BMB 515 Electronic Course Pack Sessions 21 - CA

Post-translational Modifications
In this lecture we will describe post-translational modifications and intracellular trafficking of proteins,
using primarily eukaryotic models.
Suggested Reading:
Ferrier: Chapter 14: Glycosaminoglycans, Proteoglycans, and Glycoproteins - from Oligosaccharide

Structure through Glycoproteins SynthesisChapter 19: Amino Acids: Nitrogen Disposal - Overall
Nitrogen Metabolism - Protein Turnover only; Chapter 32: Protein Synthesis - Co-And
Posttranslational Modifications only.
Objectives:
1. Describe the processes used to synthesize membrane-bound and secreted proteins (signal hypothesis,
signal peptide, signal recognition particle, signal peptidase, and stop-transfer sequence).
2. Understand the mechanisms of the different types of post-translational modifications (amino acid
modification - phosphorylation, acetylation, hydroxylation, ADP-ribosylation, and covalent
attachment of fatty acids; glycosylation - O-linked and N-linked; and site-specific proteolysis -
insulin activation, zymogens and polyproteins) and how these modifications can change protein
functions.
3. Explain how protein degradation is accomplished in the cells (ubiquitin, lysosome, proteasome).
Measurable Outcomes: What you should be able to do...
I. Protein Localization
1. List the sequence of events (and cellular locations) proposed by the "signal hypothesis" for
synthesis of membrane-bound and secreted proteins. (Obj. 1)
2. Define and describe the roles of signal peptide, signal recognition particle, ER, ribosome, signal
peptidase, and stop-transfer sequence. (Obj. 1)
3. Describe the mechanisms used for targeting proteins to lysosomes, nucleus and mitochondria.
(Obj. 1)
II. Post-translational Processing

1. Describe the structures and biological roles of the following chemical modifications of amino
acids: (Obj. 2)
A. phosphorylation.
B. acetylation.
C. hydroxylation.
D. ADP-ribosylation.
E. acylation (myristylation, palmitoylation).
2. Compare and contrast O-linked and N-linked glycosylation with respect to (Obj. 2)
A. amino acids that are modified.
B. structure of oligosaccharides.
C. mechanism of sugar attachment.
3. Identify where the different types of glycosylation takes place in the cell. (Obj. 2)
4. Describe the role of proteolysis in maturation of proteins (as for insulin and digestive enzymes).
(Obj. 2)
III. Protein Degradation
1. Differentiate between the two main protein degradation methods (lysosome v. proteosome). (Obj.
3)
2. Describe the roles of secreted proteases and the lysosome in degrading extracellular proteins.
(Obj. 3)
3. Describe the roles of ubiquitin and proteasomes in degrading intracellular proteins. (Obj. 3)
I. Protein Localization
Topology: what it means to be effectively inside or outside" of the cell!
Anything that crosses an intact

membrane from the cytoplasm is
topologically equivalent to "outside
o f the cell.
(S. Triezenberg and J. Wang, MSU)

A. Secreted Proteins and the signal hypothesis:
Pushing a protein across a membrane during translation.
1. Signal peptide (or signal sequence) consists of the

first 20 or so amino acids of the peptide and are hydrophobic in nature.
2. Signal recognition particle binds to the signal peptide as it is synthesized while it is

still attached to the mRNA/ribosome and docks on the RER membrane
3. Co-translational transport: As the ribosome continues translation, it pushes the

nascent polypeptide through the lipid bilayer of the ER on the basis of
hydrophobicity.
4. Signal peptidase cleaves off the signal peptide by proteolysis in the lumen of the ER
5. Result: Protein in lumen of the ER = outside the cell. It can then traverse the
endomembrane secretion pathway (ER. Golgi, secretory vesicles, plasma membrane)
during which it can be glycosylated, transported, and sorted.
B. Integral Membrane Proteins: the signal hypothesis revisited
1. Uses a signal peptide, SRP, and peptidase just as above
2. stop transfer signal: A sequence of very hydrophobic amino acids that stops the
extrusion through the lipid bilayer.
Cytosol
Open
translator*
Signal
p&ptidase
ER Inman
Figure 13-11
M a fa m la r C#iVBiology, ShtFJi
< 2CC6W.Hlwnun .indComfvmy
3. Result: part of the protein is in the lumen of the ER (will face the outside of the cell),
part of the protein is in the cytoplasm (cytoplasmic domain)
part remains within the membrane (transmembrane domain)
This is the definition of a RECEPTOR!
C. Peripheral (tethered) Membrane Proteins

Utilize covalently attached lipids.
D. Targeting Signals for localization to different subcellular compartments.

