primary tumours do
not necessarily exert an effect on disseminated cancer
cells. Rather, direct analysis of single disseminated cancer
cells promises to uncover novel molecular targets for
effective adjuvant therapies.
References
1. Braun S et al (2000) Cytokeratin-positive cells in the bone
marrow and survival of patients with stage I, II, or III
breast cancer. N Engl J Med 342:525–533
2. Cole BF, Gelber RD, Gelber S et al (2001) Polyche-
motherapy for early breast cancer: an overview of the
randomised clinical trials with quality-adjusted survival
analysis. Lancet 358:277–286
3. Iscove NN et al (2002) Representation is faithfully
preserved in global cDNA amplified exponentially from
sub-picogram quantities of mRNA. Nat Biotechnol
20:940–943
690 Genomic Clone
4. Klein CA et al (1999) Comparative genomic hybridiza-
tion, loss of heterozygosity, and DNA sequence analysis
of single cells. Proc Natl Acad Sci USA 96:4494–4499
5. Klein CA et al (2002) Combined transcriptome and
genome analysis of single micrometastatic cells. Nat
Biotechnol 20:387–392
6. Klein CA (2003) The systemic progression of human
cancer: A focus on the individual disseminated cancer cell
– the unit of selection. Adv Cancer Res 89:35–67
7. Telenius H et al (1992) Degenerate oligonucleotide-
primed PCR: general amplification of target DNA by a
single degenerate primer. Genomics 13:718–725
8. Zhang L et al (1992) Whole genome amplification from a
single cell: implications for genetic analysis. Proc Natl
Acad Sci USA 89:5847–5851
Genomic Clone
Definition
Genomic clone denotes a fragment of cloned DNA
originating from the genome of the organism of interest
rather than from a reverse-transcript of an RNA.
▶YAC and PAC Maps
Genomic Control
Definition
Genomic control describes a method to control for
population stratification in association studies. The
degree of genotype-phenotype association for a large
number of neutral polymorphisms is measured and
used to correct associations with candidate causal
polymorphisms.
▶COPD and Asthma Genetics
Genomic Imprinting
TAKUYA I MAMURA , A NDRAS PALDI
Ecole Pratique des Hautes Etudes, Evry, France
paldi@genethon.fr
Definition
Genomic imprinting, also called parental or gametic
imprinting, is a process of epigenetic marking that
allows the cells to differentiate the alleles of paternal
and maternal origin of a gene without changing their
DNA sequence. The phenomenon of imprinting was
explicitly recognized at the beginning of the 1980s
on the basis of two types of observations. First,
pronuclear transplantation studies on mouse zygotes
demonstrated that monoparental conceptuses were
not viable, suggesting that biparental contribution is
necessary for mammalian development (1, 2). Second,
systematic genetic studies of mice with chromo-
somal translocation showed that some chromosomal
regions must be inherited from both parents for
normal development (3). These pioneer studies led to
the construction of a low-resolution chromosomal
imprinting map of the mouse genome. It was postulated
that the requirement for both parental genomes to be
present in the same zygote was a consequence of
differential epigenetic marks on a fraction of the
paternal and maternal alleles. The parental origin-
specific imprints on the two alleles of the same gene
lead to their differential expression. Typically, one
parental allele is silenced and only the other remains
functional. It is precisely this property that has been
used to identify imprinted genes. A gene is considered
imprinted if it is expressed monoallelically and its
allelic expression depends on its parental origin.
However, the real situation is more complicated. The
analyses of the known imprinted genes have demon-
strated that most of them are expressed biallelically in
some tissues, at least at some developmental stages. As
a consequence, we face a paradoxical situation. It is
quite difficult to prove that a gene is not imprinted,
unless its allelic expression is examined in all tissues at
all stages of the life. The ambiguity of the definition
might be the reason for the difficulty estimating the
number of imprinted genes in the genome. On the basis
of the relatively low frequency of mutations with
parental origin-dependent phenotype, the number of
imprinted genes was initially estimated as not more
than 300. However, a systematic transcriptome analysis
suggested that there might be more than 2,000 genes
with differential parental origin dependent expression
in the mouse.
Characteristics
Characteristics of Imprinted Genes
In order to understand the phenomenon of imprinting,
current research is focused on the following major
questions: 1) How are the parental alleles of a gene
marked without changing the DNA sequence? The
imprint has to be sufficiently stable to be inherited
through mitosis, but reversible during the meiosis. 2)
When do the genes acquire their parental-specific
imprints? 3) What is the biological significance of this
phenomenon?

The first two imprinted genes, Igf2r and Igf2, were
identified in 1991 on the basis of the parental origin-
dependent phenotypes of heterozygous mutants. More
than 70 genes have been identified in the mouse and
human genome so far. (We quote here two compre-
hensive databases of imprinted genes that can be found
on the web: ▶http://www.mgu.har.mrc.ac.uk/research/
imprinting/imprinting2.html and ▶http://cancer.otago.
ac.nz/IGC/Web/home.html). Detailed analyses of sev-
eral of these genes made it possible to determine
general characteristics of imprinted genes in addition to
their monoallelic expression:
1. Imprinted genes are frequently associated with
regulatory regions that carry differential ▶DNA
methylation on the two parental alleles. Several
imprinted genes were identified on the basis of
systematic search for ▶differentially methylated
regions (DMRs) in the genome. In general, DNA
methylation upstream of genes, especially in the
promoter region, is associated with attenuation of
the expression.
2. It is impossible to classify the imprinted genes on
the basis of the products they encode. Peptide
hormones, growth factors, transcription factors,
metabolic enzymes, cell surface receptors and many
other proteins, but also several non-coding RNAs
can be found among the products of imprinted
genes.
3. Another characteristic feature of imprinted genes
discovered so far is their non-random distribution in
the genome. They are frequently clustered in well-
defined genomic regions of up to several hundred
kilobases. These clusters usually contain imprinted
genes with either maternal or paternal monoallelic
expression, but also genes that have been found to be
biallelically expressed in all tissues analyzed so far.
4. Interestingly, many antisense transcripts are also
detected in the imprinted genomic regions.
5. An important feature of the clusters is their
▶asynchronous replication, which occurs in com-
mon as far as we know, suggesting that this
phenomenon is one of the useful criteria that help
determine imprinted genes (4). The paternal and
maternal copies of the whole clusters replicate
differentially during the mitotic cell cycle, including
imprinted genes, intergenic sequences and the genes
that are expressed biallelically. This characteristic is
independent of the expression state of the genes in
the cluster and is detected in all cell types. They all
suggest that cluster level of imprinting regulation
might be assigned in the context of chromatin
structure as described below.
6. Imprinted chromosomal regions display strikingly
different recombination frequencies during male
and female meiosis. However, many non-imprinted
Genomic Imprinting 691
regions also show recombination with different
frequencies during male and female meiosis.
Molecular Mechanisms
As indicated above, DNA methylation is a part of the
mechanisms that differentiate the parental alleles of
imprinted genes. Methylation of cytosines in CG
dinucleotides (CpG methylation) is a well-known
▶epigenetic modification of the DNA that regulates
chromatin structures such as ▶heterochromatin in
concert with covalent ▶histone modifications. The
DMRs are usually relatively short, CG-rich DNA
segments located at a distance from genes, but
sometimes located in the promoter region or in the
coding or intronic sequences. The best characterized
DMRs in the human and mouse genome include those
located in the regions of the Igf2r, H19, Igf2, Snrpn,
G
U2af1-rs1, Gnas and Gtl2 genes. The functional
importance of these elements for the establishment
and maintenance of the methylation imprint has been
demonstrated by extensive targeted mutagenesis stu-
dies. Their deletions frequently perturb the function of
the whole imprinted domain. These elements are
usually called imprinting centers for their central role
in the imprinting of a whole region. They most
probably act as a structural organizer affecting gene
expression over the whole imprinting cluster. This
action is presumably mediated by recruiting various
proteins, for instance the CTCF protein, that play a role
in making up highly-ordered chromatin structure.
Studies of chromatin structure around imprinted genes
revealed differences in nucleosome positioning, his-
tone acetylation or nuclease sensitivity between the
parental alleles of imprinted genes. In general,
methylated sequences are associated with hypoacety-
lated histones, whereas the unmethylated sequences are
associated with hyperacetylated histones. The differ-
ences observed between the two alleles of an imprinted
gene in the same tissue are similar to those typically
observed between active and inactive copies of the
same gene in different tissues. Naturally occurring, or
experimentally induced mutations that alter the enzy-
matic mechanisms responsible for epigenetic modifica-
tions such as DNA methylation and histone
modifications frequently disturb the normal imprinting
process and modify the allelic expression of imprinted
genes. In addition to the epigenetic modifications, the
observation of several ▶antisense RNA transcriptions
in imprinted regions suggests that non-coding RNAs
might be also involved in the maintenance of the
characteristic chromatin structure and monoallelic
expression of imprinted genes. A role of non-coding
RNAs in this process has been suggested by analogy
with the function of the Xist RNA in the inactivation of
one of the X-chromosomes in females, although the
role remains unknown.
692 Genomic Imprinting
Genomic Imprinting. Figure 1 The schematic representation of the cycle of acquisition/erasure of genomic
imprinting in the germ line. Note that, in the germ cell lineage, the epigenetic marks should be erased and then
re-established according to the sex of the individual.
Establishment of the Imprint
Each individual inherits a paternal and a maternal copy
of every gene in the genome. However, both alleles are
transmitted to the offspring either as paternal or as
maternal copies depending on the individual’s sex.
Therefore, the parental imprint of a gene or a gene
cluster has to be erased in the germ line of the
individual and re-established according to its sex in
mature gametes (Fig. 1). In order to follow this process,
changes in CpG methylation pattern of DMRs were
extensively studied at various imprinted loci. In
general, the differences in CpG methylation pattern
are erased from the parental alleles in early ▶primor-
dial germ cell (PGC) differentiation. The methylation
pattern typical for the paternal or maternal alleles is
established in meiotic cells. At the moment of
fertilization, many DMRs are already differentially
methylated and conserve their allele-specific methyla-
tion profiles at all subsequent stages of development
while the bulk of the genome undergoes important
methylation changes. However, some DMRs acquire
their methylation profiles gradually during the somatic
cell division, suggesting that ▶CpG methylation is not
the only molecular mechanism that plays a role in
marking the two parental alleles.
Other characteristics of the imprinted genomic regions
follow different kinetics during development. For
example, asynchronous replication of the two parental
copies is maintained during the proliferation of PGC,
when all methylation differences are already erased,
suggesting that differences between the parental copies
are still there even in the absence of methylation.
Clinical Relevance
The biological significance of the functional none-
quivalence of the parental genomes is not yet known.
Many hypotheses were proposed to explain why
imprinting has evolved in mammals. The most popular
hypothesis is the so-called parental conflict model.
According to this model, imprinting has evolved in
mammals because of the conflicting evolutionary
interests of the paternal and maternal genomes over
the allocation of parental resources. This hypothesis is
based on the assumption that fetal imprinted genes

regulate resource transfer from the mother to the fetus.
Therefore, parents are able to modulate the use of
resources by transmitting epigenetically modified
versions of imprinted genes to their offspring. Since
the fetuses develop within the maternal uterus, the
paternal investment in the offspring is obviously much
lower than the maternal investment. This asymmetry
leads to an asymmetry of the imprints on the resource
usage-regulating genes.
Another possible explanation is that maintaining
the differential chromosomal structure of the im-
printed regions could be important for the coordinated
replication of the genome and the correct segregation of
the chromosomes during mitosis (5). The monoallelic
expression of imprinted genes might be a byproduct of
this process. Indeed, some experimental observations
indicate that the parental copies of imprinted regions
interact with each other during the somatic cell cycle in a
way that is reminiscent of some trans-sensing phenom-
ena observed in Drosophila or plants.
Whatever the biological function of parental imprint-
ing, perturbations of the process lead to severe
hereditary disorders that develop because of mutations
in the active allele of imprinted genes, the normal but
silenced allele being unable to compensate for the
mutated copy (6). Biallelic expression of usually
monoallelically expressed imprinted genes has also
been implicated in various cancers. For example,
Prader-Willi syndrome patients often display hypoto-
nia, hyperphagia, obesity, hypogonadism and develop-
mental delay. Angelman syndrome patients frequently
show ataxia, tremulousness, sleep disorders, seizures
and hyperactivity. Both syndromes may also show
mental retardation and map to the imprinted gene
cluster in human chromosome 15q11-13. Beckwith-
Wiedemann syndrome maps to 11p15 and is character-
ized by general overgrowth with symptoms such as
hemihypertrophy, macroglossia and visceromegaly.
▶G-Proteins and G-Protein Mutations in Human
Diseases
▶Microdeletion Syndromes
▶Prader Willi and Angelman Syndromes
References
1. McGrath J, Solter D (1984) Completion of mouse
embryogenesis requires both the maternal and paternal
genomes. Cell 37:179–183
2. Surani MA, Barton SC, Norris ML (1984) Development
of reconstituted mouse eggs suggests imprinting of the
genome during gametogenesis. Nature 308:548–550
3. Cattanach BM, Kirk M (1985) Differential activity of
maternally and paternally derived chromosome regions in
mice. Nature 315:496–498
4. Kitsberg D, Selig S, Brandeis M et al (1993) Allele-
specific replication timing of imprinted gene regions.
Nature 364:459–463
Genomic Information and Cancer 693
5. Paldi A (2003) Genomic imprinting: Could the chromatin
structure be the driving force? Curr Top Dev Biol 53:
115–137
6. Lalande M (1997) Parental imprinting and human disease.
Annu Rev Genet 30:173–195
Genomic Information and Cancer
T RAVIS D UNCKLEY, K EITH D. C OON ,
D IETRICH A. S TEPHAN
The Translational Genomics Research Institute,
Phoenix, AZ, USA
dstephan@tgen.org G
Synonyms
Genomics
Definition
Genomics represents the systematic study of the entire
genetic complement (DNA and RNA) of an individual
or population of individuals. Since uncontrolled growth
of cancerous cells results from inherited or somatically
acquired mutations scattered throughout the comple-
ment of our chromosomes and often affects mRNA
expression, the holistic tools of genomics are particu-
larly relevant to understanding the molecular mechan-
isms underlying this set of diseases. As such, genomics
has played a major role in advancing tumor subclassi-
fication, diagnosis and individualized treatment of
patients.
Characteristics
Cancer results from the accumulation of genetic
damage superimposed upon inherited predisposition.
This damage manifests itself in the form of DNA
mutations, chromosomal aberrations or epigenetic
alterations in the chromatin structure. Elucidating the
specific genetic events involved in the pathogenesis of
different cancer types will be critical for the develop-
ment of effective diagnostics and treatments. Initially,
genetic studies in cancer focused primarily on heritable
rare and highly penetrant alleles of cancer predisposi-
tion genes. Examples include the ▶tumor suppressors
Rb1 and p53. However, heritable predisposition alleles
account for a small percentage of cancer causing
events. Multiple combinations of weak genetic variants
may have a much larger impact on the development of
cancer in the general population, the majority of whom
do not have inherited alleles of the known highly
penetrant cancer predisposing genes. Thus, cancer can
be viewed as a multigenic disease. The challenge of
694 Genomic Information and Cancer
cancer genomics is to identify the multiple genetic
variants that are involved in the development of cancer
and to determine their effects on the molecular
pathways of the premalignant cell type.
Identifying the genetic determinants of such a complex,
multigenic disease as cancer necessitates the develop-
ment and use of high throughput methods of genomic
analysis. Toward that end, high throughput genomic
DNA scanning technologies (sequencing, LOH, CGH
and FISH) as well as microarray-based technologies
have been critical. Microarray technology involves the
fixation of DNA molecules to a slide or wafer. These
DNA molecules can be placed on the microarray slides
at very high densities, allowing for high throughput
genome-wide analyses. Various microarray technolo-
gies exist, including ▶single nucleotide polymorphism
(SNP) arrays for linkage and LOH studies, ▶compara-
tive genomic hybridization (CGH) arrays, DNA
sequencing arrays and perhaps most widely recog-
nized, gene expression microarrays.
Single Nucleotide Polymorphism Genotyping for
Linkage and LOH Studies
Traditionally, linkage analyses and ▶loss of hetero-
zygosity (LOH) have been done on a genome-wide
scale using ▶microsatellite markers at a density of

10 cM (Mb) intervals. This is a tedious methodology.
The resolution is appropriate for linkage studies but
inadequate for LOH in the majority of cases. New
information from the human genome project (HGP) has
resulted in a high resolution SNP map of the human
genome, as well as new technologies for rapidly
genotyping these SNPs. Single nucleotide polymorph-
isms represent DNA base pair variations within a
population of individuals. They can either have no
effect on gene expression or may have subtle effects
that, when combined, may lead to disease phenotypes.
An SNP occurs, on average, once in every 1300 base
pairs of the human genome and they account for the
majority of genetic variability between individuals
within a population. Although SNPs are biallelic and
thus less informative, their density of
every 30 kb (on
the new Affymetrix 100k SNP array) allows larger
haplotype block content to be inferred. Thus SNPs
are likely to be equally informative over multiple
adjacent SNPs as well as having the ability to identify
smaller hemizygous deletions. SNP array technology
has great potential for discovering multigenic contribu-
tions to cancer development. For example, by compar-
ing the SNP profiles from a group of individuals
that have a certain cancer type to the SNP profiles
of unaffected individuals, one can identify a set of
SNPs that are uniquely associated with that form of
cancer (1). Since the sequence information for the SNPs
is known, one can rapidly move towards identifying the
relevant genes. Importantly, this technology is generally
applicable to the study of any disease with a genetic
basis.
DNA Sequencing
DNA sequencing represents the ultimate in high-
throughput cancer analysis. Once we fulfill the
mandate of the HGP for rapid, whole-genome sequen-
cing at minimal cost, we will revolutionize the ability to
understand and diagnose cancer. Early attempts include
massive parallel DNA arrays for hybridization-based
sequencing. These arrays, designed by Perlegen
Sciences, Inc. (CA, USA) consist of overlapping
oligonucleotide probes that span the entire genome.
This makes possible direct and rapid sequencing of the
entire genome. This has clear advantages, particularly
when an unidentified disease gene has been mapped
previously using standard positional cloning strategies.
Comparative Genomic Hybridization
Comparative genomic hybridization (CGH) allows one
to visualize gross chromosomal losses or gains by a
modification of traditional karyotyping. The entire
genomic complement of a normal individual is labeled
and compared to a genomic sampling from a tumor that
has been labeled with a different fluorophore. While
revolutionary, the typical resolution of traditional CGH
is
10 Mb. Array CGH provides the advantage of
higher resolution compared to traditional CGH methods
(up to 0.5 Mb) and is generally useful for identifying
large deletions, insertions, amplification events or
overall changes in ploidy. Importantly, array CGH can
be used as an adjunct to expression microarrays (see
below) since the molecular events detected by CGH will
have effects on gene expression levels.
Expression Microarrays
Gene expression microarrays are used to rapidly assess
the gene expression profile of different cell types. For
genetic mutations to affect cell proliferation, and hence
the development of cancer, they must alter the function
of at least some of the signaling pathways inside the
affected cell. These effects can be seen as altered mRNA
expression profiles (even members of phosphorylation
cascades can be dysregulated) in malignant cell types
and can be identified using expression microarrays.
There are two main variations of expression arrays
currently in use, cDNA and oligonucleotide micro-
arrays. Oligonucleotide arrays provide the benefits of
greater specificity since the probes used are of shorter
sequence (
25–70 nucleotides) than those used for
cDNA arrays (200–2000 nucleotides). cDNA arrays
have greater sensitivity but cannot, for example,
discriminate between splice variants. For each type of
array, sequences of DNA that are homologous to
different genes of interest are attached to a glass slide
at different locations. Each probe (or probe set for

