Chap.

5 Molecular Genetic Techniques

(Part C)
Topics
• Identifying and Locating Human Disease Genes
• Inactivating the Function of Specific Genes in Eukaryotes

Goals
Learn genetic and
recombinant DNA
methods for isolating
genes and
characterizing the
functions of the
proteins they encode.

Use of RNA interference (RNAi) in analysis

of planarian regeneration
 Overview of Identifying & Locating
Human Disease Genes
Human geneticists use multiple
approaches to find genes
responsible for inherited
diseases. Typically, the family
inheritance pattern of the
disease initially is determined
(Fig. 5.35). Subsequently,
researchers try to find genetic
markers (e.g., SNPs, Fig. 5.36)
that consistently are linked to
the disease. This information
and sometimes linkage
disequilibrium analysis (Fig. 5.37)
further helps investigators home
in on the gene's approximate
chromosomal location. Finally,
DNA sequencing and other
procedures are used to pinpoint
the location of the responsible
gene. The relationship between
the cytogenetic and physical
maps of a human chromosome is
illustrated in Fig. 5.38.
 Common Hereditary Human Diseases
Most inherited diseases
are caused by preexisting
mutant alleles that have
been passed down from
one generation to the
next. Examples of common
autosomal recessive,
autosomal dominant, and *
X-linked recessive human
diseases of monogenic
origin are listed in Table
5.2. At least some
fraction of diseases such
as cancers, diabetes,
obesity, and heart disease *
are hereditary and
polygenic in origin. The *
molecular bases of these
diseases are even harder
to solve than those of
monogenic diseases.
 Human Disease Inheritance Patterns
One of the first procedures
performed in identifying a
disease gene is to establish its
inheritance pattern in affected 50%
families (Fig. 5.35). Analysis
of inheritance patterns can
quickly establish if the disease
gene is carried on an autosome
or a sex chromosome.
Examples of inheritance 25%
patterns (family segregation)
for autosomal dominant
(Huntington's disease),
autosomal recessive (cystic
fibrosis), and X-linked 50%
recessive (Duchenne's muscular
dystrophy) genetic diseases are
illustrated in the diagram. In autosomal dominant diseases,
male and female children each have a 50% chance of developing
the disease. In autosomal recessive diseases, both sexes have
a 25% chance of developing the disease. In X-linked recessive
diseases, males have a 50% chance of developing the disease.
In this case, the defective allele is inherited from the mother.
Human Karyotype: Metaphase Chromosomes
Linkage Mapping Using Molecular Markers
The next step in gene ID is to genetically map its position with
respect to known genetic markers in the genome. This method
can be performed by breeding studies in simple experimental
organisms in which genetic markers confer readily detectable
phenotypes. However such phenotypic markers are uncommon in
humans, and instead DNA-based molecular markers are used.
Molecular markers caused by DNA polymorphisms (sequence
differences) occur at a frequency of about 1/1,000 nucleotides.
Polymorphisms are used as landmarks in locating the position of
a disease gene. In some cases, polymorphisms change the
locations of restriction sites. This results in restriction
fragment length polymorphisms (RLFPs) which can be used in
linkage studies. Other DNA polymorphisms do not affect
restriction sites. These molecular markers--called single
nucleotide polymorphisms (SNPs) and simple sequence repeats
(SSRs)--can be identified and studied by PCR amplification and
sequencing of genomic DNA.
 SNP Analysis of Pedigrees
The use of SNP analysis in following disease gene inheritance in a
family is illustrated in Fig. 5.36. In the family shown, the region
of the chromosome being studied occurs in 3 forms based on the 3
different SNPs observed via sequencing of this region (A, T, or
C). The analysis indicates that the disease trait segregates with a
C at the SNP site.
Currently, about 104 DNA
polymorphisms have been
mapped in the human
genome. Researchers screen
DNA from individuals with
genetic diseases to try to
find polymorphisms that are
linked to a disease gene.
Pooled data from multiple
families helps in identifying
polymorphisms linked to the Key: Circles indicate females; squares
indicate males. Blue indicates
gene. SNP analysis also is unaffected individuals; orange
used in diagnostics and indicates individuals with the disease.
genetic counseling.
Linkage Mapping of Disease Gene Location
by Recombination Analysis
Although it is not commonly used in
analysis of human diseases, it is
instructive to consider the procedure
known as recombination analysis (Fig.
5.10), which is often applied in
linkage analysis in simple model
organisms. This method relies on the
facts that, phenotypic traits that
segregate together during meiosis
more frequently than expected based
on random segregation typically are
specified by genes residing on the
same chromosome. In addition, the
less frequently recombination occurs
between two markers on a
chromosome, the more tightly they
are linked and the closer together
they are. One genetic map unit is
defined as the distance between two
genes along a chromosome that
results in a 1% (1/100 gametes)
recombination frequency (1
centimorgan, cM). In humans, 1 cM
corresponds to a physical distance of
~750,000 bp.
Linkage Disequilibrium Analysis of Disease
Gene Location
In linkage disequilibrim
analysis, investigators map
the location of a disease gene
on a chromosome by looking
for regions that maintain the
same grouping of linked
genetic markers as in the
ancestral chromosome in
which the disease gene arose
(Fig. 5.37). Markers that are
unlinked to the mutant gene
recombine extensively over
generations, whereas markers
closely linked to the disease
allele do not. Linkage
disequilibrium analysis can
sometimes improve the
resolution of genetic mapping
studies to 0.1 cM.
 Final Steps of Mutant Gene Isolation
Often mutations can be
mapped only to 1 cM regions
of human DNA using the
methods discussed above (Fig.
5.38). Regions of this length
can contain dozens of genes.
The final identification of the
disease gene typically involves
sequencing and mapping of all
SNPs, etc. in a long region of
DNA. The responsible gene is
likely to be located in regions
where SNPs associated with
the disease consistently are
found in a number of affected
individuals. The mutation itself
eventually is identified by
DNA sequencing. The analysis
of gene expression by
Northern blotting and in situ
hybridization in affected
tissues also may help in
identifying a disease gene in
cases where grossly defective
mRNA transcripts are
produced from a gene.
Leptin Receptor Knockout Mice