These signals can be based on sequence, 3-D structure, or post-translational
modifications! High level of diversity creating many possible signals.
Targeting Signal Chemical Nature Receptor for Signal Activitv

Nuclear Localization Basic residues im portin-a im port into nucleus
Signal (NLS) (e.g.PPKKKRKV) importin-p
Nuclear Export leucine-rich Exportin-1 export from nucleus

Signal (NES) (e.g. LPPLERLTL)
Mitochondrial amphipathic helix involves receptors, im port into mitochondrial

signal peptide peptidases, ATP, and membrane or matrix
(Matrix Targeting Electrochemical
Signal) Gradient
Signal Sequence stretch of (~15) Signal Recognition im port into ER lumen

hydrophobic Particle (a ribonucleo-
amino acids protein)
Lysosome Targeting mannose 6- Mannose 6-phosphate im port into lysosomes

Signal phosphate Receptor
II. Post Translational Processing
There are MANY post-translational modifications that can be performed on the polypeptide.
These can occur in combinations, resulting in a great variety of different and complex structures.
Some of these events can occur even as the newly synthesized polypeptide comes off the ribosome.
Alternatively, some of these events can wait until a protein is properly localized (e.g. secreted out
of the cell).
A. Amino acid modifications: A variety of chemical groups can be covalently attached to proteins.
Attachment of different chemical groups may lead to activation or inactivation of the protein.
1. Phosphorylation: addition of
phosphate groups (P04) to hydroxyl side
groups of:
- serine, threonine
- tyrosine
Other ammo acids: P04-His; P04-Arg (s Triezenberg and j. Wang, Msu)
Protein kinases put P04groups on proteins; protein phosphatases take them off.
Example: kinase cascades (signal transduction in receptor as a tyrosine kinase pathway).
AcCoA CoA
r
2. Acetylation: addition of acetyl ?
(Lys-H3 , ^ -----
groups to: Lys-NH2-C-CH3
- amino terminus 1
- amino group of lysine ^ ......... ........J HAc HzO
(S. Triezenberg and

Acetyltransferases put acetyl groups on proteins; deacetylases take them off. J. Wang, MSU)
Example: histone acetylation key mechanism in determining tightly packed

euchromatin (regulation of gene expression) Prolyl residue
\
3. Hvdroxvlation: introduction of hydroxyl group (OH) to: C CO / w v w '
2 2 h c ch
- proline CH2
- lysine * 'Nl/' a-Ketoglutarate

Prolyl ascorbate, Fe?T
hydroxylase
a. It is catalyzed by specific hydroxylases: prolyl hydroxylase Succinate + C 0 2
and lysyl hydroxylase [Both are vitamin C (ascorbic acid) - C CO V 'V v w '
dependent enzymes]. h 2c _c h 2
CH
b. Hydroxylation of collagen takes place within the lumen of
^ OH
the rough endoplasmic reticulum (RER).
Hydroxyprolyl residue
Example: hydroxyproline and hydroxylysine collagen (Ferrier7_F4.6_p45)