oligonucleotide arrays) on a slide corresponds to a single
gene and each slide can hold thousands of probes. One
then hybridizes labeled cRNA from the cell type of
interest to the microarray, rapidly generating an mRNA
expression profile for thousands of genes.
Integration of LCM into ▶Expression Profiling
One important limitation of expression profiling has
been that the tissues used often contain multiple cell
types in addition to the diseased or cancerous, cells.
Additionally, cancers are almost always mosaic with
respect to acquired somatic changes. Thus, they are
heterogeneous with respect to the clinical and histo-
pathological trait under study. This adds unwanted
expression signatures to the global expression profile
obtained and may generate misleading results. ▶Laser
capture microdissection (LCM) is increasingly being
used to overcome this limitation. LCM uses an infrared
laser to select only cell types of interest from a thin
section of tissue sample. In the study of cancer, this
allows the analysis of a nearly homogeneous population
of malignant cells, generating a cleaner expression profile
that is more indicative of the cancerous state. Because the
volume of cells harvested using this technique is low,
RNA amplification techniques must be used to generate
enough RNA for expression profiling.
Clinical Relevance
Clearly, identification of the underlying DNA or RNA
defects leading to cancer development and progression
will lead to a greater understanding of tumorigenesis
and will translate directly into drug design. SNP
analyses provide the potential for early and reasonably
noninvasive cancer diagnosis by detecting heritable
cancer specific mutations in peripheral tissues, such as
blood. As a result, treatments may be commenced
earlier than was previously possible (2). Additionally,
SNP analysis could become a routine screening
procedure to identify individuals with SNP haplotypes
that place them at risk for developing specific forms of
cancer. This information could be used to direct at risk
individuals to appropriate prevention strategies. As the
technology matures, diagnosis and screening using this
methodology may become economically feasible for
general practice. In addition to providing a diagnosis
method, SNP analyses will be useful for identifying
causative mutations and affected molecular pathways
in various cancers. Following validation of these
pathways, new targets for therapy will emerge.
Gene expression profiling has numerous important
clinical applications. First, expression profiling already
has been used to predict the prognosis of disease course
in ▶breast cancer (3). In breast cancer susceptibility
screening, genetic testing to identify individuals
carrying known predisposition alleles has been used
to indicate when more extreme prevention strategies,
Genomic Information and Cancer 695
such as surgical resection of at risk tissue, are needed.
However, this approach suffers from the limitation that
not all individuals carrying susceptibility genes will
develop cancer. Further subclassifying a person’s
cancer risk based on additional genetic determinants
will restrict such surgical prevention strategies to only a
subset of individuals with the highest likelihood of
developing particularly aggressive forms of disease.
The above example with breast cancer illustrates the
expectation that, as more tumors are profiled and
subcategorized, expression profiling could become a
general tool for predicting the course of cancer
progression and for guiding prevention strategies in
unaffected individuals. In the future, subcategorizing
tumors based on their expression profiles may aid in
patient-specific therapies that are designed to be most G
effective in the clinic on a real-time basis for the
treatment of particular forms of cancer.
Microarrays are also important for identifying dysre-
gulated genes and signaling pathways that are involved
in tumor development and progression (4). Identifica-
tion of these genes and pathways will have important
implications for the development of novel anticancer
therapies since they provide novel targets for treatment.
As expression profiling in the field of cancer biology
moves forward, a critical goal will be to translate the
vast amounts of biological data into meaningful clinical
advances. This will require large collaborative efforts
pooling the combined knowledge and expertise of
different institutions to accomplish successfully all of
the required goals from tumor sample acquisition, to
genomic analysis, to target identification and valida-
tion, to drug design and discovery.
Summary
Analysis of SNPs and gene expression profiling are
valuable methods for identifying the genetic determi-
nants of cancer. However, to realize the value of these
techniques fully, it will be critical to translate the
findings into practical applications that can benefit
individuals who are suffering from cancer and those
who are at risk of developing particular forms of
cancer. Knowledge of an individual’s innate suscept-
ibility to various cancer types can be used to guide the
course of prevention strategies that focus, for example,
on lifestyle changes such as diet and exercise. In
addition, SNP and expression profiles can be used both
to diagnose cancers accurately and to subcategorize
tumor types based on the severity of the malignant
phenotype. This information could then be used to
target the most aggressive therapies to patients with the
most severe or invasive forms of disease. Ultimately,
the information gleaned from these powerful genomics
techniques will be used to identify novel targets for
therapeutic intervention with the eventual endpoint of
preventing tumor growth and metastasis.
696 Genomic Instability
References
1. Hoque MO, Lee CC, Cairns P et al (2003) Genome-wide
genetic characterization of bladder cancer: a comparison
of high-density single-nucleotide polymorphism arrays
and PCR-based microsatellite analysis. Cancer Res
63:2216–2222
2. Sidransky D (2002) Emerging molecular markers of
cancer. Nat Rev Cancer 2:210–219
3. van ’t Veer LJ, Dai H, van de Vijver MJ et al (2002)
Expression profiling predicts outcome in breast cancer.
Breast Cancer Res 5:57–58
4. Mac Donald TJ, Brown KM, La Fleur B et al (2001)
Expression profiling of medulloblastoma: PDGFRA and
the RAS/MAPK pathway as therapeutic targets for
metastatic disease. Nat Genet 29:143–152
Genomic Instability
Definition
Genomic instability describes a phenotypic feature of
the cell, in which the genetic material mutates at a faster
rate than normal as a consequence of a deficiency in
proteins that function in ▶DNA repair, cell cycle
checkpoints, chromosome structure maintenance, and
chromosome segregation, etc.
▶Bloom Syndrome
▶DNA Helicases
▶DNA Repair Mechanisms
Genomics
Definition
The mapping, sequencing, and analysis of an organ-
ism’s genome.
▶Protein Databases
Genomics
▶Genomic Information and Cancer
▶Functional Genomics, the Systematic Analysis of the
Function of All Genes and Gene Products in Parallel
Genotoxin
Definition
A genotoxin is a chemical, or another agent, which
damages cellular DNA resulting in mutations and/or
cancer.
▶Chromosomal Instability Syndromes
Genotype
Definition
Genotype refers to the genetic constitution of an
organism or cell; be it the alleles at a given locus, or
those of several loci. For an autosome, the genotype for
a specific chromosomal location would be 2 alleles.
▶COPD and Asthma Genetics
▶Diabetes Mellitus, Genetics
▶Familial Dilated Cardiomyopathy
▶Large-Scale ENU Mutagenesis in Mice
▶Schizophrenia Genetics
Genotype-Driven Approach
Definition
Genotype-driven approach describes a plan of action
based on the hypothesis that a specific gene is
responsible for a specific function. To this end, the
specific gene is mutated and the resulting phenotype
(appearance) of the organism provides information
about the function of the gene.
▶Mouse Genomics
Genotype-Phenotype Correlations
Definition
Genotype-phenotype correlations describe the relation-
ship between genotype (polymorphisms, sequence,
variants, and mutations) and phenotype (their clinical
expression).
▶Heritable Skin Disorders
 GFAP 697

cell lines have been formed by mutations. The period of

Genotyping development at which this mutation is formed
determines the cell type of mosaicism: somatic or
germline cells. The germline of one individual consists
Definition of two or more populations of cells, due to mutation(s)
Genotyping is the determination of the specific allelic in one or more clonally expanded cell(s) in the
composition of a genome, a gene or a set of genes. population of germline cells. Germline mosaicism
▶Cell Polarity may vary between only a few cells or about 50% cells,
▶SNP Detection and Mass Spectrometry and is the reason why genetically unaffected parents
may have children with more than one X-linked or
dominant genetic disorder.
▶Heritable Skin Disorders
Geranylgeranyl Pyrophosphate ▶Neurofibromatosis Type 1 (NF1), Genetics

Definition G
Geranylgeranyl is a 20 carbon unit made up of four
Germline Mutation
isoprene (dimethyl allyl) units, and in the form of the
pyrophosphate, is a precursor molecule in cholesterol
biosynthesis. Definition
▶Protein Prenylation Germline mutation denotes a mutation that affects the
▶Tangier Disease complete organism including the germ cells, and thus is
passed on to the progeny of the affected individual.
▶Microarrays in Pancreatic Cancer

Germ Cells
Germline Transmission
Definition
Germ cells are pre-meiotic or post-meiotic sperm cells Definition
and egg cells. Germline transmission refers to a process where the ES
▶Mutagenesis Approaches in the Zebrafish derived cells of a chimera contribute to the reproductive
cells of a mammal (germ cells) and are genetically
passed to its offspring.
▶Large-Scale Homologous Recombination Ap-
Germinal Vesicle proaches in Mice

Definition
Germinal vesicle is the meiotic prophase nucleus of an GFACT Expression screening
amphibian oocyte.
▶Xenopus as a Model Organism for Functional
Genomics ▶Genome Functionalization by Arrayed cDNA Trans-
duction (GFACT) Expression screening

Germline (Gonadal) Mosaicism

Definition
The term mosaicism in general refers to an organism
that is composed of two or more cell lines, which
originate from only one zygote and differ in their
genotype or chromosomal constitution. The different
GFAP
Definition
The glial fibrillary acidic protein (GFAP) is an
intermediate filament protein that is characteristic
for astrocytes, but it is also expressed in certain
698 GFFKR Domain
populations of ▶nestin-expressing neural stem cells. It
has thus become an ambiguous marker.
▶Neural Stem Cells
GFFKR Domain
Definition
GFFKR domain refers to a conserved amino acid motif
of the cytoplasmic tail of α integrin chains. It serves to
stabilize the α and β integrin subunits in close spatial
contact. This keeps the extracellular, ligand binding
part of the integrin in a folded inactive form. Lysins,
amino acids proximal to the GFFKR motif, are
essential for interaction of the integrin α chain with
RAPL. Binding of RAPL contributes to spatial
separation of the cytoplasmic integrin chains, allowing
unfolding of the extracellular part into a ligand binding
integrin.
▶Focal Complexes/Focal Contacts
GFP
▶Green Fluorescent Protein
GGA Proteins
Definition
GGA proteins (Golgi-associated, γ-adaptin homolo-
gous, ARF-interacting proteins) constitute a conserved
multidomain protein family involved in traffic between
the Golgi complex and endosomes. They are recruited
to membranes by GTP-bound ▶ARF. They can interact
with trafficking motifs present on certain cargo, e.g. the
mannose–6–phosphate receptor, and also interact with
▶clathrin, making them functionally analogous to
▶adaptor complexes.
▶Vesicular Traffic
Giga-Seal
Definition
Giga-seal denotes the tight connection between the tip
of a patch clamp pipette and the cell membrane during
the patch clamp analysis. The giga-seal is characterized
by a large electrical resistance that reaches values in the
giga-ohm range.
▶Patch Clamping
Glanzmann’s Thrombasthenia
Definition
Glanzmann’s Thrombasthenia is an autosomal reces-
sive disorder, characterized by the absence of dysfunc-
tion of the ▶GPIIb/IIIa complex, resulting in defective
platelet aggregation.
▶Hereditary Hemostatic Defects and Recombinant
Proteins for Treatment
GLI
Definition
GLI comprise a family of zinc finger transcription
factors involved in both developmental regulation and
human diseases. Zinc-finger transcription factors of the
GLI family play critical roles in the mediation and
interpretation of Hedgehog signals. The Drosophila
homologue is Cubitis interruptis (Ci).
▶Hedgehog Signalling
▶Wnt/Beta-Catenin Signaling
Glial Cells
Definition
Glial Cells are the non-neuronal cells of the nervous
system. Glial cells do not carry nerve impulses (action
potentials) but do have essential supportive functions,
including physical support, provision with nutrients
and trophic factors (astrocytes), insulation of axons
(oligodendrocytes and Schwann cells), and phagocytic
functions (astrocytes and microglia). During develop-
ment, radial glial cells provide a scaffold for neuronal
migration, and they function as neuronal progenitors.
▶Glial Cells and Myelination

Glial Cells and Myelination
H AUKE W ERNER , K LAUS -A RMIN N AVE
Department of Neurogenetics, Max Planck Institute of
Experimental Medicine, Goettingen, Germany
hauke@em.mpg.de
nave@em.mpg.de
Definition
The large majority of non-neuronal cells in the nervous
system are ▶glial cells. In general, glial cells serve
supportive functions for neurons, are not electrically
excitable but may respond to neurotransmission. This
overview focuses on two types of glial cells that
myelinate axonal processes, ▶oligodendrocytes in the
central nervous system (CNS), and ▶Schwann cells in
the peripheral nervous system (PNS). ▶Myelin enables
▶axons to conduct action potentials much more
rapidly, by insulating the axonal membrane, decreasing
its capacitance and restricting ion fluxes to the ▶nodes
of Ranvier. Myelin is largely made during early
postnatal life. Thus, developmental disorders of
myelination or loss of myelin are severe neurological
diseases, either inherited (▶leukodystrophies, neuro-
pathies) or acquired. One clinically important myelin
disorder is multiple sclerosis, an inflammatory ▶de-
myelination of the CNS.
Characteristics
The best understood function of oligodendrocytes and
Schwann cells is to enwrap axonal processes with an
insulating myelin sheath. Myelination occurs largely
during early postnatal life and can be divided into
several steps: (i) establishing contact between glial cell
and axon (oligodendrocytes engulf multiple axons), (ii)
spiral enwrapping of axonal segments with up to 50
layers of membrane, (iii) compaction of myelin by tight
association of the intracellular and extracellular mem-
brane surfaces, (iv) formation of functional nodes of
Ranvier and paranodal structures. Although some of the
molecules involved in myelination have been identi-
fied, the cellular mechanisms are not well understood.
In humans, myelination begins around birth, with a
peak in the first five years of life and is mostly
completed by 11 years of age. Some active myelination
has been observed until the 5th decade, possibly related
to neuronal plasticity. During the peak of myelination,
i.e. within a few days, oligodendrocytes produce a large
amount of membrane material that exceeds their own
weight several fold.
The highly periodic structure of myelin is best
visualized in cross section under the electron micro-
scope (Fig. 1). Proteins at the condensed cytoplasmic
Glial Cells and Myelination 699
membrane surface form the electron-dense major dense
line (MDL), those at the condensed extracellular
surface form the intraperiod line (IPL). The membrane
itself is electron-lucent. The ultrastructure of CNS and
PNS myelin is remarkably similar. Once myelinated,
many axons become dependent on oligodendrocyte
support. Thus, when oligodendrocytes or myelin
degenerate in the course of a demyelinating disease,
some axons will degenerate as well. The mechanism of
this axon-glia interaction is not known.
Myelinated axons of the human PNS exhibit nerve
conduction velocities (NCV) between 5 and 100 m/s,
increasing with the axon diameter. In non-myelinated
axons, NCV measures 0.5–5 m/s. To exhibit the same
NCV, a non-myelinated axon would have to be of much
larger diameter. Thus, myelin has evolved in verte- G
brates as means to achieve high NCV and reduced
space requirements.
Molecular Interactions
Only axons with a diameter >1 μm are myelinated,
smaller axons may be engulfed (i.e. in the PNS by
non-myelin forming Schwann cells) but are not
enwrapped. Axon diameter and myelin sheath thick-
ness show a constant relationship (g-ratio). In the PNS,
the axonal growth-factor neuregulin-I (Nrg1) plays a
critical role in signaling size information to receptor
tyrosine kinases erbB2/erbB3 on the Schwann cell and
in regulating myelin membrane growth.
Molecularly, compact myelin is comprised of lipids and
myelin proteins that differ in composition between
CNS and PNS. Targeted gene inactivation in (‘knock-
out’) mice has been used to explore the function of
myelin-specific proteins systematically. Many of them
are abundant integral or membrane-associated proteins
with functions in membrane adhesion. Their CNS- or
PNS-specific expression correlates well with the
clinical picture of leukodystrophies and neuropathies
respectively. Regions of non-compact myelin exist at
the innermost myelin membrane, adjacent to the nodes
of Ranvier and as Schmidt-Lanterman incisures in the
internodal region. They provide radial connections
between the periaxonal space and the glial cell soma,
form a tight seal at the node of Ranvier (paranodal
loops) and help organize the distribution of ion
channels by interacting with proteins of the axonal
membrane. Some structural proteins of non-compact
myelin are quite abundant. For updated information on
these genes and diseases refer to the McKusick entries
of the Online Mendelian Inheritance in Man (▶OMIM)
database (▶http://www3.ncbi.nlm.nih.gov/Omim/).
Myelin Lipids
Myelin has a lipid content of 70–80% (compared to less
than 50% in other ▶biological membranes) and lipids
contribute to its insulating function. How myelin lipids
700 Glial Cells and Myelination

Glial Cells and Myelination. Figure 1 Cross section of a myelinated axon at the electron microscopic level
(upper left), the ultrastructure of compact myelin (lower left), and schematic depictions of structural proteins in
myelin. The condensed cytoplasmic membrane surfaces form the electron-dense major dense line, extracellular
membrane adhesion forms the intraperiod line. The membrane itself is electron-lucent. Membrane proteins
associated with human myelin diseases are depicted in red. PLP/DM20, proteolipid protein; MBP, myelin basic
protein; P0, protein zero; PMP22, peripheral myelin protein of 22kD; Cx32, connexin of 32 kD; MAG,
myelin-associated glycoprotein; CNP, cyclic nucleotide phosphodiesterase.

are enriched in the cell membrane is not understood.
Myelin is particularly rich in cholesterol. Galactosyl-
cerebroside (GalC) and its sulfated form (sulfatide) are
nearly myelin-specific. Absence of GalC and sulfatide
in mutant mice lacking UDP-galactose:ceramide ga-
lactosyl-transferase leads to progressive demyelination
and early death. Thus, both lipids are essential for
normal myelination, although glucosylcerebroside (an
alternative product) may compensate for some func-
tions of GalC. Patients with the Smith-Lemli-Opitz
syndrome (McKusick #270400), a genetic disorder of
cholesterol biogenesis, also have myelin abnormalities.
The critical requirement for cholesterol in myelin
assembly has been shown with conditional mouse
mutants deficient in squalene synthase, a critical and
specific enzyme of cholesterol synthesis.
Proteolipid Protein (PLP, McKusick *300401)
In the CNS, the most abundant protein of compact
myelin is a hydrophobic integral membrane protein
(proteolipid) with four transmembrane domains and its
smaller splice isoform (DM20). PLP/DM20 may form
homo-oligomers and associate with alpha(v)-integrin,
but the function of these interactions is speculative. The
tight association of PLP/DM20 with cholesterol may be
required for membrane ▶raft formation and normal
membrane trafficking in oligodendrocytes. The ultra-
structure of CNS myelin lacking PLP/DM20 in
▶knockout mice suggests that the extracellular portion
of PLP acts as a strut, organizing the extracellular
apposition of myelin layers at the IPL, but myelination
is possible in the absence of PLP. For comparison, point
mutations in this gene (or PLP gene duplications)
cause severe ▶dysmyelination in Pelizaeus-Merzbacher
disease (PMD) and in rodent PMD models. This is due
to ER retainment of the mutant protein, unfolded
protein response and oligodendroglial apoptosis
Myelin Protein Zero (MPZ, P0, McKusick *159440)
Myelin protein zero is the most abundant protein of
compact PNS myelin, expressed exclusively by
Schwann cells. With a single transmembrane domain
and an extracellular Ig-like domain, P0 is a member of
the Ig-superfamily of cell adhesion proteins. The
crystal structure of the Ig-like domain, when combined
with the analysis of P0-deficient mice, indicates that
homo-tetrameric P0 engages in homophilic interactions
with the opposing membrane layer, mediating mem-
brane adhesion and formation of the IPL. Additionally,
positive charges in the cytoplasmic domain contribute
to the establishment of the MDL by direct interaction
with negatively charged head groups of membrane
phospholipids. Mutations of the human P0 gene cause a
peripheral neuropathy (CMT1B).
Myelin Basic Protein (MBP, McKusick *159430)
Myelin basic protein refers to a group of related cellular
proteins associated with both CNS and PNS myelin.
The MBP gene encodes at least 5 splice isoforms,
ranging from 14 to 21 kD in size. Positively charged
amino acids interact with negatively charged head
groups of membrane phospholipids causing MBP to
mediate and stabilize myelin compaction at the MDL.
The MBP gene is partially deleted in the natural mouse
mutant shiverer, which presents with a severe demye-
linated phenotype. The overall lack of myelin assembly
is not yet fully explained. Shiverer mice provided the
first opportunity to analyze the consequences of a
missing myelin protein before transgenic knockout
techniques became available. An involvement of the

MBP gene in a human leukodystrophy has not yet
been demonstrated.
Peripheral Myelin Protein 22K (PMP22,
McKusick *601097)
Peripheral myelin protein 22K is a glycosylated
integral membrane protein of PNS myelin. By topology
and hydrophobicity PMP22 is related to the proteoli-
pids of CNS myelin. PMP22 interacts with myelin
protein P0 in the myelin membrane and may stabilize
the myelin architecture. Experiments carried out
in vitro suggest that PMP22 also regulates Schwann
cell proliferation and apoptosis, but its in vivo function
is poorly understood. The PMP22 gene has captured
interest because a gene duplication in humans underlies
the most frequent peripheral neuropathy (CMT1A).
Myelin-associated Glycoprotein (MAG,
McKusick *159460)
Myelin-associated glycoprotein is a member of the Ig-
superfamily of cell adhesion proteins, with a single
trans-membrane domain and 5 extracellular Ig-like
domains. Its localization at the innermost (adaxonal)
membrane of both CNS and PNS myelin suggested that
MAG is engaged in adhesion and signaling events
between glial cell and axon. The diameter of PNS
axons is reduced in mice lacking MAG, suggesting that
MAG-mediated myelin-to-axon signaling regulates the
phosphorylation status of the axonal cytoskeleton.
Axonal binding partners of MAG include the NoGo
receptor (NoGoR / reticulon 4 receptor, McKusick
*605566), and sialic acid residues of sialo-glycopro-
teins or sialo-glycolipids (gangliosides).
Connexin-32 (Cx32, McKusick *304040)
Connexin-32 is a member of the connexin family of
gap junction proteins, permeable to molecules <1kD.
The protein is expressed in many cell types, including
oligodendrocytes and Schwann cells, where it is
localized to Schmidt-Lanterman incisures and para-
nodal loops. The exact function of Cx32 in PNS myelin
is not known, but its involvement in the non-classical
gap junctions that form a connection between different
myelin lamellae of the same Schwann cell is most
likely. Radial transport may be required for second
messenger molecules that are generated at the inner-
most myelin membrane. Mutations in the human Cx32
gene cause a peripheral neuropathy (CMT1X).
Claudin11/Oligodendrocyte-specific Protein (OSP,
McKusick *601326)
Claudin11/oligodendrocyte-specific protein is a mem-
ber of the claudin family of ▶tight junction-specific
integral membrane proteins with four transmembrane
domains. Knockout mice revealed that OSP is an
essential constituent of the radial component in CNS
Global Genome Repair 701
myelin that stabilizes myelin through intramembranous
tight junctions. The finding that OSP interacts with
tetraspanin-3/OAP-1 suggests that other tight junction
proteins have yet to be identified in myelin.
Nodal and Paranodal Specializations
For ▶saltatory nerve conduction, axonal voltage gated
sodium (Na+) channels must be clustered at the node of
Ranvier, separated from fast potassium (K+) channels that
assemble beneath the myelin sheath at the juxtaparanode.
K+ channels are associated with Caspr2, a member of the
neurexin family of ▶adhesion molecules, probably via a
PDZ domain protein adapter. Caspr2 in turn is associated
with TAG-1, a GPI-anchored cell adhesion molecule of
the Ig-like superfamily on the glial adaxonal membrane.
Knockout experiments in mice demonstrated that axonal G
Caspr2 is required to maintain K+ channel clusters at the
juxtaparanode. More devastatingly, in the absence of
TAG-1, axonal Caspr2 and K+ channels are unclustered at
the juxtaparanode. Na+ channel distribution is unaffected
by deletion of juxtaparanodal proteins.
The assembly sites of axonal Na+ and K+ channels are
divided by the paranode, a region devoid of ion channels.
The paranodal axon is tightly attached to the glial
paranodal loops by a septate-like junctional structure that
seals against ion flux and separates Na+ from K+
channels. The paranodal axon is molecularly defined by
a complex of the Ig-like GPI-anchored cell adhesion
molecule F3/contactin associated with the neurexin
Caspr1. This complex interacts with neurofascin155 on
the paranodal loop. Disruption of individual components
in knockout mice impedes the septate-like junction.
References
1. Arroyo EJ, Scherer SS (2000) On the molecular
architecture of myelinated fibers. Histochem Cell Biol
113:1–18
2. Werner H, Jung M, Klugmann M et al (1998) Mouse
models of myelin diseases. Brain Pathol 8:771–793
3. Salzer JL (2003) Polarized domains of myelinated axons.
Neuron 40:297–318
4. Schwab ME (2004) Nogo and axon regeneration. Curr
Opin Neurobiol 14:118–214
5. Colognato H, ffrench-Constant C (2004) Mechanisms of
glial development. Curr Opin Neurobiol 14:37–44
Global Genome Repair
Definition
Global genome repair designates the branch of
nucleotide excision repair that occurs in all regions of
702 Glomerular Filtrate
DNA, with the exception of actively transcribed genes,
and removes damage that could block DNA replication.
▶Nucleotide Excision Repair
Glomerular Filtrate
Definition
Urine formation in the kidney begins when the fluid
portion of the blood leaves the glomerulus and enters the
glomerular capsule as glomerular filtrate. Glomerular
filtrate consists of water and small size components of
blood, separated from blood cells. The glomerular
filtrate flows into the tubules, where further water is
extracted from the filtrate, and minerals and other body
chemicals are absorbed from or secreted into the filtrate.
▶Diabetes Insipidus, a Water Homeostasis Disease
Glomerulonephritis
Definition
Glomerulonephritis (GN) refers to inflammation of the
capillary loops of the glomeruli.
▶Morbus Wegener
▶SLE Pathogenesis Genetic Dissection
Glomerulus
Definition
Glomerulus is the network of blood capillaries in the
cup-like end (Bowman’s capsule) of the nephron. It is
where waste products are filtered from the blood into
the kidney tubule.
▶Kidney
Glucocorticoid/Mineralocorticoid
Receptors
Definition
Glucocorticoid receptor (GR) and mineralocorticoid
receptors (MR) are members of a nuclear receptor
superfamily that mediate an organism’s response to
glucocorticoids or mineralocorticoids, respectively, by
changing the transcription rates of glucocorticoid- or
mineralocorticoid-responsive genes.
▶Steroid Hormone Receptor Defects, Molecular
Basis
Glucocorticoid/Mineralocorticoid
Resistance
Definition
Glucocorticoid/mineralocorticoid resistance are patho-
logic conditions that demonstrate several manifesta-
tions caused by partial insensitivity of tissues to
glucocorticoid or mineralocorticoid hormones. These
are frequently due to inactivating mutations in the
glucocorticoid or mineralocorticoid receptors.
▶Steroid Hormone Receptor Defects, Molecular
Basis
Glucocorticoids
Definition
Glucocorticoids are steroid hormones that are synthe-
sised from cholesterol by cytochrome P450 dependent
steroidhydroxylase, mainly in the adrenal cortex.
▶Mendelian Forms of Human Hypertension and
Mechanisms of Disease
Glutamate
Definition
L-Glutamate is an excitatory amino acid neurotrans-
mitter. It influences almost all neurons in the brain.
Glutamatergic neurotransmission has been associated
functionally with a number of physiological and
pathophysiological processes related to neuronal plas-
ticity and memory.
▶Addiction, Molecular Biology