db/db DB/DB
Gene Replacement by Homologous
Recombination
In bacteria and yeast, specific genes
can be replaced or disrupted by
homologous recombination methods
(Fig. 5.39). To disrupt yeast genes, a
disruption construct consisting of the
kanMX antibiotic resistance marker
fused to 20-nt sequences that flank
the targeted gene is made by PCR
and transformed into diploid yeast.
Recombinants in which the disruption
construct has replaced one wild type
allele are selected by plating cells on
G-418. On sporulation, half of the
haploid spores receive the disrupted
gene. If the gene is essential, these
two spores will be nonviable. This
method has been used to show that
only 1,500 of the total 6,000 yeast
genes are essential at least under
laboratory conditions. Synthetic
lethality screens are now being
conducted to group genes with
redundant functions.
Knockout Mice: Stem Cell Manipulations I
In knockout mice, both copies
of a gene of interest are
disrupted by targeted
homologous recombination.
First, a DNA construct
containing a disrupted allele
of a particular gene (X) is
introduced into embryonic
stem (ES) cells (Fig. 5.40a).
In a small percentage of
cells, homologous
recombination occurs and the
target gene is replaced with a
copy of the neo r gene (G-
148 resistance). In most
cells, the DNA construct
recombines nonhomologously
and inserts at another site in the genome. The entire DNA
construct typically inserts into the genome via nonhomologous
recombination. This results in cointegration of neo r and a copy
of the herpes simplex virus thymidine kinase gene (tkHSV).
Knockout Mice: Stem Cell Manipulations II
ES cells carrying the neo r
gene then are selected by
growing the mixed
population of cells on G-
148 (positive selection)
(Fig. 5.40b).
Subsequently, cells
obtained by nonhomologous
recombination are
eliminated (negative
selection) by growing cells
in the presence of the
nucleoside analog,
ganciclovir. Ganciclovir is
converted into a toxic
nucleotide by the tkHSV
gene product which blocks
DNA replication in these
cells. The resulting
culture contains only ES
cells with a targeted
disruption in one copy of
gene X (genotype: X-/X+).
Knockout Mice: In Vivo Manipulations I
In the next step, the ES
cells are injected into the
blastocoel cavity of a 4.5-
day old mouse embryo (Fig.
5.41). The embryo then is
introduced into a
pseudopregnant female
(foster mother) who gives
birth to some chimeric
progeny containing the
knockout gene. In chimeras,
different tissues have
different genotypes.To
facilitate isolation of
genetically pure lines of
knockout mice, the original
ES cells are obtained from
dominant brown (A) mice of
the A/A, X-/X+ genotype. A
recessive black (a) mice of
the a/a, X+/X+ genotype is
the source of the blastocyst.
Homozygous knockout mice
are isolated after performing
the crosses shown on the
next two slides.
Knockout Mice: In Vivo Manipulations II
In the first cross, a chimeric
mouse is mated with a black
mouse that is homozygous for
the wild type X allele. Possible
germ cells from the chimeric
mouse are shown in the figure.
The brown progeny of this
cross will be derived from the
ES cells. Repeated matings and
genotype screenings are
performed until X-/X-
homozygous knockout mice are
obtained.
In many cases, germ-line
knockout mice are nonviable.
Thus, alternative strategies for
knocking out a gene of interest
in a later stage of life have
been developed (Fig. 5.42, not
covered).
Transgenic Mice
In knockout mice, both copies of
a wild type allele are inactivated
to observe a recessive trait. In
transgenic mice, an allele
specifying a dominant negative
mutation is introduced into the
germ line DNA. Animals need
express only one copy of such
an allele to exhibit a phenotype.
The method for introduction of
transgenes into mice is shown in
Fig. 5.43. A copy of the
dominant negative allele typically
inserts into the genome via
nonhomologous recombination.
Often the transgene is
controlled by a regulated
promoter, allowing it to be
expressed in certain tissues at
certain times.
 Example of a Dominant Negative Allele
Only one copy of a
dominant negative
allele is required to
cause a loss-of-
function phenotype in
a diploid organism. A
classic example of
this type of mutation
is shown in Fig. 5.44.
Certain mutant forms
of "small GTPases"
form extremely long-
lived complexes with
GEF proteins (guanine
nucleotide exchange factors). GEF proteins catalyze exchange of
GTP for GDP and activation of GTPases. In cells expressing both
the wild type and dominant-negative GTPase alleles, all copies of
GEF become complexed with the mutant GTPase, blocking the
switching function of the wild type GTPase.
 RNA Interference (RNAi)
In RNA interference, short
interfering RNAs (siRNAs, ~21
nts) produced from longer dsRNAs
specifically block gene expression
by binding to a target mRNA and
triggering its degradation.
dsRNAs can be transcribed in
vitro and injected into an embryo,
for example, where processing by
the enzyme known as dicer
produces the siRNA (Fig. 5.45 a
& b). Alternatively, dsRNA can be
expressed in vivo in response to
some signal. Subsequent
processing to siRNA by dicer then
triggers mRNA degradation (Fig.
5.45c). RNAi-mediated gene
inactivation is commonly applied to
silence gene expression in C.
elegans, Drosophila, plants, and
even mice. The mechanism by
which siRNAs cause mRNA
degradation is covered in Chap. 8.