structure [hydroxyproline maximizes interchain hydrogen bond formation, therefore it is
important in stabilizing the triple-helical structure of collagen].
Clinical relevance: vitamin C deficiency (scurvy) prolyl hydroxylase and lysyl
hydroxylase are unable to function -> impaired interchain H-bond formation and
formation of stable triple helix collagen fibrils cannot
be cross-linked great decrease in tensile strength of
the assembled collagen fiber.
- bruises on the limbs of patients with scurvy are a
result of subcutaneous extravasation of blood due to
capillary fragility
4. ADP-ribosvlation
- diphtheria toxin, cholera toxin, pertussis toxin are
enzymes that transfer ADP-ribose groups from
NAD+to His residues (of translation elongation
factors or ion channels like the cystic fibrosis
CFTR).
ADP-ribosylation: N of arg, gin; S of cys
O O NHo
II II H II (Ferrier7_F4.8_p47)
c h 2- o - p - o - p - o - c h 2 N C CH2 CH2 CH;
arg
1
aHO OH HO OH
(Lieberman4_F6.13)
Clinical relevance: diphtheria toxin catalyzes the ADP-ribosylation of EF-2 -
inactivation of EF-2 and inhibition of eukaryotic protein synthesis cell death.
Plasma membrane
5. Covalent attachment of fatty acids
- myristic acid (C14), palmitic acid (Ci6),
isoprenoid (C2o)
- attached to (processed) amino terminus
or internal Cys residues
Function: to anchor the protein to membranes
Example: receptor and signal transduction
proteins (S Triezenberg and J. Wang, MSU)
B. Glvcosvlation - is the process by which sugars are attached to proteins. Glycosylation can
present in two main modes: Glycosylation
1. O-l inked
Sugars are attached to hydroxyl groups mainly on serine and
threonine, but can also attach to the hydroxyl groups on hydroxy-
lysine and hydroxy-proline
a. Monosaccharides are added one at a time
b. Each is catalyzed by its own glycosyl transferase (e.g. galactosyl
transferase or fucosyl transferase...)
c. Takes place in the Golgi
d. Saccharide structure is key for cell surface recognition (e.g.
blood group A,B,0)
/"
Ala
\
Leu
/
Ser-O- GalNAc
\
Gly
Val Sialic
acid (S. Triezenberg and J. Wang, MSU)
(Ferrier7_F32.16_2>259)
Examples:
- Olygosaccharides on surface of red blood cells ABO blood group
- Collagen: O-linked glycosylation of the hydroxyl group of hydroxylysine.
(Ferrier7_F4.7_p46)
2. N-linked
Sugars are attached to the amide nitrogen of Asparagine
a. A "core oligosaccharide rich in mannose is added all together
b. Takes place in the Endoplasmic Reticulum (addition of mannose core)
c. The glycolipid dolichol pyrophosphate is the en bloc sugar donor (14 monosaccharide
units added all together)
d. Terminal glycosylation takes place in the Golgi
a. Some core is cut out
b. Terminal glycosylation adds specific sugars one at a time
e. Glycosylation is important for changing the half life of proteins in circulation
Example: Erythropoetin (Epo) treatment for anemic patients
(Ferrier7_F14.16_pl68)
f. Glycosylation sites are specific and are determined by the amino acid sequence of a
polypeptide. There are consensus sequences that mark a protein for either 0- or N-
glycosylation. (e.g. for N-glycosylation, Asn-X-Ser/Thr-)
C. Site-specific proteolysis
Many secreted proteins are synthesized as precursor molecules that
are functionally inactive. The active protein is released when parts
of the polypeptide chain are removed by specialized endoproteases.
1. Insulin
a. A 51 amino acid polypeptide hormone. It has two
polypeptide chains (A and B) connected by disulfide bonds.
b. Produced by the p cells of the islets of Langerhans of the
pancreas.
i. Preproinsulin - inactive form in the ER during synthesis. (Chandarl_F11.4)
ii. Proinsulin - inactive form in the ER after synthesis.
iii. Insulin - active form in the Golgi and when secreted (half-life of ~ 6 min in plasma).
+ C-peptide (longer half-life in plasma, used as indicator of insulin production and
secretion).
Zymogens - is an inactive enzyme precursor which requires the removal of polypeptides to

become active. Zymogens become activated when
trypsinogen
they reach their site of action. One example is the
pancreatic zymogen trypsinogen which is involved in
digestion. Trypsinogen is released by the pancreas i trypsin
into the small intestine. The enzyme enteropeptidase 1
(released by intestinal mucosal cells) will cleave a hexapeptide
from the N-teminus of trypsinogen to produce the activated
trypsin. Proteases are synthesized as zymogens to protect the cells from being digested by
its own enzymes.
Pro-opiomelanocortin (POMC)
Polyproteins - Although each __________ ;
eukaryotic mRNA has ONLY ONE 1 ----
ORF, the resultant polypeptide can be 1 i 1
fragmented into little pieces, each of V /A IIMITTTTTTTITTTTTTII
y-MSH ^C orticotropin ^ P-Lipotropin ^
which carries a specific biological
activity. Htittl |_ ~~n i i
o: MSH '-.n o tre o n -rd o rp h n
Clinical relevance: poliovirus, HIV and hepatitis A and C
viruses use polyproteins to produce proteins required for EZU
viral function. (S. Triezenberg and J. Wang, MSU)
III. Protein Degradation (or protein turnover)
Proteins that are old (no longer needed), damaged, or abnormal (misfolded) are targeted for
degradation. Approximately 300 - 400 g of protein is recycled per day in a human. This takes
place through two major mechanisms
ubiquitin
target protein
A. ATP-dependent ubiquitin-proteasome system of the
cytosol
o Responsible for turnover of endogenous proteins
o Ubiquitin protein units are added to target the
recycle
protein to the proteosome i- PPi
o Proteosome degrades targeted protein and

recycles ubiquitin
B. ATP-independent degradative enzyme system of the

lysosome
o Responsible for turnover of exogenous proteins
o Acid hydrolase enzymes of the lysosome 26S
fragment the targeted protein proteasom e
com plex AD P + F
PHAGOCYTOSIS
RECEPTOR-MEDIATED
ENDOCYTOSIS \ short polypeptides
and am ino acids
PINOCYTOSIS %
(Pawlina7_F2.24_p44)
(Pawlina7_F2.21_p41)
In both cases, the amino acids are recycled for use by the cell.
Gene Expression and Regulation
It may be obvious, upon even a casual reflection, that not all genes of an organism will be
expressed at all times. Different sets of genes must be expressed in particular types of cells, at various
developmental stages, or in response to changing environmental conditions.
We will illustrate some general principles of gene regulation using selected examples from E.
coli and eukaryotes. We will make no attempt to be comprehensive in covering principles or in
providing examples. Instead, we will simply introduce you to the array of intricate and elegant strategies
by which cells modify expression of genes in response to environmental and developmental signals.
Suggested Reading:
Ferrier: Chapter 33: Regulation of Gene Expression