Glutathione Peroxidase
Definition
GPx defines a family of homologous proteins that is
characterized by a catalytic triad composed of a
(seleno) cysteine, a glutamine and a tryptophan. These
enzymes reduce H2O2 and other
▶Free Radicals
Glycan
Definition
Glycan is a general term for a polymer of mono-
saccharide units joined by glycosidic bonds. It may or
may not have other components.
▶Biochemical Engineering of Glycoproteins
▶Glycosylation of Proteins
Glycated Protein
Definition
Glycated protein designates a protein containing
carbohydrate that was added by a nonenzymatic,
chemical modification, usually through a Schiff-base
reaction with the amino group of the side chain of
lysine, and subsequent Amadori rearrangement, to give
a stable conjugate.
▶Glycosylation of Proteins
Glycine
Definition
Glycine is an amino acid that is derived from dietary
sources, but is also generated endogenously from
glyoxylate. Glycine serves as an important inhibitory
neurotransmitter, predominantly in the spinal cord,
brain stem and retina.
▶Peroxisomal Disorders
Glycohemoglobin 703
Glycoconjugate
Definition
Glycoconjugate refers to a compound that is composed
of an oligosaccharide which is linked to a protein or
lipid.
▶Biochemical Engineering of Glycoproteins
Glycoform G
Definition
Glycoform describes various forms of a particular
species of glycoproteins that differ in the structures
and/or types of glycans.
▶Glycosylation of Proteins
Glycogen Synthase Kinase–3
Definition
Glycogen synthase kinse–3 (GSK3) is a constitutively
active kinase that undergoes inhibition by hormones
and growth factors. One of its functions is to inhibit
Wnt/β-catenin signaling by phosphorylation of β-
catenin thereby targeting it for degradation. Also called
zeste white 3/shaggy in Drosophila.
▶Wnt/Beta-Catenin Signaling Pathway
Glycohemoglobin
Definition
Glycohemoglobin stand for glycosylated hemoglobin.
The ratio of glycohemoglobin and total hemoglobin is
indicative of a person’s average blood glucose level
over the last months.
▶Affinity Chromatography and In Vitro Binding
(Beads)
704 Glycolysis
Glycolysis
Definition
Glycolysis is the metabolic pathway that occurs in the
cytoplasm of cells, and by which glucose is broken
down to pyruvic acid.
▶Limb Girdle Muscular Dystrophies
Glycoprotein
Definition
Glycoprotein defines a protein with one or more
carbohydrate moieties that are covalently bound to it.
▶Affinity Chromatography and In Vitro Binding
(Beads)
▶Biochemical Engineering of Glycoproteins
▶Glycosylation of Proteins
▶Protein Databases
Glycoproteomics
Definition
Glycoproteomics refers to the science of defining the
structures of glycoproteins, and the sites of attachment
and structure of glycans to proteins.
▶Glycosylation of Proteins
Glycosaminoglycan
Definition
Glycosaminoglycan refers to polysaccharide side-
chains of proteoglycans or free complex polysacchar-
ides that are composed of linear disaccharide repeating
units, each composed of a hexosamine and a hexose or
a hexuronic acid (heparin, heparan sulfate, chondroitin
sulfate, dermatan sulfate, and hyaluronan).
▶Glycosylation of Proteins
Glycosidic Linkage
Definition
Glycosidic linkage describes the linkage of a mono-
saccharide to another residue via the anomeric hydro-
xyl group. The linkage generally results from the
reaction of a hemiacetal with an alcohol (e.g. a
hydroxyl group on another monosaccharide or amino
acid) to form an acetal.
▶Glycosylation of Proteins
Glycosylase
Definition
Glycosylase is an enzyme that catalyzes the cleavage of
an N-C1' glycosylic bond, which links a DNA base to
the deoxyribosephosphate backbone of DNA.
▶Base Excision Repair
Glycosylation of Proteins
R ICHARD D. C UMMINGS
University of Oklahoma Health Services Center,
Department of Biochemistry and Molecular Biology,
Oklahoma City, Oklahoma, USA
richard-cummings@ouhsc.edu
Definition
▶Glycoproteins are proteins that contain one or more
covalently attached carbohydrates, including ▶mono-
saccharides and ▶oligosaccharides. The attached car-
bohydrates are also termed ▶glycans. In typical
glycoproteins the glycans can contribute up to
20%
of the total weight. ▶Proteoglycans are a special class
of glycoproteins that contain at least one large-
sized (typically >5 kD), acidic polysaccharide (▶glyco-
saminoglycan) attached to protein. ▶Mucins are another
special class of glycoproteins with a repeating peptide
motif that usually contains multiple Ser and Thr residues
to which relatively small-sized (usually <3 kD) glycans
are attached. The mucin glycans can contribute more than
25% of the total weight. The ▶storage polysaccharides
starch and glycogen, which contain glucose polysacchar-
ide linked to a core protein, also represent a special class
of glycoprotein. In these cases the glycan portion

contributes more than 90% of the total weight. Another
type of glycoprotein is the glycosylphosphatidylinositol-
anchored or ▶GPI-anchored glycoprotein. These contain
a C-terminal amino acid that is linked to ethanolamine,
which is linked to the glycans of the GPI anchor. GPI-
anchored glycoproteins, which may also contain cova-
lently attached N- and/or O-glycans at other residues
within the polypeptide, are usually anchored to plasma
membranes of cells by insertion of the acyl chains of the
GPI moiety into the membrane outer leaflet. Thus,
glycoproteins are found in many different sizes, ranging
from several thousand Daltons to millions of Daltons.
The presence of carbohydrate on protein is a type of
▶post-translational modification, which along with
phosphorylation constitutes one of the most common
types of such modifications. The majority of the nearly
30,000 proteins expressed in human cells are glyco-
proteins (1). The carbohydrates of glycoproteins are
added enzymatically to specific sites on a protein. This
contrasts with what is seen for ▶glycated proteins in
which carbohydrate addition occurs through the
chemical or non-enzymatic addition of a free mono-
saccharide, usually glucose or galactose, to amino
groups in proteins through formation of a Schiff base
and rearrangement to a stable oxoamine adduct known
as an Amadori product. This process is termed
glycation and is often seen in patients with diabetes
and children with galactosemia. Animals, plants, fungi,
Protoctista, archaea and bacteria synthesize glycopro-
teins and many animal viruses contain glycoproteins. In
animals, most of the proteins that are on cell surfaces
and those that are secreted by cells are glycosylated.
Glycoproteins in membranes occur as integral or
intrinsic membrane glycoproteins and may contain
one or more transmembrane domains. GPI-anchored
glycoproteins are also considered to be integral
membrane proteins. Glycoproteins can also occur as
extrinsic membrane glycoproteins that associate with
the membrane through other mechanisms. Many
proteins within the cytosol of eukaryotes are also
glycoproteins. In animal cells two of the major classes
of intracellular glycoproteins are the storage polysac-
charides and those that contain O-linked GlcNAc or
O-GlcNAc (termed O-GlcNAcylated) modifications.
The attached glycans in different glycoprotein species
often exhibit tremendous diversity in size and structure.
Within a single glycoprotein species these structural
differences are often denoted by the term microheter-
ogeneity and the varied forms of a single glycoprotein
species are termed ▶glycoforms. Different glycopro-
teins from the same cell may be glycosylated very
differently, depending on the primary, secondary,
tertiary and/or quaternary structure, association with
other proteins and subcellular localization. However,
glycosylation differences are greater among glycopro-
teins from different cell types within an organism; the
Glycosylation of Proteins 705
differences are often even greater in glycoproteins
between different organisms. Identifying the structures
of the glycans made by different organisms is known as
the field of glycomics and identifying sites of glycan
attachment in glycoproteins is known as the field of
▶glycoproteomics.
Characteristics
Glycans on glycoproteins can vary in the types of
sugars that are attached, the ▶anomeric configuration
and structure of the attached sugars, the numbers of
attachments and the sites of attachments (2). The
linkage of one monosaccharide to another is typically
via a ▶glycosidic linkage, characterized by the acetal
structure. The part of the glycan linked to protein is
termed the reducing end and the opposite, unattached G
end(s) of the glycan is termed the non-reducing end or
terminal region. Although there are many types of
sugar-protein linkages, which are also glycosides, most
sugar-protein linkages are of two types, N-glycosides,
in which the amide of Asn (and in some organisms Arg)
forms the linkage group (-C-N-C-) and O-glycosides,
in which the hydroxy group of hydroxyamino acids,
such as Ser, Thr, Tyr, hydroxylysine (Hyl) and
hydroxyproline (Hyp), forms the linkage group (-C-
O-C-). C-glycosides are an exception to this general-
ization, here a -C-N-C- bond links the sugar to an
amino acid, as seen in Man-C-Trp. Acetal or glycosidic
linkages between sugars are stable to alkali. By
contrast, the linkage of sugar to protein via Asn, Ser
or Thr residues is labile to relatively mild alkali. The
sugar-protein linkages via Tyr, Hyl and Hyp are
resistant to alkali. The glycosidic linkages within a
glycan and all glycosidic linkages of sugars to proteins
can be hydrolyzed by treatment with strong acid. An
exception to this generalization is the C-glycoside
linkage, which cannot be hydrolyzed by treatment with
either alkali or acid.
Table 1 lists some of the common sugar-protein
linkages found in glycoproteins from animals, plants,
fungi, archaea and bacteria. Many dozens of linkages
are now known, but mammalian cells appear to
generate about a dozen or so. In most cases the
monosaccharide residues shown in Table 1 are
extended by the addition of other sugar residues. For
examples see the composite animal cell glycoproteins
shown in Fig. 1. The typical mammalian glycoprotein
may contain N- and/or O-glycans of the types shown.
One exception to the generalization that glycans are
extended by addition of other sugars is found in
eukaryotic cytosolic proteins that contain O-linked
GlcNAc (GlcNAcβ-O-Ser/Thr), where this residue is
not further modified. It is also one of the few examples
where the sugar addition is reversible, i.e. the GlcNAc
residue is removed and added back multiple times on a
mature protein. This probably serves an important
706 Glycosylation of Proteins

Glycosylation of Proteins. Table 1 Examples of Sugar-Protein Linkages in Glycoproteins from Different

Organisms

Kingdom Sugar-Protein Lionkage

Animalia GlcNAcβ-N-Asn*
GalNAcα-O-Ser/Thr
GlcNAcβ-O-Ser/Thr
Manα-O-Ser/Thr
Xylβ-O-Ser
Fucα-O-Ser/Thr
Glcβ-O-Ser/Thr
Glcα-O-Tyr (in glycogen)
Galβ-Hyl
Man-C-Trp
Plantae GlcNAcβ-N-Asn
Galα-O-Hyp
Galα-O-Ser
Arafβ-O-Hyp
Glcβ-Arg (in starch)
Fungi GlcNAcβ-N-Asn
Manα-O-Ser/Thr
Protoctista (algae, sea-weeds and protozoa) GlcNAcβ-N-Asn
Arafβ-O-Hyp
GlcNAcα-O-PO-3-Ser
GlcNAcα-O-Ser/Thr
GlcNAcα-Hyp
Archaea Glcβ-N-Asn
GalNAcβ-N-Asn
Rha-N-Asn
Gal-O-Thr
Bacteria (Eubacteria) Galβ-O-Tyr
Galβ-O-Ser/Thr
GalNAcβ-O-Ser/Thr
Glcα-O-Ser

*
Abbreviations: GlcNAc, N-acetylglucosamine; Asn, Asparagine; GalNAc, N-acetylgalactosamine; Ser, Serine; Thr, Threonine;
Man, Mannose; Xyl, Xylose; Fuc, Fucose; Glc, Glucose; Tyr, Tyrosine; Gal, Galactose; Hyl, Hydroxylysine; Trp, Tryptophan; Hyp,
Hydroxyproline; Ara, Arabinose; Arg, Arginine; Rha, Rhamnose; Pro, Proline; Cys, Cysteine; Gly, Glycine; Xaa, any amino acid,
except as indicated

regulatory function for cytosolic glycoproteins, akin glycosylation is found in glucosylation of N-glycans
to the action of protein phosphorylation, which is as part of the ▶quality control system for glycoprotein
also reversible. (The other example of reversible folding, as discussed below.)
 Glycosylation of Proteins 707

Glycosylation of Proteins. Figure 1 Examples of different types of protein glycosylation. Shown are examples
of composite membrane glycoproteins in animals that may contain one or more O- or N-glycans and a GPI-anchor
or a transmembrane domain. Glycoproteins in the cytosol may also contain O-GlcNAc residues. The key
for the symbols and abbreviations of monosaccharides is indicated and used in other figures.

N-glycans in higher animals typically contain 7–20
monosaccharide residues, whereas O-glycans typically
have 2–10 residues. However, in yeast, many N-
glycans contain mannan, a polysaccharide of mannose,
which can contain hundreds of mannose residues. In
proteoglycans the attached glycosaminoglycans can be
hundreds of residues in length. Some examples of
common types of N- and O-glycans in mammals are
shown in Fig. 2. Mammalian glycoproteins are largely
composed of the ten sugars or building blocks shown,
which are the ▶hexoses galactose (Gal), glucose (Glc)
and mannose (Man), the ▶deoxyhexose fucose (Fuc),
the ▶hexosamines N-acetylglucosamine (GlcNAc) and
N-acetylgalactosamine (GalNAc), the ▶uronic acids
glucuronic acid (GlcA) and iduronic acid (IdoA), the
pentose xylose (Xyl) and the 9-carbon carboxylated
amino sugar ▶sialic acid (Sia) and its multiple
derivatives. In humans Sia occurs primarily as N-
acetylneuraminic acid (NeuAc). Other organisms use
many of these same monosaccharide residues, but also
use novel ones not found in animals. For example, both
Gal and GlcNAc are commonly found in glycoproteins
from all the known kingdoms. Rhamnose (Rha) and
arabinose (Ara) are found in plant, but not animal,
glycoproteins, whereas sialic acids are found in animal,
but not plant, glycoproteins. Some bacteria synthesize
sialic acid, however it has not yet been identified on
bacterial glycoproteins. Some glycoproteins from
nematodes contain tyvelose (3,6-dideoxy-D-arabino-
hexose - Tyv), which is not found in vertebrate
glycoproteins. Some of these monosaccharide residues
may themselves be further modified before or after
incorporation into the glycan moiety of the glycopro-
tein to provide even more diversity of structure, as seen
for NeuAc, which may be O- and/or N-acetylated at
various positions, GlcA and IdoA, which can be N-
sulfated and O-sulfated at various positions, and
GlcNAc and Gal, which may be O-sulfated. Thus, the
number of possible glycan structures is astronomically
large and the upper limit of the number of structures is
not known for any organism. It is important to note that
polypeptides are typified by a linear structure in which
two amino acids (L-amino acids in animals) are linked
by a peptide bond. By contrast, glycans in glycopro-
teins are usually branched structures, where the
monosaccharides are linked to each in multiple ways,
such as different anomeric configuration (α versus β) to
various positions on a residue (position C-2, C-3, C-6,
etc.). In addition, the sugars in glycoproteins can be
in either pyranose (6-membered ring) or furanose
708 Glycosylation of Proteins

Glycosylation of Proteins. Figure 2 Examples of different types of N- and O-glycans. Animal cell N-glycans shown
on the left side have a common pentasaccharide core (shown in the boxed structure), which is composed of a
trimannosyl sequence linked to a chitobiosyl disaccharide, which is in turn linked to an Asn residue through N-
glycosidic linkage. The N-glycans are generally classified as high mannose-, hybrid- or complex-type as shown. The
complex-type N-glycans may have multiple branches or antennae, described as mono-, bi-, tri-, or tetra-antennary,
etc. Animal cell mucin-type O-glycans shown on the right side have a common structure of GalNAc linked to either
Ser or Thr. This GalNAc residue may be modified in various ways to generate a variety of core structures (shown in
the boxed structures).

(5-membered ring) structures and the residues may be
either or D- or L-enantiomers (mirror images). Con-
sidering all these possibilities, it is easy to see that two
identical amino acids linked together in a protein give a
single dipeptide structure, whereas two identical
hexoses may be linked together to give 64 possible
isomeric disaccharide structures. If two different
hexoses are linked together it is possible to obtain
128 different isomers (3).
The N-glycans, also called Asn- or ▶N-linked
oligosaccharides, in animals, plants, fungi and protista
contain the common trimannosyl core structure that is
linked via a chitobiosyl core (-GlcNAc-GlcNAc-) to
Asn, as highlighted in Fig. 2. There are various types of
N-glycans in animal cells, distinguished by the outer or
terminal sugar structure, as seen for ▶high mannose-
type, hybrid-type and ▶complex-type sequences. The
O-glycans in mucins of animal cells, which are also
called Ser/Thr- or O-linked oligosaccharides, are
characterized by the linkage to Ser/Thr residues via
GalNAc, as shown in Fig. 2. The GalNAc residue
may be modified in different ways by linkage to other
sugars to give various core structures. Altogether there
are at least 8 different core structures in mucin-type
O-glycans of animals. Some of the more common ones
are highlighted in Fig. 2 as cores 1–4.
The attachment of N-glycans to Asn residues of
secreted and membrane-bound glycoproteins occurs
within a ▶consensus sequence or sequon –Asn-X-Ser/
Thr- (or Cys) (Table 2), although not all Asn residues
within the sequon of such glycoproteins are always
used. Asn residues outside this sequon are not N-
glycosylated. In addition, cytoplasmic proteins with the
N-glycosylation sequon are not N-glycosylated, be-
cause the pathway of N-glycosylation occurs within the
lumen of the ▶endoplasmic reticulum (ER), as
discussed below. Although the N-glycosylation sequon
is the most well known glycosylation sequon, a few
other sugar-amino acid linkages also occur in definable
sequences of proteins, as seen for addition of O-Glc, O-
Fuc, C-Man and O-Gal (collagen) (Table 2). For most
other attachments of sugars to proteins however, there
are no clearly predictable consensus sequences,
although the probability of attachment of some sugars
to protein, such as GalNAc or GlcNAc to Ser/Thr
residues in mucins and in cytosolic glycoproteins,
appears to be enhanced by clusters of Ser and/or Thr
residues and nearby amino acids (e.g. Pro) (Table 2).
Some mathematical algorithms have been deve-
loped based on this information to predict sites of
addition of GalNAc to Ser/Thr residues in animal
mucins.
 Glycosylation of Proteins 709

Glycosylation of Proteins. Table 2 Some Protein Consensus Sequences for Sugar Addition
*
Sugar-Protein Linkage Consensus Sequence
GlcNAcβ-N-Asn -Asn-Xaa-Ser/Thr- (where Xaa ≠ Pro)#
-Asn-Xaa-Cys- (where Xaa ≠ Pro) rare (animals, plants, fungi, Protoctista)
Fucα-O-Ser/Thr -Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys- (animals)
Glcβ-O-Ser/Thr -Cys-Xaa-Ser-Xaa-Pro-Cys- (animals)
Galβ-Hyl -Gly-Xaa-Hyl-Gly- (animal collagen)
Man-C-Trp -Trp-Xaa-Xaa-Trp- (animals)
Xylβ-O-Ser -Ser-Gly- (or Ala) (indefinite) (animals)
GalNAcα-O-Ser/Thr clustered Ser/Thr near Pro (indefinite) (animals)

*Amino acids to which sugars are linked are in bold G

#
Abbreviations: See Table 1

Glycoprotein Biosynthesis UDPGlcNAc to generate GlcNAc-P-P-dol (4). This

N-Glycan Biosynthesis
In animal cells glycoprotein biosynthesis occurs in
several cellular compartments. The primary sites for
N-glycan biosynthesis are the ER and ▶Golgi
apparatus. N-glycans are generated by a unique
pathway, involving the addition of a preformed glycan
to Asn residues in the consensus sequence –Asn-X-Ser/
Thr- in nascent or forming polypeptides during their
translation (▶co-translational modification) through
the translocon Sec61p, the pore-forming protein in
the ER associated with ribosomes on the cytoplasmic
face of the ER. The preformed glycan added to newly
synthesized glycoproteins occurs as a lipid-linked
donor ▶dolichol-pyrophospho-oligosaccharide (dol-
P-P-oligosaccharide), which in vertebrates contains
14 monosaccharide residues in the formula Glc3Man9-
GlcNAc2-dolichol (Fig. 3). This large oligosaccharide
can be eventually converted, as described below, to the
high-mannose-, hybrid- and complex-type N-glycans
discussed above. The enzyme that transfers this
preformed glycan is called the ▶oligosaccharyltrans-
ferase (OST) and occurs in all eukaryotes as a complex,
hetero-oligomeric enzyme associated with the ER
membrane. Dolichol (dol) is a polyisoprenoid alcohol
(prenol) whose general formula can be seen from
the structure of dolichol-phosphate (P-dol) (Fig. 3).
Dolichol contains 75–95 carbons and is one of the
largest, and most unusual lipids found in animals. It is
synthesized from the same initial precursors and using
the same early enzymatic steps that are used to generate
sterols, such as cholesterol.
The synthesis of the dol-P-P-oligosaccharide also
occurs in the ER and is initiated by the addition of N-
acetylglucosamine from the sugar nucleotide donor
step in the biosynthesis of N-glycans is blocked by the
naturally occurring inhibitor ▶tunicamycin, a transi-
tion state analog of UDP-GlcNAc, which was origin-
ally identified in the fungus-like soil bacterium
Streptomyces lysosuperificus. (The name tunicamycin
derives from its discovery as an antiviral agent that
blocked viral coat (tunica) formation.) Treatment of
animal cells with tunicamycin blocks N-glycosylation
of proteins by blocking formation of the precursor dol-
P-P-oligosaccharide) and results in cell death, due to
the inability to synthesize and correctly fold glycopro-
teins, as discussed below. Interestingly, the synthesis of
GlcNAc-P-P-dol and several other steps beyond this
occurs on the cytoplasmic side of the ER. Following
this first step to synthesize GlcNAc-P-P-dol, additional
GlcNAc and mannose (Man) residues are added
stepwise from their respective sugar nucleotide donors
(Fig. 3). After the formation of Man5GlcNAc2-P-P-dol,
which faces the cytoplasm, the Man5GlcNAc2-P-P-dol
is “flipped” across the membrane of the ER by an
unknown mechanism so that it now faces the lumen or
inner region of the organelle. This Man5GlcNAc2-P-P-
dol is then further elongated in the lumen of the ER by
donation of Man residues from the dolichol inter-
mediate dol-P-Man (Fig. 3). In vertebrate cells,
following completion of the mannose addition to give
Man9GlcNAc2-P-P-dol, 3 glucose residues are added by
the intermediate donor dol-P-Glc, to generate the final
product Glc3Man9GlcNAc2-dol. Each step from the
formation of GlcNAc-P-P-dol to the formation of
Glc3Man9GlcNAc2-P-P-dol is catalyzed by a distinct
enzyme. Mutations in any steps in the pathway usually
result in an inability to add other sugars, thus truncated
dol-P-P-oligosaccharides are generated, which are often
710 Glycosylation of Proteins
Glycosylation of Proteins. Figure 3 Biosynthesis of
dolichol-P-P-oligosaccharide in higher animals. The
pathway shown occurs in the cytosolic and lumenal
regions of the endoplasmic reticulum (ER). A separate
enzyme catalyzes each step in the pathway shown. The
mechanism by which the intermediate dolichol-P-P-
oligosaccharide is reoriented in the ER membrane by
“flip-flop” is not yet understood.
not efficiently utilized by the OST. The blockage in the
addition of Dol-P-man, because of an inability to
synthesize it, was first identified in a cultured mamma-
lian cell line and resulted in the accumulation of
Man5GlcNAc2-P-P-dol, leading to the addition of
Glc3Man5GlcNAc2 instead of Glc3Man9GlcNAc2 to
proteins. Such mutant cell lines, which are viable
in vitro, helped to elucidate one of the key intermediate
steps in this complex pathway of N-glycan biosynthesis.
The addition of Glc3Man9GlcNAc2 to proteins occurs
on the nascent polypeptides and multiple Glc3Man9-
GlcNAc2 residues can be added to Asn residues within
the N-glycosylation sequon as they emerge translation-
ally into the ER through the translocon Sec61p (Fig. 4).
But protein folding can also begin with polypeptide
intermediates and such folding may interfere with
glycan addition to Asn residues. Thus, while Asn
residues in some N-glycosylation sequons are quanti-
tatively and efficiently N-glycosylated, Asn residues
within other sequons may be only partly or inefficiently
N-glycosylated. This partial glycosylation at some
Asn residues may not be accidental however, and may
be under metabolic control and help to regulate
glycoprotein function. Following the addition of Glc3-
Man9GlcNAc2 to protein, the glucose residues are
removed sequentially from nascent polypeptides and
completely translated glycoproteins by two different α-
glucosidases (I and II) in the ER to generate Man9-
GlcNAc2-Asn (Fig. 4). Glucosidases are examples
of enzymes termed glycosidases, which are able
to cleave glycosidic linkages. The actions of the
α-glucosidases are the first steps in glycoprotein
biosynthesis termed processing, where specific sugars
are removed from a newly synthesized glycoprotein in
an orderly fashion. These α-glucosidases are inhibited
by some sugar analogs, such as australine, which
inhibits α-glucosidase I and castanospermine and
1-deoxynorijirimycin, which inhibit both α-glucosi-
dases I and II.
Following removal of the three Glc residues, a single
glucose residue can be added back to Man9GlcNAc2-
Asn to generate Glc1Man9GlcNAc2-Asn (Fig. 4). This
re-glucosylation is catalyzed by the enzyme UDPGlc:
glycoprotein glucosyltransferase (UGGT), a protein
found in the ER (5). UGGT adds Glc from the sugar
nucleotide donor UDPGlc, and generates a UDP
byproduct. The action of UGGT is part of a quality
control process in which glycoprotein folding in the ER
is regulated. Improperly folded proteins either fail to
leave the ER and are degraded there or leave to be
degraded by the ▶proteasome machinery located in the
cytoplasm. Mature proteins have a specific shape that
results from folding the polypeptide backbone and
chemical cross-linking between Cys residues to form
disulfide bonds. While protein folding is spontaneous,
it is relatively slow and inefficient. Protein folding
during biosynthesis is rapid and this is usually achieved
through the assistance of ▶molecular chaperones.
Chaperones are proteins that assist other proteins in
acquiring their mature and active forms. This is often
associated with proper protein folding and prevention of
 Glycosylation of Proteins 711

Glycosylation of Proteins. Figure 4 Biosynthesis of N-glycans in glycoproteins in higher animals. The pathway
shown occurs in the lumenal regions of the secretory organelles the ER and the Golgi apparatus. The latter is
generally partitioned into separate regions known as cis, medial, and trans. Lysosomal acid hydrolases may acquire
Man-6-P residues on their N-glycans, which are recognized by the Man-6-P receptors, which can deliver the
lysosomal enzymes to late endosomes. Following the completion of N-glycan structures, glycoproteins may stay in
the secretory organelles and endosomes as either soluble or membrane-bound glycoproteins, be secreted into the
extracellular space or be incorporated into plasma membrane.

the undesirable protein oligomerization that can occur in
the concentrated protein environment of the ER.
Glycoproteins that are partly or improperly folded are
transported back into the cytoplasm by a process
termed retrograde transport, which appears to occur
through translocon Sec61p. UGGT binds its acceptor
substrate Man9GlcNAc2-Asn with high affinity only on
partly unfolded proteins, and thus has the potential for
dual recognition of protein and glycan determinants.
The Glc1Man9GlcNAc2-Asn generated by this enzyme
is recognized by two different ER lumenal molecular
chaperones, ▶calnexin and ▶calreticulin. Calnexin and
calreticulin are examples of animal lectins, which are
generally defined as non-immune proteins that recog-
nize and bind to specific glycan structures without
catalyzing a chemical modification. Calnexin/calreti-
culin bind in a reversible manner and their binding is
probably associated with binding of other chaperones
that recognize specific peptide features of the protein.
Once released from calnexin/calreticulin, the Glc1-
Man9GlcNAc2-Asn is subject to action of α-glucosi-
dase II, resulting in reformation of Man9GlcNAc2-Asn.
If a glycoprotein is still not properly folded, the UGGT
adds back Glc to regenerate Glc1Man9GlcNAc2-Asn.
UGGT participates in quality control of N-glycan
biosynthesis and protein folding through this cycle of
712 Glycosylation of Proteins
glucosylation/deglucosylation, which is repeated until
a glycoprotein assumes a conformation that blocks its
interaction with UGGT. Blocking the action of α-
glucosidase with castanospermine or other glucosidase
inhibitors can result in glycoprotein accumulation in
the ER, due to inefficient protein folding. This single
Glc residue serves as a type of ER-retention signal,
preventing glycoprotein exit from the ER.
Following the formation of Man9GlcNAc2-Asn on a
folded glycoprotein, the ER α-mannosidase removes
one of the mannose residues of Man9GlcNAc2-Asn to
form Man8GlcNAc2-Asn (Fig. 4). Interestingly, some
cells contain an endomannosidase that can remove Glc-
Man disaccharide from Glc1Man9GlcNAc2-Asn in an
α-glucosidase II-independent pathway to form an
alternative Man8GlcNAc2-Asn. The endomannosidase
can also act on Glc1-3Man9GlcNAc2-Asn derivatives.
Following formation of Man8GlcNAc2-Asn on ER
glycoproteins, they usually exit to the Golgi appar-
atus by vesicular transport involving COP-coated
vesicles. The Golgi apparatus is recognized as a multi-
compartment organelle, with cis, medial and trans
compartments and a terminal compartment called the
transGolgi network (TGN). The Golgi apparatus is
usually positioned in the cell so that the cis-Golgi
is nearest or proximal to the ER and the trans-Golgi is
away from or distal to the ER.
Upon reaching the cis-Golgi the Man8GlcNAc2-Asn
in glycoproteins is subjected to further processing by
α-mannosidase I (Fig. 4). This enzyme removes 3
additional α-linked mannose residues from Man8-
GlcNAc2-Asn to generate Man5GlcNAc2-Asn. Not all
the high-mannose N-glycans, however, are susceptible
to α-mannosidase I. The Man8GlcNAc2-Asn in some
glycoproteins are not very accessible to α-mannosidase
I, leading to formation of mature glycoproteins having
Man8GlcNAc2-Asn or partly processed forms such as
Man7-, Man6-, or Man5GlcNAc2-Asn. The action of α-
mannosidase-I can be inhibited by several drugs,
including the plant alkaloid kifunensine and the
mannose derivative 1-deoxymannojirimycin.
Acid hydrolases that are destined to enter lysosomes
are subject to the action of an alternative pathway in
which they acquire phosphorylated mannose residues
(Man-6-P) that are recognized by the mannose-6-
phosphate (Man-6-P) receptors (Fig. 4). These recep-
tors help to deliver lysosomal enzymes to endosomes,
from which the ▶lysosomal acid hydrolases can enter
mature lysosomes and the Man-6-P receptors recycle to
the Golgi for additional rounds of delivery (6). The
high mannose-type N-glycans of lysosomal acid
hydrolases in the early Golgi apparatus are recognized
by the UDPGlcNAc:lysosomal enzyme phosphotrans-
ferase. Many of the lysosomal acid hydrolases have
unique 3-dimensional structures that generate a surface
patch, which is a basic region that includes
Lys residues. The phosphotransferase recognizes the
signal patch and adds GlcNAc-1-P from the donor
UDPGlcNAc to nearby Man residues on the high
mannose-type N-glycans to generate the phosphodie-
ster GlcNAc-1-P-6-Man5-8GlcNAc2-Asn. Following
formation of GlcNAc-1-P-6-Man5-8GlcNAc2-Asn on
lysosomal enzymes, they are subjected to the action
of the α-N-acetylglucosamine-1-P phosphodiesterase
(“uncovering” enzyme or UCE), which removes the α-
linked GlcNAc residue, resulting in formation of the
phosphomonoester structure P-6-Man5-8GlcNAc2-Asn.
Thus, lysosomal enzymes acquire one or more Man-6-
phosphate- (Man-6-P) phosphomonoester residues on
high mannose-type N-glycans. The presence of Man-6-
P blocks action of α-mannosidases on the specifically
phosphorylated Man residues. Thus, as described
below, phosphorylated glycans cannot be converted
to complex-type N-glycans, although they can be
converted to Man-6-P-containing hybrid-type N-gly-
cans.
It is important to note that sugar nucleotides, which are
important donors for glycosyltransferases in the lumen
of the ER and Golgi apparatus, are synthesized in the
cytosol. The sugar nucleotides are imported into the
lumen of the ER and Golgi apparatus by specific
transporters. Most of these transporters function as
antiporters; they move a sugar nucleotide into the
lumen and the cognate nucleoside monophosphate into
the cytosol for reutilization. Defects in transporter
function are associated with human genetic diseases, as
discussed below.
The recognition of lysosomal hydrolases by the
phosphotransferase is dependent on the folded structure
of lysosomal enzymes and unfolded proteins are not
recognized by the phosphotransferase (6). As discussed
below, mutations in the phosphotransferase can result
in lack of addition of GlcNAc-1-P to the more than 50
lysosomal enzymes. Alternatively, the phosphotrans-
ferase can stochastically fail to add the GlcNAc-1-P to
a fraction of the lysosomal acid hydrolases. Such non-
phosphorylated glycans of lysosomal acid hydrolases
are subject to further processing and modification in the
Golgi apparatus and can acquire complex-type N-
glycan structures that contain sialic acid and other
terminal sugars.
The Man5GlcNAc2-Asn structures are potential accep-
tors for the enzyme N-acetylglucosaminyltransferase
I (GNT-I), which adds GlcNAc from the donor
UDPGlcNAc to form GlcNAcMan5GlcNAc2-Asn
(Fig. 4). This is a hybrid-type N-glycan, that contains
non-reducing terminal Man residues and other terminal
sugars, such as GlcNAc. In vertebrate cells the product
GlcNAcMan5GlcNAc2-Asn is usually acted upon by
α-mannosidase II, which specifically recognizes
GlcNAcMan5GlcNAc2-Asn and removes two mannose
residues to form GlcNAcMan3GlcNAc2-Asn. The

concerted action of α-mannosidases and GNT-I, which
appears to occur largely in the cis-Golgi region,
generates the trimannosyl structure common to all
complex-type N-glycans (7). This GlcNAcMan3-
GlcNAc2-Asn is usually acted upon by N-acetylgluco-
saminyltransferase II, which adds GlcNAc from
UDPGlcNAc to form the product GlcNAc2Man3-
GlcNAc2-Asn. This is an example of a biantennary
complex-type N-glycan, which is characterized by the
lack of non-reducing terminal Man residues and the
presence of other terminal sugars. The biantennary
nature refers to the presence of two branches of the
complex-type N-glycan. But within the cis-Golgi
additional N-acetylglucosaminyltransferases (GNT-III
through VI) can add additional GlcNAc residues to the
Man residues to form bisected N-glycans, or multi-
antennary N-glycans, such as tri-, tetra-, penta- and
hexa-antennary structures. Within the more distal
trans-Golgi apparatus, the GlcNAc2Man3GlcNAc2-
Asn is subjected to modification by galactosyltrans-
ferases, causing addition of Gal residues to GlcNAc
residues from the donor UDPGal to form Galβ4Glc-
NAc-R sequences. This disaccharide terminal sequence
is termed N-acetyllactosamine (LN). In vertebrates the
pituitary glycoprotein hormones, such as lactating
hormone and follicle stimulating hormone, acquire
biantennary N-glycans but are subject to addition of
GalNAc residues from the donor UDPGalNAc to form
the sequence GalNAcβ4GlcNAc-R; this terminal dis-
accharide is termed lactosamine-di-N-acetyl (Lacdi-
NAc or LDN). This formation of LDN sequences on
pituitary glycoprotein hormones requires the action
of a specific N-acetylgalactosaminyltransferase that
appears to recognize primary sequences within the
hormones, and does not generally act on other
glycoproteins within the pituitary (8). But less specific
N-acetylgalactosaminyltransferases are also expressed
in other cells to generate the LDN structure on non-
pituitary glycoprotein hormones. The LDN termini
of pituitary glycoprotein hormones are sulfated at the
C-3 position of the terminal GalNAc residues by a
PAPS:GalNAc 3-O-sulfotransferase to form S-3-GalNAc
moieties. The resultant formation of (S-3-GalNAc)2
GlcNAc2Man3GlcNAc2-Asn on pituitary glycoprotein
hormones promotes their recognition and clearance
from the blood circulation by a liver receptor for
S-3-GalNAc.
In most glycoproteins following the addition of Gal
residues to GlcNAc residues, the complex-type N-
glycans can acquire other modifications in the trans-
Golgi and TGN. These include addition of sialic acid
from CMPNeuAc, fucose from GDPFuc, and other
residues. Each addition or modification is catalyzed by
a separate enzyme. A tremendous variety of modifica-
tions are possible depending on a wide variety of
factors, such as expression of the modifying enzymes
Glycosylation of Proteins 713
and the availability of the glycoprotein glycans to the
enzymes. Following these modifications of the N-
glycans in the Golgi apparatus, secretory glycoproteins
are released into the extracellular space by secretory
vesicles, while membrane-bound glycoproteins may be
targeted to the plasma membrane or lysosomes.
GPI-Anchor Biosynthesis
Many glycoproteins in eukaryotes contain a novel C-
terminal modification of a glycosylphosphatidylinosi-
tol lipid anchor, the GPI anchor (9). The addition of the
GPI-anchor to proteins occurs in the ER and involves
recognition of a C-terminal domain of newly synthe-
sized protein (Fig. 5). A preformed lipid-linked
precursor is generated from phosphatidylinositol by
initial reactions in the cytosolic face of the ER. An G
intermediate containing glucosamine is then reoriented
(“flip-flop”) by translocation across the ER membrane
to allow further elongation of the precursor by addition
of Man residues from dol-P-Man. Ethanolamine
phosphate is added to Man residues by donation from
phosphatidylethanolamine to generate a GPI precursor.
In all organisms this GPI precursor is characterized by
having glucosamine linked to inositol and the triman-
nosyl sequence linked to ethanolamine in a “core”
structure. But this core structure may be differentially
modified in a tremendous variety of ways, depending
on the organism, by addition of other sugars, e.g. Man
and Gal residues, additional ethanolamine residues,
addition of phosphate, fatty acylation of the sugars and
further acylation of inositol.
The GPI precursor is the substrate for a transamidation
reaction by the GPI transamidase complex. This
enzyme complex recognizes the C-terminal lipophilic
portion of some proteins with a GPI anchor sequence,
causing the cleavage of the polypeptide bond and
transfer of the GPI precursor to the new C-terminal
amino acid. The GPI transamidase forms a carbonyl
intermediate with the substrate protein. The signal
sequence for GPI anchor addition is a C-terminal
region with an amino acid to which the anchor is
eventually attached that is termed the ω site. The amino
acids that are two residues to the carboxyl side of ω
residue (the ω + 2 site) have small side chains, whereas
the residues at the ω + 1 site can have large side chains.
In all cases the ω + 2 site is followed by a stretch of
5–10 hydrophilic amino acids and then 15–20 hydro-
phobic residues at or very near the carboxyl or
C-terminus of the protein. Following the addition of
the GPI anchor, the GPI-anchored glycoproteins move
to the plasma membrane. These glycoproteins usually
have other sugar residues attached to other amino acids,
such as N-glycans, that may or may not be processed
within the ER and Golgi apparatus. It is interesting that
the formation of N-glycans and GPI-anchored glyco-
proteins uses a common intermediate, i.e. dol-P-Man.
714 Glycosylation of Proteins

Glycosylation of Proteins. Figure 5 Biosynthesis of GPI-anchored glycoproteins. The pathway shown is for
human GPI anchor biosynthesis from phosphatidylinositol, which occurs in the cytosolic and lumenal regions of the
ER. Following generating of the GPI anchor precursor, the GPI anchor is added en bloc to the C-terminal region of an
ER protein by the transamidase complex, resulting in the cleavage and release of a C-terminal peptide.

Some individuals have a mutation in the gene (termed
PIG A) encoding the first enzyme of the pathway for
GPI anchor biosynthesis that normally adds GlcNAc
from UDPGlcNAc to phosphatidylinositol. Thus, these
individuals are defective in generating the mature GPI
anchor precursor and are deficient in generating GPI-
anchored glycoproteins. Such individuals are often
clinically recognized as having paroxysmal nocturnal
hemoglobinuria (PNH), a form of hemolytic anemia.
GPI-anchored glycoproteins also occur in many
protozoans, and have been especially well character-
ized in African trypanosomes, where the GPI-anchored
glycoprotein is recognized as a highly antigenic variant
surface glycoprotein (VSG).
Mucin-Type O-glycan Biosynthesis
Many glycoproteins within the Golgi apparatus are
modified to contain GalNAcα1-Ser/Thr residues,
typically found in animal mucins, by the action of a
family of UDPGalNAc:polypeptide α-N-acetylgalacto-
saminyltransferases (ppGalNAcTs). While mucins may
contain hundreds of such linkages, some glycoproteins,
such as the transferrin receptor, contain only a single O-
glycan. Yet, all such linkages are categorized as mucin-
type. The ppGalNAcTs recognize Ser and Thr residues
in glycoproteins and add GalNAc in O-glycosidic
linkage from the donor UDPGalNAc to these amino
acid side chains to form GalNAcα1-Ser/Thr, which is
also called the Tn antigen (10). Well over a dozen
different ppGalNAcTs are known and many of these
are expressed simultaneously within cells. Some of
these enzymes may have unique, but partly over-
lapping, recognition of Ser/Thr residues within the
polypeptide sequence. Interestingly, many ppGalNAcTs
are dual function enzymes containing a catalytic
domain that transfers GalNAc and a lectin domain
(ricin- or R-type) that binds to GalNAc residues.
Thus, addition of GalNAc to some Ser/Thr sites may
promote further modification by attracting more
ppGalNAcTs. Such concerted actions of ppGalNAcTs
in the Golgi apparatus may promote the relatively
efficient modifications of hundreds of Ser/Thr
residues within some very large mucin polypeptides,
some of which have over 10,000 amino acids.
Following the formation of GalNAcα1-Ser/Thr residues,
glycoproteins are subjected to the action of a β3-
galactosyltransferase, also called the T-synthase, to form
the disaccharide Galβ3GalNAcα1-Ser/Thr, which is
called the Thomsen-Friedenrich, TF or simply T antigen,
using the donor UDPGal. The T antigen disaccharide is
also the simplest core 1 O-glycan structure (Fig. 2).
However, occasionally the GalNAcα1-Ser/Thr residues
may be sialylated to generate the disaccharide NeuA-
cα6GalNAcα1-Ser/Thr (sialyl Tn antigen), which cannot
be further modified. Upon formation of Galβ3GalNA-
cα1-Ser/Thr, the core 1 structure may be modified by an
Glycosylation of Proteins 715
N-acetylglucosaminyltransferase (the core 2 GlcNAcT)
to generate the trisaccharide Galβ3(GlcNAcβ6)GalNA-
cα1-Ser/Thr (core 2 O-glycan) from the donor UDPGlc-
NAc. This core 2 O-glycan can be subsequently modified
by addition of other sugars, such as galactose, fucose and
N-acetylneuraminic acid, and/or sulfate residues on
selected sugars to generate a wide variety of O-glycan
structures.
Biosynthesis of Other O-Glycans
The biosynthesis of non-mucin type O-glycans is
incredibly varied depending on the cellular compart-
ment. O-GlcNAc residues are added to proteins in the
cytoplasm, as discussed below. Glycosaminoglycan
addition is initiated by UDPXyl:core protein β-D-
xylosyltransferases I and II, which transfer Xyl from G
UDPXyl to specific Ser residues in proteoglycan
core proteins in the ER. The Xyl residue is subse-
quently modified by addition of Gal and GlcA residues
by galactosyltransferases and glucuronyltransferases
respectively, to form the core linkage tetrasaccharide of
glycosaminoglycans, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-
Ser, which occurs in all proteoglycans. This synthesis
of the glycosaminoglycan core region may be com-
pleted in the ER, while the subsequent elongation of the
glycans and sulfation and epimerization, which is an
orchestrated and incredibly complex series of reactions,
appear to occur primarily in the Golgi apparatus. The
elongation of glycosaminoglycans on proteoglycans
can be partly averted by feeding cells β-xylosides, that
act as acceptors for addition of Gal, thus effectively
decreasing elongation of glycosaminoglycan within the
proteoglycan acceptors. Remarkably, β-xylosides ap-
pear capable of penetrating the ER and possibly the
Golgi apparatus of cells. This inhibition by competition
can result in synthesis of free glycosaminoglycans on
the β-xyloside and reduced addition of glycosamino-
glycan to proteoglycans. O-Mannosylation of proteins
in yeast in initiated in the ER by transfer of Man from
the donor dol-P-Man using a specific O-mannosyl-
transferase. Further elongation to generate mannose-
containing polysaccharides in yeast occurs by Man
donation from GDPMan in the Golgi apparatus by
additional mannosyltransferases. An equivalent en-
zyme in animals, termed POMT1, may initiate O-
Man formation on selective Ser/Thr residues in
glycoproteins in the ER using dol-P-Man as the donor,
while further elongation and addition of other sugars
may occur in the Golgi apparatus. O-fucosylation and
O-glucosylation of EGF-like domains on glycoproteins
are catalyzed by specific enzymes that transfer Fuc or
Glc from GDPFuc or UDPGlc, respectively, in the
Golgi apparatus. Collagen is glycosylated in the ER
following hydroxylation of Lys residues to generate
hydroxylysine (Hyl). The addition of Gal to Hyl is
catalyzed by a collagen-specific enzyme UDPGal:
716 Glycosylation of Proteins
procollagen-5-hydroxy-L-lysine D-galactosyltransfer-
ase, which adds Gal to Hyl residues on procollagen in
the ER during procollagen biosynthesis and concomi-
tantly with Hyl formation on nascent polypeptides
catalyzed by lysyl hydroxylase activity.
Glycosylation in the Cytosol
Many cytosolic proteins in animals (and probably
plants) contain one or more residues of β-linked
GlcNAc in O-glycosidic linkage to Ser/Thr residues
(11). These O-GlcNAcylated proteins (O-GlcNAc-
containing glycoproteins) are generated by the action
of the UDPGlcNAc:polypeptide O-acetylglucosami-
nyltransferase (O-GlcNAc transferase), which transfers
GlcNAc from UDPGlcNAc to selected Ser/Thr resi-
dues of cytosolic proteins. Some of the more pro-
minent O-GlcNAcylated glycoproteins include RNA
polymerase II, c-myc and the estrogen receptor.
O-GlcNAcylation is one of the only types of
glycosylation that is reversible. The O-GlcNAc may
be selectively removed by the action of an O-GlcNAc
specific acetylglucosaminidase (O-GlcNAcase) in the
cytosol. This alternating addition and removal of
O-GlcNAc by these two enzymes is akin to reversible
phosphorylation and dephosphorylation of cytosolic
proteins. O-GlcNAcylation may serve to regulate many
metabolic pathways and is required for animal and
plant cell growth.
The storage polysaccharide glycogen, which is a
glycoprotein in animals, is generated on the core
protein glycogenin within the cytosol of animals, by its
autocatalytic “self-glucosylation” of a Tyr residue at
position 194 using UDPGlc as a donor. The Glc-O-Tyr
is then elongated by addition of other Glc residues
(up to
10) from UDPGlc by glycogenin activity. The
Glc-containing oligosaccharide on glycogenin is then
elongated by glycogen synthase. A similar type of
activity may occur on the starch protein amylogenin.
Plant Glycoproteins
Many plant glycoproteins contain N-glycans, which are
also synthesized via the dolichol pathway in the ER.
They can also be subsequently modified by processing
reactions and addition of other sugars to generate high
mannose-, hybrid- and complex-type N-glycans. Many
plant wall proteins are typically glycoproteins rich in
the amino acids hydroxyproline (▶hydroxyproline-rich
glycoprotein, HRGP), proline (proline-rich protein,
PRP), and glycine (glycine-rich protein, GRP). The O-
glycans in HRGPs may account for up to 95% of the
glycoprotein weight and the glycans can range in size
from a single attached Ara residue to large ▶arabino-
galactans containing nearly 100 residues of Ara and
Gal. Many of these glycoproteins form rods (HRGP,
PRP) or β-pleated sheets (GRP). Extensin is one of the
best-studied HRGPs. HRGP expression is increased by
plant wounding and pathogen attack. The pistil and
pollen tube extracellular matrix are enriched in these
highly glycosylated proteins.
Bacterial Glycoproteins
Glycoproteins are also found in prokaryotes and in
archaebacteria, although the general structures of the
attached glycans and sugar residues are very different
from those found in animals and plants. Among the
best studied prokaryotic glycoproteins are the cell
surface or S-layer glycoproteins (12). Such S-layer
glycoproteins can assemble into ordered lattice-like
structures on the cell surface. Each S-layer glycoprotein
may contain more than one attached glycan, which can
be linked via Asn or other amino acid residues (Table
1). In many cases the bacterial S-layer glycan chains are
linear or branched homo- or hetero-saccharides having
20–50 identical repeating units. By contrast, archaeal
S-layer glycoproteins have shorter glycans, generally
lacking repeating units. Although the exact mechan-
isms of S-layer glycoprotein biosynthesis are not yet
defined, it appears that most sugar residue addition
occurs in the outer membrane following protein
translocation.
Many Factors Regulate Protein Glycosylation
As discussed above, two of the major factors regulating
protein glycosylation are the sequence motifs within the
primary structure of glycoproteins and the site of
biosynthesis. But many other factors also contribute to
regulation of protein glycosylation. These include the
expression of glycosyltransferases, expression of gly-
cosidases, secondary, tertiary and/or quaternary struc-
tures of proteins, availability of donor substrates, e.g.
sugar nucleotides and dolichol, cations, e.g. magnesium
and manganese, temperature and membrane lipid
composition and structure. Many of these factors,
especially expression of glycosyltransferases, vary
tremendously between cell types. Dozens of different
glycosyltransferase genes encoding enzymes that act on
glycoproteins exist in the genomes of most multi-
cellular organisms. Together, these many factors help to
explain the huge differences in glycosylation observed
between different cells and tissues.
Glycoproteins Have Many Biological Functions
Because glycoproteins are so common in all cells, it is
not surprising that the glycan moieties have many
different functions. Although many of the specific
functions of glycoproteins are being defined, it is likely
that the complete picture of glycoprotein functions will
take many years to complete. Some of the known
functions of glycoproteins and their attached glycans
include cell-cell adhesion, cell-matrix interactions,
glycoprotein targeting to organelles and cell signaling.
For example, glycoproteins regulate many different

types of cell adhesion, including sperm-egg adhesion,
leukocyte-platelet-endothelial cells adhesion, recog-
nition and phagocytosis and neuronal cell-matrix
adhesion. Some of the non-specific functions of
glycoprotein glycans include protein folding and
assembly, protein protection and stability against
proteases, control of the circulatory half-life of
glycoproteins, regulation of protein conformation and
thermal stability and control of enzyme kinetics. Many
glycoprotein glycan functions are generated by glycan
recognition through carbohydrate-binding proteins or
lectins. Lectins are made by all organisms, including
animal, plants, bacteria and viruses.
Human Disorders Associated with Defective
Protein Glycosylation
There are many human disorders associated with
an altered ability to add carbohydrate residues to
glycoproteins. One of the first defined examples of this
was ▶I-cell disease, where patients were found to have a
recessive genetic mutation of the gene encoding the
phosphotransferase activity that helps to generate Man-
6-P residues on lysosomal acid hydrolases. Conse-
quently, their cells are unable to synthesize lysosomal
acid hydrolases with Man-6-P residues efficiently (13).
Most of the non-phosphorylated lysosomal acid hydro-
lases from these patients become processed within the
Golgi apparatus, acquire sialic acid and other sugar
residues on hybrid- and complex-type N-glycans and are
secreted into body fluids. The patients accumulate
undegraded macromolecules in lysosomes due to lack of
acid hydrolases and these accumulations are recognized
microscopically as inclusion or I-cells, hence the name
I-cell disease. Another historically important defect in
glycoprotein glycosylation associated with human
disease is PNH. Patients with PNH have reduced ability
to generate the GPI anchor, due to mutation in the
X-linked PIG A gene. Hemolytic anemia results due to
deficiencies in the normally GPI-anchored glycopro-
teins termed decay accelerating factor (DAF or CD55)
and membrane inhibitor of reactive lysis (MIRL or
CD59), which function to decrease autolysis of
erythrocytes by activated complement.
Many of the genetic defects in the ability to glycosylate
proteins are now recognized within the broad category
of ▶congenital disorders in glycosylation (CDGs) (14).
The CDGs are highly varied depending on the glycan
structures made and are recognized as different types,
such as Type 1a, 1b, 1c, 1d, 1e, and IIa. Each type of
CDG results from mutations in one of the many genes
encoding proteins involved in N-glycosylation via the
dolichol pathway or subsequent processing and
glycosylation reactions, or in genes regulating orga-
nelle trafficking and biosynthesis. CDGs are often
diagnosed by examining the N-glycosylation pattern of
serum glycoproteins, such as transferrin, where altered
Glycosylation of Proteins 717
N-glycosylation can affect mobility upon isoelectric
focusing chromatography. CDG patients, depending on
the altered gene, exhibit a variety of changes in
physiognomy and suffer from neurological, liver and/
or intestinal problems. Children with CDG, depending
on the type of genetic mutation, exhibit impairments in
cognitive ability, speech and balance and motor skills.
Other disorders where altered protein glycosylation is
observed include several forms of congenital muscular
dystrophy, such as Fukuyama congenital muscular
dystrophy, ▶limb-girdle muscular dystrophy, muscle-
eye-brain disease and Walker-Warburg syndrome (15).
Many of these diseases are associated with mutations in
genes encoding glycosyltransferases that add GlcNAc or
Man residues to generate O-linked Man-containing
glycans on α-dystroglycan, which is a membrane- G
associated glycoprotein that helps to link neuronal cells
and their cytosolic signaling machinery to extracellular
matrix molecules such as laminin. Patients with proger-
oid-type Ehlers-Danlos (E-D) syndrome have defects in a
galactosyltransferase that is required to synthesize the
common linkage region of glycosaminoglycans. Another
disorder where altered protein glycosylation is observed
is leukocyte adhesion deficiency type II (LAD II), where
patients lack the ability to add fucose to glycoproteins.
This deficiency in fucosylation results in a lack of
leukocyte adhesion to selectins, a group of carbohydrate-
binding proteins that recognize fucose-containing O-
glycans and serve to regulate leukocyte trafficking from
the bloodstream. Some LAD II patients have mutations in
the gene encoding the Golgi transporter for GDPFuc, thus
preventing normal movement of GDPFuc from its site of
synthesis in the cytosol into the Golgi apparatus for
utilization by fucosyltransferases. Finally, defects in
glycoprotein glycosylation are also seen in patients with
some autoimmune diseases, such as may occur in
congenital dyserythropoietic anemia type II, where a
defect in N-glycosylation may occur due to deficiency of
α-mannosidase II activity and in IgA nephropathy, where
a subset of IgA molecules lack appropriate O-glycan
structures within the hinge region. These are just a few of
the many examples where protein glycosylation is
essential to biological processes.
▶Biochemical Engineering of Glycoproteins
▶Protein Databases
▶Recombinant Protein Production in Mammalian Cell
Culture
References
1. Apweiler R, Hermjakob H, Sharon N (1999) On the
frequency of protein glycosylation, as deduced from
analysis of the SWISS-PROT database. Biochim Bio-
phys Acta 1473:4–8
2. Spiro RG (2002) Protein glycosylation: nature, distribu-
tion, enzymatic formation, and disease implications of
glycopeptide bonds. Glycobiology 12:43R–56R
718 Glycosylphosphatidylinositol (GPI) Anchors
3. Laine RA (1994) A calculation of all possible oligosac-
charide isomers both branched and linear yields 1.05 × 10
(12) structures for a reducing hexasaccharide: the Isomer
Barrier to development of single-method saccharide
sequencing or synthesis systems. Glycobiology 4:759–767
4. Helenius J, Aebi M (2002) Transmembrane movement of
dolichol linked carbohydrates during N-glycoprotein
biosynthesis in the endoplasmic reticulum. Semin Cell
Dev Biol 13:171–178
5. Parodi AJ (2000) Protein glucosylation and its role in
protein folding. Annu Rev Biochem 69:69–93
6. Kornfeld S (1987) Chromatography: A review of clinical
applications. FASEB J 1:462–468
7. Schachter H (2000) The joys of HexNAc. The synthesis
and function of N- and O-glycan branches. Glycoconj J
17:465–483
8. Baenziger JU, Green ED (1988) Pituitary glycoprotein
hormone oligosaccharides: structure, synthesis and
function of the asparagine-linked oligosaccharides on
lutropin, follitropin and thyrotropin. Biochim Biophys
Acta 947:287–306
9. McConville MJ, Menon AK (2000) Recent develop-
ments in the cell biology and biochemistry of glycosyl-
phosphatidylinositol lipids (review). Mol Membr Biol
17:1–16
10. Brockhausen I (1999) Pathways of O-glycan biosynth-
esis in cancer cells. Biochim Biophys Acta 1473:67–95
11. Wells L, Vosseller K, Hart GW (2001) Glycosylation of
nucleocytoplasmic proteins: signal transduction and O-
GlcNAc. Science 291:2376–2378
12. Schaffer C, Messner P (2004) Surface-layer glycopro-
teins: an example for the diversity of bacterial glycosyla-
tion with promising impacts on nanobiotechnology.
Glycobiology 14:31R–42R
13. Raas-Rothschild A, Cormier-Daire V, Bao M et al
(2000) Molecular basis of variant pseudo-hurler poly-
dystrophy (mucolipidosis IIIC). J Clin Invest 105:673–681
14. Marquardt T, Denecke J (2003) Congenital disorders of
glycosylation: review of their molecular bases, clinical
presentations and specific therapies. Eur J Pediatr
162:359–379
15. Endo T, Toda T (2003) Glycosylation in congenital
muscular dystrophies. Biol Pharm Bull 26:1641–1647
Glycosylphosphatidylinositol (GPI)
Anchors
Definition
In the lumen of the endoplasmic reticulum, the GPI
anchor is covalently attached to the C terminus of
proteins destined for the plasma membrane, and the
transmembrane segment of the protein is cleaved off.
As proteins are only attached to the exofacial leaflet of
the plasma membrane by the GPI anchor, they can be
released in soluble form from the cell surface by the
action of specific phospholipases.
▶Epithelial Cells
▶Glycosylation of Proteina
Glycosyltransferase
Definition
Glycosyltransferase is a member of a large family of
enzymes expressed in the endoplasmic reticulum and
Golgi apparatus, which catalyze the transfer of a
monosaccharide unit from a sugar-nucleotide donor,
typically to the non-reducing terminus of an oligosac-
charide chain in glycoproteins and glycolipids.
▶Glycosylation of Proteins
▶Limb Girdle Muscular Dystrophies
▶Methylation of Proteins
Glyoxylate
Definition
Glyoxylate is a toxic compound, generated in vivo,
which needs to be eliminated by conversion into
glycine via the peroxisomal enzyme alanine glyoxylate
aminotransferase.
▶Peroxisomal Disorders
Glypidation
Definition
Glypidation describes the attachment of a glycosyl-
phosphatidylinositol-(GPI)-anchor to certain integral
membrane proteins. The anchor is composed of the
lipid phophatidylinositol to which a carbohydrate and
an ethanol and phosphate moiety is linked. The GPI-
anchor is attached post-translationally in the lumen of
the endoplasmic reticulum, thereby replacing a tran-
sient transmembrane region of the modified protein.
The ▶GPI-anchor attaches proteins to the exoplasmic
leaflet of membranes, possibly to certain subdomains
such as caveolae and lipid-rafts.
▶Fatty Acid Acylation of Proteins
▶Glycosylation of Proteins

Golgi Apparatus (Golgi Complex)
Definition
Golgi apparatus (Golgi complex) refers to a cytoplasmic
organelle in eukaryotes consisting of stacked, flattened
membrane cisternae, surrounded by vesicles, which is
involved in transport and post-translational modifica-
tion (especially glycosylation) of proteins on their
journey through the secretory pathway. The Golgi
complex is also a central sorting station in the secretory
pathway; on the trans- or exit-side of the Golgi complex,
proteins get sorted into several distinct vesicle types for
transport to different final destinations.
▶Biochemical Engineering of Glycoproteins
▶Exocytotic Pathway
▶Glycosylation of Proteins
▶Limb Girdle Muscular Dystrophies
▶Rho, Rac, Cdc42
▶Vesicular Traffic
Gomori Trichrome
Definition
Gomori trichrome is a mixture of chemical compounds
that stain mitochondria red.
▶Mitochondrial Myopathies
Gonadal Mosaicism
▶Germline (Gonadal) Mosaicism
Gonadotropin Deficiency
Definition
Gonadotropin deficiency describes the absence, de-
creased production or dysfunction of anterior pituitary
hormone (LH and/or FSH), which results in a
decreased or lack of testosterone in males and estrogen
Gorlin’s Syndrome 719
in females. This causes delayed or no pubertal
development and infertility.
▶Hypothalamic and Pituitary Diseases Genetics
Gonadotropins
Definition
Gonadotropins are pituitary hormones that influence
the functions of the ovary: ▶follicle stimulating
hormone (FSH) and ▶luteinizing hormone (LH). G
▶SRY – Sex Reversal
▶Hypothalamic and Pituitary Disease, Genetics
Gordon’s Syndrome
Definition
Gordon’s syndrome is also known as type 2 Pseudo-
hypoaldosteronism (PHA2).
▶Mendelian Forms of Human Hypertension and
Mechanisms of Disease
▶Type 2 Pseudohypoaldosteronism
Gorlin’s Syndrome
Definition
Gorlin’s syndrome (also known as naevoid basal cell
carcinoma syndrome (NBCCS)) is a rare autosomal
dominant cancer disorder characterised primarily by a
predisposition to several tumours, most commonly
basal cell carcinoma (BCC). In addition to cancer
susceptibility, this syndrome is also associated with a
range of defects resulting from abnormal embryonic
development. Gorlin’s syndrome results from mutation
of the patched gene which functions in the hedgehog
signalling pathway. The developmental defects are
believed to result from ▶haploinsufficiency, with
subsequent mutation of the remaining allele resulting
in tumour formation.
▶Hedgehog Signalling
720 GPCRs
GPCRs
▶G-Protein Coupled Receptors
G-Phase
▶Cell Cycle – Overview
GPI (-Anchored) Protein
Definition
A GPI-anchored protein is a protein that is anchored
in the membrane by glycosylated derivatives of
phosphatidylinositol (GPI). The carboxyl group of the
C-terminal amino acid is connected through an amide
link to phosphoethanolamine, which is attached to a
core tetrasaccharide composed of three mannose sugars
and a single glucosamine sugar. The tetrasaccharide is
in turn attached to phosphatidylinositol embedded in
the membrane. Proteins with this type of lipid anchor
are only found on the extracellular face (outer leaflet)
of the membrane, and can be released in soluble form
on the cell’s surface by the action of specific
phospholipases.
▶Biological Membranes
▶Epithelial Cells
▶Glycosylation of Proteins
GPIIb/IIIa Complex
Definition
The GPIIb/IIIa complex is a platelet membrane
glycoprotein complex mediating platelet aggregation
and adhesion to endothelial cells. The complex is an
integrin that recognises the arginine-glycine-aspartic
acid (rgd) sequence present on several adhesive
proteins. The GPIIb/IIIa complex functions as a
receptor for fibrinogen, von Willebrand Factor
(vWGF), fibronectin, vitronectin, and thrombospondin.
Deficiency of GPIIb/IIIa causes ▶Glanzmann’s
Thrombasthenia.
G-Protein Coupled Proteolytic Site
A G-protein coupled proteolytic site is a peptide
sequence found in a number of G-protein coupled
receptors that acts as the target for specific proteolytic
cleavage, which releases the extracellular portion of the
receptor from the rest of the molecule.
▶Autosomal Dominant (Inherited Disorder)
▶G-Proteins and G-Protein Mutations in Human
Diseases
▶Polycystic Kidney Disease, Autosomal Dominant
G-Protein Coupled Receptors
Definition
G-protein coupled receptors (GPCRs) comprise of the
largest family of cell surface receptors, which commu-
nicate their signal through G-proteins. A common
structural feature of GPCRs is the presence of seven
hydrophobic transmembrane helices. Several hundred
subtypes exist, which bind a huge variety of ligands,
such as hormones and neurotransmitters. About 50% of
the currently used drugs are directed at GPCR’s.
▶Cardiac Signaling: Cellular, Molecular and Clinical
Aspects
▶Cytokine Receptors
▶G-Proteins and G-Protein Mutations in Human
Diseases
▶Growth Factors
▶Methylation of Proteins
▶Photoreceptors
▶Seven-Transmembrane Receptors
▶Wnt/Beta-Catenin Signaling Pathway
G-Proteins
Definition
▶Diabetes Insipidus, a Water Homeostasis Disease
▶G-Proteins and G-Protein Mutations in Human
Diseases
▶Molecular Motors

G-Proteins and G-Protein Mutations
in Human Diseases
T HOMAS G UDERMANN
Phillips-University Marburg, Marburg, Germany
gudermann@staff.uni-marburg.de
Synonyms
Heterotrimeric guanine nucleotide-binding proteins
Definition
A plethora of extracellular signaling molecules like
hormones, neurotransmitters, autacoids and growth
factors convey information between cells of a living
organism. The majority of these extracellular signaling
molecules transmit their signal by interacting with a
three-protein transmembrane signal transduction sys-
tem composed of receptors, G-proteins and cellular
effectors. All mammalian cells are endowed with a
complement of G-protein-coupled receptors and sev-
eral types of heterotrimeric G-proteins. Over the last
few years systematic biochemical, cell biological and
structural studies have laid a solid foundation for our
understanding of G-protein dependent signal transduc-
tion processes. The characterization of genetically
engineered mice carrying mutations in different G-
protein genes as well as the clinical phenotype of
patients affected by mutated G-proteins have greatly
furthered our understanding of the biological functions
of heterotrimeric G-proteins as central processors of
information.
Characteristics
Basic Structures, Mechanisms and Classifications
The vast majority of extracellular signals interact with
transmembrane receptors which couple to heterotri-
meric guanine nucleotide-binding proteins (G-proteins)
acting as transducers and signal amplifiers (1).
Activated G-proteins then modulate the activity of
cellular effectors. A comprehensive analysis of the
human and mouse genomes defined a repertoire of
G-protein-coupled receptors (GPCRs) for endogenous
ligands comprising close to 400 genes (2, 3). GPCRs
form a large and functionally diverse superfamily,
participate in a variety of physiological processes and
are prime targets for pharmaceutical drugs. They are
integral membrane proteins, characterized by 7 α-
helical transmembrane domains arranged in an anti-
clockwise bundle (as viewed from the extracellular
side) and connected by alternating extracellular and
intracellular loops of variable lengths. The crystal
structure of the prototypical GPCR ▶rhodopsin in the
G-Proteins and G-Protein Mutations in Human Diseases 721
inactive ground state has been resolved, by and large
confirming the overall architecture deduced from
analogy-based biomodelling and various mutagenesis
approaches (4). As yet, it is only rudimentarily
understood how ligand-induced conformational change
of a GPCR is translated into G-protein activation.
G-Protein Composition and Structural Aspects
G-proteins are heterotrimers composed of α, β, and γ
subunits. The α subunit is responsible for guanine
nucleotide binding and GTP hydrolysis; the β and γ
subunits are associated in a tenaciously linked βγ
complex and can be regarded as one functional unit. To
date 16 distinct genes for α, 5 for β and 12 for γ
subunits have been identified and characterized
functionally (Table 1). Access to a deeper under- G
standing of G-protein structure and function at the
atomic level has been granted by solving crystal
structures of G-protein α subunits in the GDP- and
GTP-bound forms as well as in the transition state (5).
In addition, atomic structures of G-protein βγ dimers
and of αβγ heterotrimers are also available. Gα proteins
are principally composed of a ▶Ras-like GTPase and
an α-helical domain, both forming a deep cleft
harboring the guanine nucleotide. Gβ subunits fold
into a highly symmetrical β propeller with an
approximate 7-fold symmetry. Gγ binds to Gβ in an
extended conformation devoid of intrachain tertiary
interactions. Those segments in the Gα protein that
undergo structural changes upon GTP hydrolysis are
named switch I, II, and III regions. The most obvious
changes occur in the switch II region. A mechanistic
explanation for subunit dissociation and reassociation
contingent upon the guanine nucleotide bound to the α
subunit can be derived from the observation that the
most extensive contact area between Gα and βγ
comprises the protein surface around the switch II
region of Gα. So far, structural information does not
provide an obvious cue as to the specificity in the
pairing of a particular α subunit with a defined βγ
dimer.
Because effector contact sites of Gα have also been
mapped to areas around the switch II region, Gβγ- and
effector-interacting surfaces of Gα overlap signifi-
cantly. Thus, Gα cannot interact with cellular effectors
unless it dissociates from Gβγ. Conversely, activation
of Gβγ is a consequence of its release from Gα, which
functions as a negative regulator of free βγ subunits
within the cell. The regions of Gβγ that interact with
downstream effectors map to an N-terminal Gβ
fragment of approximately 100 amino acids. As yet,
it is not understood how an activated receptor catalyses
the dissociation of GDP from a G-protein heterotrimer.
The most clearly defined Gα contact sites with
heptahelical receptors are located in the C-terminal
region of the α subunit. However, compelling evidence
722 G-Proteins and G-Protein Mutations in Human Diseases

G-Proteins and G-Protein Mutations in Human Diseases. Table 1 G-protein α subunits

Name Expression Examples of effectors Bacterial Toxins

αs subfamily
αs ubiquitous AC ↑, VGCC, Src, RGS-PX1 CTX
(GAP, sorting nexin)
αolf olfactory epithelium, brain CTX
αi subfamily
αt-r retinal rods cGMP PDE ↑ CTX, PTX
αt-c retinal cones cGMP PDE ↑ CTX, PTX
αgust taste cells PDE ?, PLC-β ? CTX, PTX
αi1 mainly neuronal cells AC ↓, Src, RapIGAPs PTX
αi2 ubiquitous PTX
αi3 widely expressed PTX
αo neuronal, neuroendocrine cells PTX
αz neuronal cells,thrombocytes
αq subfamily
αq ubiquitous PLC-β ↑, LARG-RhoGEF
α11 widely expressed
α14 kidney, lung, spleen
α15/16 hematopoietic cells
α12 subfamily
α12 ubiquitous RhoGEFs, E-cadherin
α13 ubiquitous

AC, adenylyl cyclase; CTX, cholera toxin; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PDE,
phosphodiesterase; PLC, phospholipase C; PTX, pertussis toxin; RGS, regulator of G-protein signaling; VGCC, voltage-gated
calcium channel

has also been presented for participation of the Gα N- G-Protein Cycle

terminus as well as the βγ dimer in receptor interaction.
Considering the proximity of the Gα N-terminus and
Gγ C-terminus on a common face of the G-protein
heterotrimer anchored to the plasma membrane via
lipid modifications, cytoplasmic receptor domains have
access to large surface areas of α, β and γ subunits.
However, according to the crystal structures available,
the distance between the posttranslationally modified
Gα N-terminus and Gγ C-terminus (estimated mini-
mum of 40 Å) appears to be too large to interact with
rhodopsin’s cytoplasmic surface area (maximal dis-
tance between interacting loop sites approximately
35 Å) at the same time, thus implying a two-step
sequential interaction mechanism. The structural basis
underlying the selectivity of receptor-G-protein inter-
action still remains only partially defined.
Binding of an agonist to a heptahelical receptor entails
the formation of a ternary complex consisting of
agonist, receptor and heterotrimeric G-protein in its
GDP-liganded form. The binding of βγ subunits to Gα
stabilizes the flexible switch regions and hence the
GDP-dependent inactive Gα conformation. The acti-
vated receptor fulfills the role of a catalytically acting
▶guanine nucleotide exchange factor that facilitates
the release of GDP (Fig. 1). The short-lived guanine
nucleotide-free G-protein heterotrimer stabilizes the
receptor in its high-affinity conformation. Due to its
high intracellular concentration, GTP is rapidly in-
corporated into the guanine nucleotide-binding pocket
in Gα, resulting in a conformational change in the α
subunit and a dissociation of GTP-bound Gα and Gβγ.
Both reaction products are intracellular signaling

G-Proteins and G-Protein Mutations in Human Dis-
eases. Figure 1 The G-protein cycle. The activated
receptor functions as a guanine nucleotide exchange
factor to release bound GDP. G-protein βγ subunits
stabilize the inactive, GDP-bound Gα conformation.
Pertussis toxin (PTX) modifies the C-terminus of some
G-protein α subunits and uncouples these G-proteins
from the receptor. Both GTP-bound α subunits and βγ
dimers are signaling proteins in their own right and
interact with effector proteins. Gα activation is termi-
nated by hydrolysis of GTP. The endogenous GTPase
activity is accelerated by effectors such as phospholi-
pase C-β or RGS (for “regulator of G-protein signaling”)
proteins. Cholera toxin (CTX) modifies a highly con-
served arginine residue in some α subunits, thereby
abolishing GTPase activity and rendering the G-protein
α subunit constitutively active. For further details see
text.
proteins in their own right, activating distinct and
overlapping portfolios of cellular effectors. In contrast
to the situation with monomeric GTPases of the Ras
family, G-protein α subunits are endowed with an
endogenous GTPase activity. A highly conserved
arginine residue in the helical domain, Arg201 in Gαs,
directly participates in GTP hydrolysis by stabilizing
the negative charge on γ-phosphoryl oxygen atoms in
the transition state. Ras proteins lack such a residue and
are essentially inactive as GTPases. The conformation
of GDP-bound Gα allows for the reassociation with the
βγ dimer and the inactive GαGDPβγ complex is now
prone to another round of activation and deactivation.
Two bacterial toxins interfere with the GTPase cycle by
covalently modifying G-protein α subunits. ▶Cholera
toxin (CTX) ADP-ribosylates the aforementioned
conserved arginine residue in the GTPase domain of
some G-protein α subunits (Table 1), thus blocking
G-Proteins and G-Protein Mutations in Human Diseases 723
GTP hydrolysis and rendering the α subunit constitu-
tively active. ▶Pertussis toxin (PTX) adds the ADP-
ribosyl moiety to a cysteine residue in the C-terminus
of most Gi proteins, thereby effectively uncoupling
modified G-proteins from the receptor. Both toxins
have been extensively used to dissect G-protein-
mediated signaling pathways.
In a physiological setting, the GTPase activity of α
subunits is controlled by a diverse family of multi-
functional signaling proteins, ▶regulators of G-protein
signaling (RGS), which bind directly to activated Gα
subunits in order to accelerate the rate of GTP
hydrolysis by several orders of magnitude. In addition,
G-protein effectors such as phospholipase C-β can also
potentiate GTPase activity of α subunits (Fig. 1). Apart
from deactivation of G-protein α subunits and termina- G
tion of downstream signals, RGS proteins may also be
viewed as bona fide effectors setting in motion various
intracellular signaling cascades by means of protein-
protein interactions mediated by a host of defined
structural signaling motifs. Furthermore, they may
profoundly shape the dynamics and determine the
efficacy of G-protein cycling. An approximately 120-
amino acid region that directly interacts with GTP-
bound α subunits, called the RGS domain, is the
structural hallmark of more than 30 RGS family
members. The majority of RGS proteins appear
to target Gi and Gq family members, whereas some
(p115-RhoGEF, PDZ-RhoGEF, LARG-RhoGEF) spe-
cifically interact either with Gα12/13 proteins or with
Gαs (RGS-PX1). The exact physiological functions of
RGS proteins are still poorly understood.
Within the past few years alternative modes of signal
input into G-protein cascades have been discovered
which are independent of heptahelical receptors. Four
proteins, called AGS1-4 (for ▶activators of G-protein
signaling), have been identified that engage G-protein-
dependent signaling pathways in the absence of a
classical receptor. AGS1 is a distinct member of the
large superfamily of Ras-related proteins and targets α
subunits. AGS2 represents a Gβγ-binding component
of the cytoplasmic motor protein dynein and has
undefined roles in cellular signaling. The third protein,
AGS3, possesses seven tetratrico peptide (TRP) repeats
and four amino acid repeats termed GoLoco motifs
(meaning Gαi/o-Loco interaction motif) also described
as G-protein regulatory (GPR) motifs. The GoLoco
motif, which can also be found in other signaling
proteins like RGS12 and 14 functions as a selective Gα
binding partner. The GoLoco/Gα interaction releases
βγ subunits and at the same time inhibits guanine
nucleotide exchange in the bound α subunit, thus
“freezing” monomeric Gα in the inactive, GDP-bound
state. Recently, an additional AGS protein, AGS4, was
identified which contains three GPR motifs and
regulates the activation state of Gαi. At present, the
724 G-Proteins and G-Protein Mutations in Human Diseases
cell physiological role of AGS proteins is still fairly
obscure. Possibly, these proteins are involved in the
regulation of basic cellular processes like the main-
tenance of cell polarity and cell division. By acting in
concert with GPCRs they may provide for a signal
amplification mechanism and at the same time allow
signal transmission via G-proteins independent of
heptahelical membrane receptors.
G-Protein Families and Effectors
Based on the primary amino acid sequence of their α
subunits, G-proteins are subdivided into four distinct
families: Gs, Gi, Gq, and G12 (Table 1) (6, 7). Concentra-
tions of Gi proteins in the cell considerably exceed those
of other families, and in brain Go may amount to 1–2%
of total membrane protein. Some G-protein α subunits
are characterized by a very restricted expression pattern
(Table 1), while others like Gs, Gi2, Gq, G12, and G13
are ubiquitously expressed. G-proteins can also be
classified on the basis of the cellular effectors to which
they couple. Gs proteins classically stimulate ade-
nylyl cyclase activity, while Gi proteins inhibit adenylyl
cyclase via their α subunits, but activate inwardly
rectifying potassium channels and inhibit P/Q-, N- and
R-type voltage-gated calcium channels via βγ subunits
released upon GTP binding to Gαi proteins. Gq proteins
activate phospholipase C-β isoforms, and G12 proteins
couple to Rho guanine nucleotide exchange factors
resulting in Rho activation and stress fiber formation
(Table 1). Both GTP-loaded α subunits and βγ dimers
are eo ipso signaling proteins exerting their action
through activation or inhibition of an ever expanding list
of cellular effector proteins (for Gα effectors see: Table
1). Effectors for G-protein βγ subunits include inwardly
rectifying potassium channels (Kir3.1–3.4), G-protein-
coupled receptor kinases (GRKs), adenylyl cyclases
(adenylyl cyclases II and IV), phospholipases C (PLC)-
β1, -β2 and -β3 and phosphatidylinositol 3-kinases
(PI3K) β and γ.
Considering that hundreds of GPCRs transduce signals
by interacting with a limited number of G-proteins, the
question of coupling specificity is worth considering.
The concept of linear G-protein-mediated signal
transduction pathways, i.e. one receptor coupling to
one distinct G-protein activating one receptor, appears
to be inadequate to describe physiology. G-protein-
mediated signal transduction is a complex signaling
network with diverging and converging transduction
steps at each coupling interface. Deciphering the
mechanism of signal specificity in living cells still
remains a scientific challenge of paramount importance.
G-Protein Mutations as the Molecular Basis
of Human Diseases
Because of their central role in controlling many
physiological functions, naturally occurring activating
and inactivating mutations in GPCRs and G-proteins
are responsible for an increasing number of human
diseases. Functional variability resulting from poly-
morphisms may underlie interindividual differences in
response to endogenous ligands as well as drugs. At
present, the Gαs gene (GNAS1) is the only G-protein
gene that has been unequivocally shown to be afflicted
with activating or inactivating mutations that cause
human diseases (8-10).
Activating Mutations in Gai
Mutations in the Gαi2 gene were diagnosed in fixed
sections of human ovarian sex chord stromal tumors
and adrenal cortical tumors. In a few affected speci-
mens, the highly conserved Arg179 corresponding to
the aforementioned Arg201 in Gαs in the helical domain
was found to be exchanged for a histidine (Arg179H)
giving rise to a Gαi2 protein devoid of any GTPase-
activity. Constitutively active Gαi2 was subsequently
referred to as the gip2 oncogene. Most notably, this
finding could not be confirmed by subsequent studies
on fresh surgically resected tumors, and transgenic
animals expressing the gip2 oncogene in selected
tissues have not been reported yet. Therefore, one has
to conclude at this point that the oncogenic potential as
well as the frequency of activating mutations of Gαi2
appear to be rather low.
Activating Mutations in Gas
In many endocrine glands, cAMP stimulates prolifera-
tion, differentiation and hormone secretion. A first hint
to the possible causative contribution of activating
mutations in GNAS1 arose from the identification of a
subset of growth hormone (GH)-secreting pituitary
tumors characterized by high intracellular cAMP
concentrations and increased adenylyl cyclase activity.
These adenomas accounting for approximately 40% of
GH-secreting tumors were shown to harbor hetero-
zygous missense mutations in GNAS1 exons 8 or 9
giving rise to Arg201Cys/His and Gln227Arg/Lys
missense mutations, respectively. Both the highly
conserved Arg and Gln residues are essential for GTP
hydrolysis to occur in the α subunit with the
requirement of Gln227 to orient and polarize the
catalytic water in the transition state. Therefore, these
missense mutations ablate the endogenous GTPase
activity of Gαs and render the α subunit constitutively
active, leading to uncontrolled, excessive cAMP
production in somatotrophs. As cAMP stimulates
proliferation and differentiation in these cells, the
GTPase-deficient Gαs mutants have been designated
gsp oncogenes.
Gsp mutations are also rarely observed in other
pituitary tumors like corticotrophs, resulting in in-
creased ACTH release (Table 2). Besides, approxi-
mately 10% of non-functional pituitary adenomas carry
 G-Proteins and G-Protein Mutations in Human Diseases 725

G-Proteins and G-Protein Mutations in Human Diseases. Table 2 Diseases associated with GNAS1 mutations

Type of mutation Disease Mode of inheritance

Gain of function
Loss of function
Loss and gain of function
GNAS1 imprinting defect
GH-secreting pituitary adenomas, thyroid somatic mutations
adenomas and carcinomas, Leydig cell
adenomas, pheochromocytoma, parathyroid
adenoma, McCune-Albright syndrome, osseous
fibrous dysplasia
Albright hereditary osteodystrophy germline mutations,
PHP Ia maternal transmission
PPHP paternal transmission
progressive osseous heteroplasia (POH) paternal transmission?
382
PHP Ib (Gαs-ΔI ) maternal transmission
PHP Ia and testotoxicosis (Gαs-A 366
S) maternal transmission G
PHP Ib maternal transmission

GH, growth hormone; PHP, pseudohypoparathyroidism; PPHP, pseudopseudohypoparathyroidism

gsp mutations. Several studies have confirmed the
presence of activating mutations in Gαs in up to 30% of
toxic thyroid adenomas and in less than 10% of thyroid
carcinomas. Sporadically, gsp mutations are found in
parathyroid and adrenocortical tumors as well as in
▶pheochromocytomas.
The ▶McCune-Albright syndrome (MAS) is classi-
cally defined by the clinical triad of cafe´-au-lait
hyperpigmented skin lesions, precocious puberty and
polyostotic fibrous dysplasia of the bone. Apart from
the gonads, other endocrine glands such as the pituitary,
adrenal cortex and thyroid that are sensitive to trophic
cAMP-dependent stimuli were also found to be
hyperfunctional in MAS. Nodular and diffuse goiters
as well as benign thyroid nodules are associated with
MAS. The sporadic occurrence of thyroid cancer
(papillary and clear cell thyroid carcinoma) in MAS
patients suggests that additional mutational or epige-
netic events in addition to gain-of-function Gαs
mutations are mandatory for thyroid carcinogenesis in
these patients. In 1986 the dermatologist Happle
suggested MAS to be caused by a dominant somatic
mutation as an early postzygotic event resulting the
mosaic pattern of clinical stigmata. Mutations in
GNAS1 have been confirmed in affected endocrine
tissues and in hyperpigmented skin lesions of all MAS
patients. Interestingly, missense mutations were de-
tected at only one position, i.e. Arg201His/Cys. The
overall clinical picture of an individual MAS patient is
determined by the distribution of cells bearing the
somatic gsp mutation. It is tempting to speculate that
germline gsp mutations are incompatible with life. In
contrast to the latter concept, one patient with severe
developmental, skeletal and endocrine abnormalities
was described to harbor a germline Arg201Leu mutation
in GNAS1.
Gαs mutations have also been found in all cases of
fibrous dysplasia (FD) of the bone. The majority of FD
patients has only a single bone defect, a small group
suffers from multiple bone lesions or has other features
of MAS. Missense mutations in GNAS1 identical to
those in MAS patients, i.e. Arg201His/Cys, were
diagnosed in nearly all forms of FD. A possible
explanation for the clinical phenotype relates to the
general concept that elevated intracellular cAMP levels
in osteogenic precursors entail increased proliferation
and decreased differentiation of these cells resulting in
benign fibrous bone lesions. Activating Gαs mutations
have also been reported to occur in isolated intramus-
cular myxomas and those that present in conjunction
with FD (Mazabraud syndrome)
Loss-of-Function Mutations in Gas
More than 60 years ago, Fuller Albright and his
colleagues described several patients presenting with
short stature, obesity, skeletal abnormalities, mental
retardation and often subcutaneous ossification. This
syndrome, now collectively called ▶Albright’s heredi-
tary osteodystrophy (AHO) frequently concurs with
resistance to parathyroid hormone (PTH) and other
hormones like GH-releasing hormone, thyrotropin and
gonadotropins acting via Gs-coupled receptors. AHO in
conjunction with this kind of hormone resistance gives
rise to the complex syndrome of ▶pseudohypopar-
athyroidism (PHP) type Ia (Table 2). Patients with
pseudopseudohypoparathyroidism (PPHP) show the
726 G-Proteins and G-Protein Mutations in Human Diseases
typical features of AHO, yet do not suffer from any
kind of hormone resistance. On the contrary, PHP Ib
patients present with symptoms of isolated PTH
resistance, but lack the typical AHO phenotype. A
mild form of thyrotropin resistance has recently been
observed in PHP Ib patients raising the possibility that
other endocrine systems may also be affected.
Subsequent systematic studies were able to allocate
the molecular defect in these three main forms of PHP
to GNAS1.
Heterozygous inactivating mutations affecting one of
the Gαs-specific exons are the molecular cause of PHP
Ia, PPHP and of progressive osseous heteroplasia
(POH). POH patients suffer from severe heterotopic
ossification involving skeletal muscle and deep con-
nective tissue. They frequently lack hormone resistance
and typical AHO features. Many of the GNAS1
mutations are deletions or insertions that give rise to
frameshifts and premature stop codons, nonsense
mutations or splice junction mutations. In addition, a
number of missense mutations adversely affect protein
stability. The latter scenario is exemplified by a
missense mutation, A366S, in the critical guanine
nucleotide binding motif of the GTPase domain,
leading to an accelerated release of GDP from the α
subunit and marked instability of the guanine nucleo-
tide-free protein at the body core temperature of 37 °C.
At lower ambient temperatures, for instance in the testis,
protein stability is not impaired and the accelerated
nucleotide exchange manifests as constitutive Gαs
activity. Therefore, the clinical phenotype of PHP Ia
and excessive testicular testosterone production (testo-
toxicosis) arise from the intriguing A366S mutation
(Table 2). In most tissues, an approximately 50%
reduction of functional Gαs activity significantly
reduces cAMP formation in the case of inactivating
Gαs mutations. However, the cAMP levels that can still
be generated are sufficient to maintain physiological
functions. Thus, there is no evidence for haploinsuffi-
ciency to explain the hormone resistance observed in
patients.
Retrospective analyses of PHP patients revealed that
the clinical phenotype was strongly influenced by the
parent transmitting the mutated allele. Any inactivating
Gαs mutation leads to AHO irrespective of the parent
transmitting the defective gene. Hormone resistance
characteristic of PHP Ia occurs only if the genetic
defect is inherited from a mother suffering from either
PHP Ia or PPHP. Conversely, there is mounting
evidence that POH is inherited from the father. This
conspicuous parent-of-origin-specific inheritance pat-
tern suggests that GNAS1 is imprinted.
The human gene for Gαs is a single-copy gene located
at 20q13.2-13.3. Gαs is encoded by exons 1–13
(Fig. 2). Alternative splicing of exon 3 produces two
long and two short forms of Gαs. There is little
evidence to suggest that these splice variants have
distinct signaling properties. During the past few years
it has become obvious that GNAS1 not only codes for
Gαs, but also for several other transcripts by using
4 alternative promoters and first exons which splice
onto a common exon 2 (Fig. 2). The most upstream
alternative promoter gives rise to transcripts coding for
the chromogranin-like neuroendocrine secretory pro-
tein 55 (NESP55) whose entire coding sequence
resides in the upstream exon, thus leaving Gαs exons
2–13 within the 3′ untranslated region of the NESP55
transcript. The NESP55 promoter is methylated on the
paternal allele, so that the NESP55 gene is exclusively
transcribed maternally. The XLαs transcript encodes a
protein with an extended N-terminus when compared
to Gαs and is transcribed from the paternal allele only
(Fig. 2). The C-terminal 348 amino acid residues are
identical to Gαs. XLαs is highly expressed in the
pituitary, is targeted to the plasma membrane, interacts
with βγ subunits and can be activated by non-
hydrolysable GTP analogs. However, there is no
evidence that XLαs is regulated by GPCRs. An
additional transcript derived from the sense strand of
the paternal allele uses exon 1A (exon A/B) as the first
exon and also splices onto exons 2–13 (Fig. 2).
However, exon 1A generates transcripts that are
presumably untranslated. Upstream of the XLαs exon,
a promoter for antisense transcripts traversing the
NESP55 exon has been identified. These NESP55
antisense transcripts are only expressed from the
paternal allele and may contribute to the imprinting
of NESP55 by silencing the NESP55 promoter on the
paternal allele.
Around 100 autosomal genes are subject to ▶genomic
imprinting. One genomic region controlled by this
epigenetic phenomenon is located in the distal portion
of chromosome 2 and encompasses GNAS1. All
imprinted genes have one or more regions in which
the cytosines within CpG dinucleotide stretches are
methylated on one parental allele only. Very often these
methylated regions coincide with gene promoters. As
described above and illustrated in Fig. 2, the promoter
regions of GNAS1 display a complex imprinting
pattern. To complicate the scenario even further, the
promoter region giving rise to Gαs transcripts does
not exhibit allele-selective methylation and in most
tissues expression occurs bialellically. This situation
notwithstanding, paternal Gαs expression is silenced in
a few tissues by a mechanism that is presently
unknown. In proximal renal tubular cells, adipocytes,
pituitary gland, thyroid and gonads, Gαs expression is
largely driven by the maternal allele. In PHP Ia
patients, renal proximal tubule cells are resistant
to PTH action, because Gαs expression is restricted
 G-Proteins and G-Protein Mutations in Human Diseases 727

G-Proteins and G-Protein Mutations in Human Diseases. Figure 2 Organization and imprinting of the
GNAS1 locus.GNAS1 is characterized by 4 alternative first exons which splice onto exon 2. Methylation patterns
(methyl) and transcriptional activation (arrows) of the maternal and paternal allele are indicated. The hatched
arrow for exon 1 of the paternal allele indicates that it does not contribute to Gαs expression in all cells.
An antisense mRNA is transcribed across the NESP55 exon on the paternal allele.

to the maternal allele that carries an inactivating
mutation. Yet PHP Ia patients are not prone to
hypercalciuria, suggesting that the anticalciuric PTH
action in the thick ascending limb is fully operative
because of biallelic Gαs expression in this part of the
nephron. Thus, tissue- and cell-specific imprinting
represents the molecular mechanism underlying the
clinical features of PHP Ia, while haploinsufficiency
alone may lead to AHO.
A first glance at the mechanism of tissue-specific Gαs
imprinting was granted by studies on patients with PHP
Ib. The vast majority of these patients who present with
renal PTH resistance, sometimes accompanied by
partial TSH resistance, exhibit a loss of methylation
at the GNAS1 exon 1A, while lacking mutations in the
exons coding for Gαs. This loss of the maternal allele-
specific methylation pattern, linked to an upstream 3 kb
deletion, makes the maternal allele look like the
paternal one, resulting in silencing of maternal Gαs
expression in renal proximal tubules. One possible
explanation is based on the hypothesis that the non-
methylated exon 1A region allows for the binding of a
tissue-specific repressor protein that hampers Gαs
expression. Alternatively, the deletion may disrupt a
cis-acting imprinting control element necessary for the
methylation imprint at exon 1A (exon A/B). As the
described deletions disrupt another gene, STX16
coding for Syntaxin 16, one may speculate that the
STX16 region comprises such an imprinting control
element. The relevance of these epigenetic changes, i.e.
the loss of maternal-specific methylation of GNAS1,
for the clinical PHP Ib phenotype is emphasized by a
patient with paternal uniparental disomy of chromo-
some 20q. In this situation both long arms of
chromosome 20q are of paternal origin resulting in
PTH resistance, but not in AHO.
A unique heterozygous 3 bp deletion causing loss of
Ile382 in the C-terminus of Gαs was detected in 3
affected boys with PHP Ib. When heterologously
expressed, the mutant Gαs was found to be unable to
couple to the PTH receptor, while interaction with the
Gs-coupled thyrotropin and luteinizing hormone re-
ceptors was unaffected. These results explain PTH-
specific hormone resistance in the affected patients.
728 GPx
The absence of any phenotype in the mother and
maternal grandfather carrying the same mutation is
commensurate with our current understanding of
paternal imprinting of the GNAS1 gene.
The Gb3-C825T Polymorphism in Multigenic Disorders
A single base substitution (C825T) in the Gβ3 subunit
leading to a truncated protein has originally been
reported in association with primary hypertension.
More recently, genetic associations with a number of
other disorders such as obesity and insulin resistance
have been suggested. So far, the underlying mechanism
by which the Gβ3 variant causes the different
phenotypes remains elusive.
Conclusions
In the past few years significant progress has been
made towards a truly molecular understanding of
receptor/G-protein-mediated signal transduction. One
crystal structure of a heptahelical receptor, rhodopsin,
and several of G-proteins provide a solid foundation
for future work on the mechanisms of receptor and
G-protein activation. An important goal will be to
determine the structural differences between the
inactive and active receptor conformations as well as
the structure of receptors in complex with heterotri-
meric G-proteins. Studies on engineered gene-deficient
mice as well as the thorough in vivo and in vitro
characterization of naturally occurring G-protein muta-
tions detected in patients have taught us invaluable
lessons on the physiology of these cardinal signaling
proteins. Studying clinical and molecular aspects of the
different forms of PHP has highlighted the complex
regulation of Gαs expression and provided remarkable
insights into the basic mechanisms of genomic
imprinting. Our understanding of receptor and G-
protein-mediated signaling processes has shifted
from studying linear signaling cascades towards the
consideration of complex signaling networks which
will require novel collaborative research initiatives to
integrate bits and pieces of knowledge into a coherent
instructive model.
▶Cardiac Signaling: Cellular, Molecular and Clinical
Aspects
References
1. Gudermann T, Kalkbrenner F, Schultz G (1996)
Diversity and selectivity of receptor-G-protein interac-
tion. Annu Rev Pharmacol Toxicol 36:429–459
2. Schöneberg T, Schulz A, Gudermann T (2002) The
structural basis of G-protein-coupled receptor function
and dysfunction in human diseases. Rev Physiol
Biochem Pharmacol 144:143–227
3. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-
transmembrane receptors. Nat Rev Mol Cell Biol 3:639–
650
4. Palczewski K, Kumasaka T, Hori T et al (2000) Crystal
structure of rhodopsin: A G-protein-coupled receptor.
Science 289:739–745
5. Wall MA, Posner BA, Sprang SR (1998) Structural basis
of activity and subunit recognition in G-protein hetero-
trimers. Structure 6:1169–1183
6. Offermanns S (2003) G-proteins as transducers in
transmembrane signalling. Progr Biophys Mol Biol
83:101–130
7. Cabrera-Vera TM, Vanhauwe J, Thomas TO et al (2003)
Insights into G-protein structure, function, and regula-
tion. Endocr Rev 24:765–781
8. Spiegel AM, Weinstein LS (2004) Inherited diseases
involving G-proteins and G-protein-coupled receptors.
Annu Rev Med 55:27–39
9. Weinstein LS, Liu J, Sakamoto A et al (2004) GNAS:
normal and abnormal functions. Endocrinology
145:5459–5464
10. Bastepe M, Juppner H (2005) GNAS locus and
pseudohypoparathyroidism. Horm Res 63:65–74
GPx
▶Glutathione Peroxidase
G-Quartet DNA
Definition
G-quartet DNA (also know as G4 DNA) defines a four-
stranded DNA structure formed by nucleic acid rich
guanine/cytosine regions. This structure is highly
stabilized by a planar array of four hydrogen-bonded
guanine bases.
▶DNA Helicases
Graafian Follicles
Definition
Graafian follicles designate cellular components of the
ovary, each consisting of a germ cell (oocyte),
surrounded by somatic cells (follicle cells), and a large

fluid-filled cavity from which the unfertilized egg
emerges.
▶Mammalian Fertilization
Grade of Malignancy
Definition
Grade of malignancy designates the histomorphologi-
cal assessment of the malignant behavior of a tumor, as
estimated by cytological criteria such as nuclear
pleomorphism and number of mitoses, and histological
criteria such as the formation of differentiated struc-
tures. Usually, three grades (G1, well differentiated;
G2, moderately differentiated; and G3, poorly differ-
entiated; with increasing aggressiveness in this order)
are distinguished.
▶Breast Cancer
Granulation Tissue
Definition
Granulation tissue defines a new connective tissue that
is formed during the wound repair process and
temporarily replaces the lost dermal part of the skin.
The name derives from the granular appearance of
numerous new capillaries.
▶Wound Healing
Granuloma
Definition
Granuloma represents a chronic inflammatory lesion
initiated by various infectious and non-infectious
agents. Granuloma consists of either small, nodular
aggregations of mononuclear inflammatory cells or of
aggregations of different cells, usually modified
macrophages surrounded by lymphocytes and multi-
nucleated giant cells. Sometimes granuloma may also
contain eosinophils and B cells, and are surrounded by
fibrotic tissue.
▶Morbus Wegener
Green Fluorescent Protein 729
Granulomatosis
Definition
Granulomatosis refers to a multisystem disease that is
characterized by an inflammation of the blood vessels
(▶vasculitis) involving the upper and lower respiratory
tracts and variable degrees of systemic, small vessel
vasculitis, which is generally considered to represent a
hypersensitivity reaction to an unknown antigen.
▶Recombinant Protein Expression in Bacteria
G
GRAS
Definition
Generally Recognised As Safe: the US Congress
established this concept and regulatory policy in 1958
as part of its food safety legislation. Judged by qualified
experts, it means that ingredients or hosts are safe when
used in food or food production to accomplish their
technical or nutritional purposes.
▶Recombinant Protein Expression in Yeast
Grb2
Definition
Grb2 stands for Growth-factor-receptor-bound protein
2. It is an adaptor protein containing src homology
domains, one of which binds to and translocates the
guanine nucleotide exchange factors ▶SOS. It is
involved in activation of Ras, but can also play a role
in other signaling pathways in mammalian cells.
▶Ras Signalling Pathway
▶Signal Transduction: Integrin-Mediated Pathways
▶Tyrosine Kinase
Green Fluorescent Protein
Definition
GFP stands for Green Fluorescent Protein. It is a natural,
27 kDa fluorescent protein, originally produced by the
marine jellyfish Aequorea victoria, and fluoresces or
730 Greig’s Cephalopolysyndactyly
glows green visible light when excited by UV light (395
nm). GFP is commonly used in the laboratory for
labeling, detecting and tracking proteins and biological
processes. It can be cloned without co-factors in most
organisms, and is used as a reporter molecule in light
microscopic imaging in co-expression assays to visua-
lize cellular structures and molecules. The protein has
been mutated to generate blue, cyan, yellow, photo-
activatable, and monomeric variants.
▶C. Elegans as a Model Organism for Functional
Genomics
▶Electron Tomography
▶FCS
▶FRAP
▶Functional Assays
▶High-Throughput Approaches to the Analysis of
Gene Function in Mammalian Cells
▶Immunochemical Methods, Localization
▶Large-Scale Homologous Recombination Approaches
in Mice
▶Medaka as a Model Organism for Functional
Genomics
▶Transgenic and Knockout Animals
Greig’s Cephalopolysyndactyly
Definition
Greig’s cephalopolysyndactyly refers to a syndrome
that affects embryonic development of the limbs, skull
and face. Major features include polysyndactyly of the
hands and feet, broad thumbs, macrocephaly and a high
prominent forehead. This disorder is due to mutation of
the gene encoding the ▶GLI3 zinc finger transcription
factor which is involved in mediation of the ▶Hedge-
hog Signal Transduction Pathway.
▶Hedgehog Signalling
Growth Factor Receptors
Definition
Growth factor receptors are cell surface molecules that
bind growth factors, and initiate an intracellular signal
that affects gene transcription in cells.
▶Growth Factors
▶Receptor Serine/Threonine Kinase
▶Tyrosine Kinases
Growth Factors
U LF H EDIN 1 , J OY R OY 2
1
Department of Surgical Sciences,
Karolinska Hospital, and 2Department of Surgery,
St Gorans Hospital, Stockholm, Sweden
ulf.hedin@kirurgi.ki.se
Definition
Polypeptide growth factors are proteins with a funda-
mental role during embryogenesis and regeneration of
tissues. In contrast to some hormones, which regulate
growth of entire organisms, growth factors are essential
for replication of individual cells and to the main-
tenance of normal cell function. Some growth factors
stimulate cell division in numerous different cell types,
while others are specific for particular cells. Growth
factors mediate their effects on cells by binding to
specific surface receptors. Binding of growth factors to
their corresponding receptors activates signaling sys-
tems inside cells, which regulate transcription of genes
involved in cellular processes such as differentiation,
proliferation, migration, protein synthesis and metabo-
lism. In adult organisms, certain growth factors are
essential for the regeneration of cells in the bone
marrow and for tissue repair, whereas other factors
regulate the development and growth of tissues in the
embryo (see ▶Limb Development). In human disease,
growth factors contribute to the abnormal regulation of
cell proliferation found in cancer but also regulate
cellular processes in cardiovascular and inflammatory
diseases. The progressive accumulation of new in-
formation regarding the function of growth factors in
human health and disease is providing new alternatives
for future treatment strategies.
Characteristics
In 1986, Rita Levi-Montalcini and Stanley Cohen were
awarded the Nobel Prize in Physiology or Medicine for
their discoveries of the first identified growth factors,
nerve growth factor (▶NGF) and epidermal growth
factor (▶EGF). NGF regulates the development and
survival of neurons whereas EGF stimulates cell
proliferation in a number of different cell types. Ever
since, numerous other growth factors have been
identified and their functions in human development,
homeostasis and disease explored. These discoveries
have to a large extent been promoted by cancer
research, especially investigations into how viruses
cause tumors. Some viruses that infect cells cause
permanent mutations in the host cell DNA, which lead
to the expression of genes that disturb the internal
control of the cell’s ability to proliferate. These so

called ▶oncogenes can promote abnormal cell pro-
liferation, a hallmark of cancer cells, by changing the
function of any protein involved in the normal control
of cell replication. Thus oncogenes can lead to
abnormal expression of a particular growth factor,
altered expression of growth factor receptors, increased
growth factor receptor activity or any other disruption
of the intracellular machinery that regulates cell
division. These insights have led to the discovery of
individual growth factors, growth factor receptors and
intracellular signaling pathways that transmit growth
stimuli to the cell nucleus. These naturally expressed
growth regulatory proteins are called proto-oncogenes
and historically, they have been named after the virus
that gave rise to the growth disturbing mutation. For
example, the proto-oncogene that encodes for platelet-
derived growth factor (▶PDGF) was named sis after
the virus simian sarcoma virus, which induces
proliferation in infected cells through over-expression
of a PDGF-like protein.
The polypeptide growth factors include a wide variety
of signaling molecules that can be categorized into
several groups or families. They can be produced and
secreted by cells in order to act locally on neighboring
cells (▶paracrine function) or actually even on the
same cells that produced them (▶autocrine function).
Some growth factors are also produced in organs but
exert their action on target cells after being transported
in the blood to a distance from the source (▶endocrine
function).
One of the first growth factors characterized, EGF, is
found in salivary glands in the gastrointestinal tract and
promotes proliferation of a large variety of cells,
epithelial cells and mesenchymal cells included.
▶Erythropoetin is a growth factor produced in the
kidney, which stimulates proliferation of immature red
blood cells in the bone marrow. Some growth factors
are stored extracellularly in tissues or in cells and can
be released to stimulate cells in the immediate vicinity.
For example, fibroblast growth factor (▶FGF), a
member of a large family of growth factors, has the
capacity to be stored in tissues by binding to sugar
residues on proteins in the extracellular matrix, the so-
called proteoglycans, and can be released after injury to
participate in tissue repair by stimulating cell prolifera-
tion. Several members of the FGF family stimulate
proliferation of endothelial cells and participate in the
formation of new vessels, angiogenesis. FGF is also an
important growth factor in the developing embryo and
mutations in receptors for these growth factors have
been associated with several different bone disorders,
for example achondroplasia (dwarfism). There are
numerous other growth factors that are involved in
angiogenesis. Vascular endothelial cell growth factor
(▶VEGF) can be produced in tissues with a deficient
blood supply, for example after myocardial infarction,
Growth Factors 731
and selectively stimulate proliferation of endothelial
cells to recruit new vessels into the anoxic tissue.
VEGF can also be released from cancer cells and
stimulate growth of vessels from the surrounding
tissues, which ensures a blood supply in the growing
tumor. PDGF is stored in blood platelets and can act on
cells after platelet degranulation in association with
tissue injury, bleeding and blood clotting. Together
with other growth factors such as EGF, which is
also stored in platelets, PDGF acts locally on cells in
the injured tissue and promotes cell proliferation in the
healing process (Fig. 1). Insulin-like growth factor-1
(▶IGF I) chemically resembles the hormone insulin
and is mainly produced in the liver under the control of
growth hormone. During development, this growth
factor participates in the regulation of skeletal growth G
and maturation after birth, but it is also involved in
tissue repair and may be important for abnormal
proliferation of cancer cells. The transforming growth
factor family (▶TGF) is a large family of different
growth factors that were initially characterized by their
ability to transform normal cells into tumor cells in
culture. They have profound effects on cell metabolism
and cellular synthesis of extracellular matrix proteins,
and in some cell types they rather prevent than
stimulate cell proliferation. ▶Cytokines are a unique
family of growth factors, which primarily act on cells in
the immune system and stimulate proliferation of
lymphoid cells. Cytokines such as the interleukins
(▶IL), a large family with more than 20 members,
regulate proliferation on a variety of lymphocytes but
also affect differentiation and growth of cells in the
bone marrow.
The identification and characterization of growth
factors have yielded potential therapeutic tools for the
management of a large variety of human diseases. In
patients with chronic kidney failure, the metabolic
dysfunction of the organ will lead to anemia due to
insufficient production of erythropoietin. Today, pa-
tients suffering from this condition receive injections of
recombinant erythropoietin that stimulate production
of red blood cells in the bone marrow. A number of
other conditions where the bone marrow does not
produce enough blood cells can also be corrected
through the addition of specific growth factors. For
example, deficient production of white blood cells
in the bone marrow, a side effect of cancer treatment
with chemotherapy, can now be corrected through
injections of growth factors that specifically stimulate
proliferation of leukocytes. In some disorders, espe-
cially in cancer, pharmacological approaches are
taken to develop drugs that prevent the effects of
growth factors, for example drugs that interfere
with the binding between specific growth factors
and their corresponding receptors on the surface of
cells.
732 Growth Factors

Growth Factors. Figure 1 Growth factors regulate healing responses. After tissue injury, bleeding and
formation of a blood clot, growth factors can be released from degranulating platelets (1) and stimulate fibroblast
proliferation or growth of new blood vessels in the injured tissue. In addition, inflammatory cells recruited from
the blood stream can produce growth factors (2); they may be released from storage pools in the tissue
(3) or they may be released from proliferating cells (4).

Regulatory Mechanisms single transmembrane domain, an extracellular domain

Growth factors are secreted into the extracellular
environment of cells where they may come in contact
with specific receptors on the same cell that secreted
them, on nearby cells or on distant cells after
transportation through the blood stream. Polypeptide
growth factors usually bind with high affinity to their
corresponding receptors. The receptors are transmem-
brane proteins with an extracellular domain that has a
specific binding site for the growth factor. Upon
binding of the ligand, a signal is sent to the intracellular
domain, which initiates an intracellular signal trans-
mitted from the receptor to the cell nucleus through
specific signaling pathways. Once the signal has
reached the nucleus, a specific response is elicited in
the regulation of gene transcription and the cell will
express specific genes, for example genes involved in
the control of proliferation.
Growth factor receptors can be classified into three
different groups, receptor tyrosine kinases (▶RTK),
▶tyrosine-kinase-associated receptors, and G-protein
coupled receptors (▶GPCR). Most growth factor recep-
tors are RTKs. These growth factor receptors have a
that binds to the ligand and a cytosolic domain, which
harbors an enzymatic activity that catalyzes addition of
phosphate residues to the amino acid tyrosine, ▶tyrosine
kinase activity. Many growth factors, such as PDGF, are
dimers, two separate polypeptides linked to each other,
which bind to two separate receptor subunits at the cell
surface. This dimerization of the receptor subunits
facilitates tyrosine kinase activity in the cytosolic
domains whereby tyrosine residues are phosphorylated,
a phenomenon termed ▶autophosphorylation. Other
monomeric growth factors, such as FGF, may instead
form pairs by binding to sugar structures at the cell
surface and thereby achieve simultaneous binding of two
corresponding cell surface receptors and receptor dimer-
ization. Autophosphorylation of tyrosine residues in the
cytosolic domains of RTKs allows binding and initiation
of catalytic activity in intracellular proteins that transmit
signals to the nucleus through different pathways. These
signaling pathways generally function by a series of
kinases that step by step are phosphorylated and activated
one after the other so that the signal is propagated to the
final activation of gene expression in the nucleus (Fig. 2).
 Growth Factors 733

Growth Factors. Figure 2 Growth factors regulate gene transcription in cells. 1) Binding of a growth factor to a
corresponding receptor (receptor tyrosine kinase; RTK) on the cell surface induces dimerization of two receptor
subunits, which elicits phosphorylation (P) of tyrosine residues on the cytosolic domains of the receptor. 2) Specific
intracellular adaptor proteins (AP) bind to the receptor and facilitate activation of catalytic proteins, such as Ras,
which therafter activate intracellular signaling through the mitogen activated protein kinase (MAPK) pathway. 3) In
this pathway, phosphorylation of amino acid residues activates a series of kinases and the last activated protein in
the pathway enters the cell nucleus (4) where it induces activation of transcription factors (tf), which promote
expression of specific genes, for example genes coding for proteins necessary for cell proliferation.

Cytokines bind to tyrosine-kinase-associated receptors
that are structurally similar to RTKs but lack intrinsic
tyrosine kinase activity in their cytosolic domains.
Instead, these growth factor receptors are associated
with molecules that have tyrosine kinase activity.
Receptor binding of cytokines also facilitates dimeriza-
tion of receptor subunits, which then mediate phosphor-
ylation of associated tyrosine kinases. Activation of
these tyrosine kinases leads to phosphorylation of
tyrosine residues on the receptor, which provides
binding sites for downstream signaling molecules and
initiation of signaling cascades. The receptors of the
GPCR family are traditionally regarded as mediators of
signaling for substances that regulate specific physio-
logical responses in differentiated cells, for example
contraction and relaxation in muscle cells. Lately, it has
been understood that these receptors may also transmit
growth signals in less differentiated and proliferative
734 Growth Hormone Deficiency
cells after stimulation by factors not normally perceived
as growth factors, for example angiotensin II which
stimulates contraction of smooth muscle cells in the
vessel wall but also initiates proliferation in de-
differentiated smooth muscle cells in diseased vessels.
GPCRs have an extracellular domain that binds the
ligand, seven transmembrane segments and an intracel-
lular domain that associates with a guanine nucleotide-
binding protein (hence the name G-protein). Ligand
binding, for example of thrombin or angiotensin, to its
specific GPCR extracellular domain, leads to a con-
formational change in the cytosolic domain of the
receptor. The altered receptor structure facilitates
binding of G-proteins to the intracellular domain, which
in turn activates an enzyme attached to the plasma
membrane. This enzyme catalyzes a reaction leading to
the release of a second messenger, which can then
activate intracellular signaling molecules that reach the
nucleus and affects gene transcription.
Cascades of intracellular signaling molecules constitute
links between growth factor binding to a growth factor
receptor and expression of genes in the nucleus that
control cell proliferation. The ▶mitogen activated
protein kinase (MAPK) pathways are the most well
studied and understood intracellular signaling path-
ways, which are activated after binding of growth
factors to their corresponding cell surface receptors.
For example, binding of PDGF to the PDGF receptor
leads directly to receptor dimerization and autopho-
sphorylation of tyrosine residues on the cytosolic
domains of the receptor. Tyrosine autophosphorylation
provides binding sites for an adaptor protein called
Grb2. Grb2 in turn binds to another adaptor protein SoS
(son of sevenless) that can activate the small GTP
binding proto-oncogene protein Ras. Ras then phos-
phorylates a Map-kinase-kinase-kinase, which in turn
phosphorylates a MAP-kinase-kinase and then finally,
the last signaling molecule of the pathway, a MAP-
kinase (MAPK) is activated. After phosphorylation,
MAPK translocates into the nucleus and activates a set
of ▶transcription factors, which promote expression of
growth-related genes (Fig. 2).
The ▶cell cycle is divided into four phases, G0, G1, S, G2
and M. Non-malignant eukaryotic cells are normally
resting in the G0 phase. In cells that harbor the capacity to
proliferate, such as fibroblasts, growth factors stimulate
the cells to leave the G0 phase and enter the G1 phase.
When cells have been stimulated with growth factors for a
specific time period in the G1 phase, further progression
in the cell-cycle and cell division will inevitably follow,
even if the growth factor is removed. This so called
▶restriction point is followed by the S-phase where DNA
replication takes place, the G2 phase when factors
necessary for the physical division of the cell are
produced and finally the M phase when mitosis occurs.
Passage through the cell-cycle is controlled by periodic
changes in the expression and activity of families of cell-
cycle regulatory proteins termed ▶cyclins and cyclin
dependent kinases (▶cdk). The expression of some
cyclins is largely dependent on growth factors and
activation of growth factor receptors. After growth factor
stimulation, cyclins are synthesized, which form com-
plexes with cdks. These complexes catalyze massive
phosphorylation of the retinoblastoma protein, ▶Rb. In
resting cells, Rb is bound to the transcription factor E2F
but upon hyperphosphorylation, E2F is released and can
activate transcription of a number of genes necessary for
S-phase initiation and further cell-cycle progression.
References
1. Oxford Reference Online. Oxford University Press.
BIBSAM, 2004. ▶http://www.oxfordreference.com
2. Bast et al (2000) Cancer Medicine, 5th edn. BC Decker
Inc, Hamilton
3. Lodish et al (2003) Molecular cell biology, 5th edn. WH
Freeman and Co, New York
4. Alberts et al (1994) Molecular Biology of the Cell, 3rd
edn. Garland Publishing, New York and London
5. Heldin and Purton (1996) Signal transduction, 1st edn.
Chapman & Hall, London
Growth Hormone Deficiency
Definition
Growth hormone deficiency is caused by absence,
decreased production or dysfunction of the anterior
pituitary hormone, which results in dwarfism or short
stature and possibly some metabolic abnormalities
(such as hypoglycemia).
▶Hypothalamic and Pituitary Diseases Genetics
Growth Plate
Definition
The growth plate is a cartilaginous structure at the end
of bones that generates the entire longitudinal growth
through proliferation and differentiation of chondro-
cytes, and the conversion of cartilage into bone.
▶Bone Disease and Skeletal Disorders, Genetics

Gamma-Secretase (Complex)
▶γ-Secretase
GSK3
▶Glycogen Synthase Kinase–3
GST
Definition
GST stands for Glutathione-S-transferase, a 26.9 kDa
protein-fragment that is used to tag recombinant proteins.
▶Recombinant Protein Expression in Bacteria
GST Pull-Down Experiment
Definition
In this kind of assay, a recombinant affinity tag ▶fusion
protein is used as bait to capture (‘pull down’) binding
partners out of a cell lysate. The cell lysate is applied to the
immobilised bait protein, or, alternatively, bait protein and
lysate are mixed in solution and complexes are captured
by affinity chromatography afterwards. Glutathion-S-
transferase (GST) is often used as the affinity tag.
▶Affinity Chromatography and In Vitro Binding
(Beads)
GTP
Definition
GTP stands for Guanosine 5′–triphosphate. It is
produced by phosphorylation of GDP (guanosine 5′–
diphosphate).
▶Rho, Rac, Cdc42
Guanine Nucleotide Exchange Factors 735
GTPase-Activating Proteins
Definition
GTPase-activating proteins (GAPs) comprise of pro-
teins that bind to a GTP-binding protein and inactivate
it by stimulating its GTPase activity so that it
hydrolyzes its bound GTP to GDP.
▶Rho, Rac, Cdc42
GTPases
G
Definition
GTPases comprise of a large group of intracellular
signaling proteins characterized by an active state when
GTP-bound, and an inactive state when GDP-bound.
Their enzymatic activity hydrolyzes GTP.
▶Cardiac Signaling: Cellular, Molecular and Clinical
Aspects
▶Diabetes Insipidus, a Water Homeostasis Disease
▶Rho, Rac, Cdc42
Guanine Nucleotide Dissociation
Inhibitors
Definition
Guanine nucleotide dissociation inhibitors (GDIs) are
protein factors that inhibit the dissociation of guanine
nucleotides (GDP/GTP) from GTPbinding proteins.
▶Rho, Rac, Cdc42
Guanine Nucleotide Exchange Factors
Definition
Guanine nucleotide exchange factors (GEFs) are
proteins that catalyze the release of guanine nucleotides
(mostly GDP) from monomeric or heterotrimeric
GTPases, thereby allowing them to bind GTP in its
place. In the latter case, heptahelical receptors serve as
GEFs.
▶G-Proteins
▶Rho, Rac, Cdc42
▶Tight Junctions
736 Gut Epithelium
Gut Epithelium
M ICHÈLE K EDINGER , J EAN -N OËL F REUND
INSERM U682, Strasbourg, France
michele.kedinger@inserm.u-strasbg.fr
Definition
The intestinal epithelium lines the gut lumen, dividing
the outside from the interior of the body. It is implicated
in most digestive and absorptive functions of the gut,
but also in the immunological and non-immunological
protection against nutritional, bacterial, viral and
parasite aggressions. Its importance in terms of
evolution is demonstrated by its presence early in the
animal kingdom long before the spinal cord or neuronal
cells. The intestinal epithelium forms invaginations
into the stroma, the crypts, and outwardly projecting
villi in the small intestine or flat cuffs in the colon.
Taking into account the length of the gut (5–6 m in
humans), these structures, and additional microscopic
foldings at the apical membrane of epithelial cells, the
microvilli, provide an overall absorptive surface of
about 300 m2. The gut epithelium exhibits a morpho-
logical and functional regionalization along the prox-
imo-distal axis, mainly obvious between the small
intestine and colon.
Characteristics
1. The gut epithelium develops in interaction with
multiple partners.
The intestinal epithelium derives from the mid and
posterior parts of the definitive endoderm (midgut and
hindgut), which itself originates from the ingression of
primitive streak cells at gastrulation. Specification of
the presumptive intestinal endoderm is driven by
interactions with lateral mesoderm. Morphogenesis of
the gut generates the single layer columnar epithelium
laid on and separated from lamina propria myofibro-
blasts by a ▶basement membrane, which is a
specialization of the ▶extracellular matrix. Intestinal
epithelial cells also interact with lymphocytes (either
intra-epithelial lymphocytes or organized into sub-
jacent nodules), and with neuronal cell processes.
On the luminal side, nutrients and the intestinal
microflora are able to signal and modulate the
epithelial cell behavior.
2. The gut epithelium is highly polarized.
Intestinal epithelial cells exhibit an asymmetrical
morphology shaped by a highly organized network
of microtubules, intermediate filaments and actin
filaments connected to the plasma membrane.
Apical-lateral ▶junctional complexes (tight and
adherent junctions) link the cells together, regulate
the barrier function, and delineate two compart-
ments of the plasma membrane, the apical and the
basolateral membranes. These complexes are com-
posed of at least 40 different proteins, among which
are transmembrane proteins such as occludin,
claudins and cadherins, and cytoplasmic proteins,
among which are some which can shuttle to the
nucleus like the transcription factor ZONA B
associated to ZO-1 and β-catenin which connects
E-cadherin to actin filaments. The apical membrane,
called the brush border, is formed by microvilli
sustained by an actin core associated to a number of
cytoskeletal proteins among which is villin, which
regulates the plasticity of the brush border. The
functional proteins (digestive enzymes, outside-in
transporters) are anchored into the brush border
membrane. The basolateral membrane carries in-
side-out transporters and ion channels involved in
the processing of nutritional metabolites, but also
receptors to basement membrane molecules. These
receptors, including ▶integrins and ▶ dystroglycan,
ensure the link with the basement membrane and
also act synergistically with growth factor receptors
to initiate intracellular signaling and to participate in
the regulation of cell functions.
3. The gut epithelium is composed of several func-
tionally different cell types.
The main cell type in the small intestine (about
90%) is a columnar absorptive cell responsible for
the digestive functions, the enterocytes. Additional
cytotypes belong to the secretory cell lineages and
comprise (i) mucus-producing goblet cells, which
produce the protective mucus layer on top of the
epithelium, (ii) entero-endocrine cells composed of
at least 15 different cell subtypes classified on the
basis of their hormonal content, which control
important physiological functions such as glycemia,
exocrine pancreatic secretion and growth/repair of
the gut epithelium and (iii) Paneth cells, which are
involved in the mucosal defense function and
produce a variety of antibiotic and antimicrobial
factors. The last cytotype is composed of M cells,
which correspond to a specialization of absorptive
cells facing lymphoid nodules and participate in the
control of intestinal immunity and tolerance. Mucous
cells represent the major cell type in the colon.
4. The gut epithelium is continually and rapidly
renewed throughout life.
The adult intestinal epithelium is compartmenta-
lized. Stem cells confined near the crypt bottom
generate proliferative transit amplifying cells, which
cycle every 12 h, and migrate vertically towards the
crypt mouth. When leaving the crypts, epithelial
cells abruptly stop proliferating and become differ-
entiated. All the differentiated cell types – except the

Paneth cells, which migrate downwards to the crypt
bottom – migrate to the villus tip in the small
intestine or to the surface of the colon cuffs, and
subsequently undergo apoptosis and exfoliation into
the gut lumen. The overall life cycle of the gut
epithelial cells is around 5 days in humans.
5. Multipotent stem cells are maintained in the adult
gut epithelium.
The continuous renewal of the gut epithelium
implies the presence of stem cells located in a
specific niche near the crypt bottom surrounded by
sub-epithelial myofibroblasts and specific extracel-
lular molecules. Although intestinal stem cells have
still not been isolated, several experimental ap-
proaches – transgenesis, mouse embryo aggregation
chimeras, mutagenesis, regeneration after X-ray
irradiation – have provided evidence that all the
differentiated cell types of the gut epithelium derive
from ▶multipotent stem cells (1, 2). Stem cells
undergo asymmetrical division to stochastically
produce one new self-maintaining stem cell and
one daughter cell that cycles and fuels a population
of potential clonogenic stem cells. These cells are
further displaced into the compartment of transit
amplifying cells that eventually go into the
differentiation process. Potential clonogenic stem
cells, unlike transit amplifying cells, can replace true
stem cells if this population is altered. The process
towards differentiation uses two minor pathways
consisting of long-lived progenitors of absorptive
and of mucous cells respectively and one major
pathway in which short-lived progenitors can
produce both absorptive and mucous short-lived
progenitors. Interestingly, glucagon-like peptide-2
produced by one of the enteroendocrine cell types –
known to prevent intestinal damage and to facilitate
repair – signals through enteric neurons to produce a
mediator that stimulates the production of long-
lived progenitors of the absorptive lineage (2).
6. Malignant epithelial tumors develop in the colon
and rectum.
Colorectal cancer is a major disease in terms of
incidence and malignancy. It results from imbal-
anced cell proliferation, differentiation, migration
and apoptosis in colonic crypts. The vast majority of
tumors are of sporadic origin while a small
proportion is familial. The major familial form,
▶Hereditary Non-Polyposis Colon Cancer
(HNPCC), is characterized by microsatellite DNA
instability resulting from germ line mutations in the
MLH1 or MSH6 genes, which cause defects in
the DNA mismatch repair system. The second
familial form, ▶Familial Adenomatous Polyposis
(FAP), is characterized by chromosomal instability
and is linked to germ line mutations in the tumor
suppressor gene ▶APC. Loss of function of
Gut Epithelium 737
APC leads to inappropriate activation of the Wnt/
β-catenin signaling pathway (3) and to chromoso-
mal instability linked to altered kinetochores. Rare
cases of FAP without germ line mutation of APC are
associated to biallelic germ line mutations in the
base excision repair gene MYH. The sporadic form
of colorectal cancer is mainly related to chromoso-
mal instability and to gradual histological changes,
the adenoma-carcinoma sequence, associated to the
accumulation of somatic alterations in a number of
tumor suppressor genes (APC, p53) and oncogenes
(K-ras, Bcl2). Malignant tumors occur almost
exclusively in the colon and rectum, while atypical
tumors develop in the small intestine in the context
of chronic inflammatory bowel disease. The pri-
mary incidence of tumors in the colon correlates G
with the colon specific expression of the anti-
apoptotic gene Bcl2 at the stem cell positions in the
crypt base.
Regulatory Mechanisms
1. Intestinal epithelial identity and homeostasis: in-
volvement of the ▶Cdx genes.
▶Homeobox genes belong to a large family of
transcription factors acting at multiple levels during
embryonic development. Cdx1 and Cdx2 are two
paralogue homeobox genes, which are expressed in
the presumptive gut endoderm and in posterior
organs in the embryos, and specifically in the gut
epithelium throughout adult life. Cdx2 displays the
homeotic function devoted to defining the intestinal
identity (4). Indeed, ectopic expression of Cdx2 in
the stomach epithelium – normally devoid of Cdx
gene expression – converts gastric mucosa to an
intestinal phenotype, whereas loss of expression in
the gut endoderm leads to a gastric transdifferentia-
tion. Cdx1 and Cdx2 are also modulators of cell
renewal via distinct and complementary functions;
Cdx1 stimulates cell proliferation, resistance to
apoptosis and eventually cell differentiation,
whereas Cdx2 reduces cell proliferation and stimu-
lates differentiation. The Cdx1 and/or Cdx2 proteins
act directly on a panel of target genes and cellular
functions including regulators of cell cycle and
apoptosis (p21WAF, Bcl2), transcription factors
(KLF4), proteins involved in cell interactions (LI-
cadherin), in calcium metabolism (vitamin D
receptor, calbindin-D9k) and in glucose metabolism
(glucagon, glucose-6-phosphatase), digestive en-
zymes (sucrase, lactase) and mucus production
(Muc2). Interestingly, the expression profiles of
Cdx1 and Cdx2 are altered in colorectal cancers
and pro-oncogenic pathways have opposite effects
on the two homeobox genes. For instance Cdx1
is upregulated by the Wnt/β-catenin pathway,
738 Gut Epithelium
whereas Cdx2 is down-regulated by the ▶PI3K/Akt
pathway. These observations suggest that Cdx1 and
Cdx2 dysfunctions contribute to cancer progression.
In accordance with this, the alteration of the Cdx2
status sensitizes the colon epithelium to carcinogen-
esis, linked to a lower capacity to switch on
apoptosis. Thus, in addition to its homeotic function
during embryonic development of the digestive
tract, the Cdx2 homeobox gene is a gut-specific
tumor suppressor gene in the adult colon.
2. The Wnt/β-catenin signaling pathway.
Cell growth and polarity are major attributes of the
gut epithelium, and the Wnt/β-catenin pathway
plays a pivotal role in this balance. β-catenin
contributes to cell polarity by cross-linking the
membrane E-cadherin to the actin cytoskeleton. In
differentiated epithelial cells, an excess of β-catenin
molecules not coupled to E-cadherin are loaded on a
complex comprising APC, Axin and the CKI and
GSK3β kinases that phosphorylate β-catenin and
target it to the proteasome degradation system.
During intestinal development as well as in the
crypts, the Wnt/β-catenin signaling pathway is
activated by secreted morphogens of the Wnt family
that bind to Frizzled receptors and activate several
downstream pathways, one of which leads to the
inhibition of GSK3β activity. In this context, β-
catenin escapes degradation and translocates into
the nucleus to bind HMG-box transcription factors
of the Tcf/Lef family. These factors play a major role
in the maintenance and self-renewal of the stem cell
stock, since Tcf4-deficient mice lack proliferative
cells in the prospective intervillous regions; in
contrast over-expression of Lef1 causes increased
stem cell apoptosis. Major targets of the activated
Wnt/β-catenin pathway are the proto-oncogene c-
myc and cyclin D1, which subsequently down-
regulates the cell cycle inhibitor p21WAF (5).
Interestingly, a transcriptomic approach has identi-
fied a set of genes of the c-myc cascade in the stem
cells/progenitors compartment, including regulators
of c-myc gene transcription and protein stability and
c-myc downstream targets (6). As mentioned above,
a direct Wnt/β-catenin tissue-specific target is Cdx1,
and an indirect target is Cdx2, through another
HMG-box factor, SOX9. Finally, the Wnt/β-catenin
pathway also controls crypt cell sorting via the
regulation of combinations of ephrin receptors
EphB2/EphB3 and their ligands EphrinB1/
EphrinB2 (5).Thus, crypt formation, cell sorting
and the mechanism of stem cell selection appear to
depend on an adequate threshold of β-catenin-
mediated signaling during normal intestinal home-
ostasis, whereas inappropriate activation of this
pathway contributes to colorectal tumorigenesis.
Cooperating with the WNT/β-catenin pathway,
BMP (Bone Morphogenetic Protein) signaling and
Hh (Hedgehog) signaling also control the prolifera-
tion/differentiation equilibrium of the gut epithelium
(7, 8, 9).
3. Molecular determinants of gut stem cells and
progenitors.Although gut stem cells and progenitors
have been described near the crypt base, molecular
markers remain elusive. Yet, a gene product, Msi1,
has been reported in intestinal crypts, specifically in
individual cells that display the theoretical location
attributed to stem cells (1). Msi1 is an RNA-binding
protein associated with asymmetrical division of
neural progenitors. Msi1 controls the translation of
several RNAs; in particular, it acts as a translational
repressor of numb, an antagonist of Notch signal
activation. The Notch pathway is involved in the
maintenance of an undifferentiated state by a lateral
inhibition mechanism by which a cell differentiating
along a given pathway produces a signal that
prevents neighboring cells from differentiation
along the same pathway. Upon activation by their
ligands, the intracellular domains of Notch receptors
are released and translocated into the nucleus where
they bind to CSL (CBF1/Suppressor of hairless/
Lag1) DNA binding proteins. This results in the
transcriptional activation of downstream targets
encoding ▶bHLH transcription factors of the Hes
family, among which is Hes1. These are negative
regulators of differentiation, by repressing other
bHLH genes that promote differentiation. Indeed, a
series of gene invalidations led to the conclusion
that cell fate commitment in the intestine depends
essentially on a genetic cascade controlled by bHLH
transcription factors. Firstly, Hes1, which is ex-
pressed in crypt cells like Math1 and Ngn3,
maintains the precursor pool expansion and pre-
vents premature endocrine and mucous cell differ-
entiation. Secondly, Hes1 antagonizes Math1,
which is required for the commitment of the three
secretory cell lineages of the gut epithelium (goblet,
endocrine and Paneth cells). This suggests that the
choice between absorptive and secretory cell
lineages is balanced by Hes1 and Math1 (2).
Thirdly, within the population of Math1 expres-
sing-cells, Ngn3 specifies the endocrine progenitors
(10), whereas NeuroD, which is expressed in the
villus cells, is required for the differentiation of a
subset of endocrine cells. Genetic modulations of
the Notch pathway have confirmed its influence on
the maintenance of undifferentiated, proliferative
cells in the crypts and on intestinal cell lineage
specification by acting on the balance between
bHLH factors (11).
In conclusion, the renewal of the gut epithelium has
long been recognized as a paradigm in cell biology as

regards well-ordered cell proliferation followed by
differentiation from self-renewing stem cells. Recent
investigations have provided new insights into the
molecular mechanisms implicated in multiple aspects
of this process, such as the determination of intestinal
identity, the cell commitment into differentiated
lineages. Emerging concepts propose integrative mod-
els involving the interplay of local and reciprocal
stimulatory and inhibitory signals between epithelial
cells and the underlying myofibroblasts, to fine-tune
the homeostatic balance between stemness, commit-
ment, proliferation and differentiation (7, 9). Further-
more, these results enlarge our knowledge of the
molecular alterations at the basis of malignant
transformation in ▶colorectal cancers. However, the
exact nature of the gut stem cells and their relationship
and regulation by neighboring elements of the stem cell
niche remain to be elucidated. Understanding the
biology of the gut stem/progenitor cells is a challenge
for the future that should open new avenues in the field
of cancer therapy, intestine regeneration and cellular
therapy of type-1 diabetes.
References
1. Booth C, Potten CS (2000) Gut instincts: thoughts on
intestinal epithelial stem cells. J Clin Invest 105:1493–
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3. Giles RH, van Es JH, Clevers H (2003) Caught up in a
Wnt storm: Wnt signaling in cancer. Biochim Biophys
Acta 1653:1–24
4. Freund J-N, Domon-Dell C, Kedinger M et al (1998) The
Cdx1 and Cdx2 homeobox genes in the intestine.
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The beta-Catenin/TCF-4 complex imposes a crypt
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features of adult mouse small intestinal epithelial
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