Rhoades and Bell: Chapter 1: Homeostasis and Cellular Signaling - Molecular Basis of Cellular
Signaling - Hormone receptors bind specific hormones to initiate cell signaling in the cells only (p.
14-15)
Optional Suggested Videos:
http://www.voutube.com/watch?v=eYrOOEhVCYA
https://www.voutube.com/watch?v=Ti 6DcUTRnM
Objectives:
1. Understand how gene expression can be regulated at different levels (e.g. transcriptional, post-
transcriptional and post-translational regulation).
2. Explain and provide key examples of how transcriptional regulation of gene expression is
accomplished at the DNA level for both prokaryotes (sigma factors and operons) and eukaryotes
(promoter, enhancer, silencer, transcription factors and chromatin modifiers).
3. Understand the basis of the receptor as a transcription factor pathway (steroid hormone receptor,
Sterol Responsive Element-binding Protein and NF-kB).
4. Describe the different mechanisms involved in transcriptional regulation of gene expression at the
chromatin level (Epigenetic modifications - DNA methylation, genomic imprinting, and post-
translational modification of core histones such as acetylation, methylation, phosphorylation,
ubiquitylation and sumoylation).
I. General Principles of Gene Regulation

1. Differentiate between transcriptional, post-transcriptional and post-translational regulation of
gene expression. (Obj. 1)
II. Strategies of Gene Regulation in E. coli

1. Explain the use of alternative sigma factors to regulate specific set of genes by recognition of
specific promoter sequences. (Obj. 2)
2. Describe how operons work to regulate gene expression in E. coli. (Obj. 2)
III. Regulation of Eukaryotic Gene Expression
1. Define promoter, enhancer, and silencer with respect to gene expression. (Obj. 2)
2. Distinguish between basal (or general) transcription factors and regulatory proteins (such as
transcriptional activators). (Obj. 2 and Obj. 3)
3. Describe the two domains of transcriptional regulatory proteins and their functions or
activities. (Obj. 2 and Obj. 3)
4. List the structural motifs of three classes of DNA-binding proteins (helix-tum-helix, zinc-
finger, and leucine zipper). (Obj. 2 and Obj. 3)
5. Explain the location and process by which DNA gets methylated. (Obj. 4)
6. List the DNA methyltransferases and their functions. (Obj. 4)
7. Describe how DNA methylation affects gene expression. (Obj. 4)
8. Define euchromatin and heterochromatin in the context of DNA methylation. (Obj. 4)
9. Discuss how genetic imprinting is involved in Prader-Willi syndrome and Angelman
syndrome. (Obj. 4)
10. Differentiate between epigenetic reprogramming in the zygote and in primordial germ cells.
(Obj. 4)
11. Understand how defects in DNA methyltransferases may lead to disease. (Obj. 4)
12. Describe how histone modifications regulate gene expression. (Obj. 4)
13. List and define the histone modifications. (Obj. 4)
14. Differentiate between Receptor as a Transcription Factor, cholesterol metabolism and
NF-kB signaling pathways. (Obj. 2 and Obj. 3)
IV. Specialized types of Recombination

1. Define the concept of transposition. (Obj. 1 and Obj. 2)
2. Describe the genetic impact of transposon insertions and of homologous recombination between
two transposons. (Obj. 1 and Obj. 2)
I. General Principles of Gene Regulation
A. Goal of gene regulation:

the right genes at the right time, in the right place, in response to the right signals
turn genes ON when theyre needed - activation
keep genes OFF when theyre not needed - repression or silencing
B. Gene regulatory mechanisms work in combinations
ON signals and OFF signals - promoters in front of specific genes are subject to both
positive and negative signals.
integration of all regulatory signals determines final output
How strong is the sum of positive and negative signals rheostat, rather than a set of switches
Where do we cross the threshold above which the gene will be turned on?
C. Transcriptional initiation is a key step in gene For most genes

the main site
regulation of control is
transcription of
DNA to RNA
assembly of the RNA polymerase complex (on
which we will focus most of our attention and
provide the most number of examples) but other
events can also be controlled
Gene expression is regulated at multiple levels:
Transcriptional regulation: transcription initiation

(main site of control)
Post-transcriptional regulation (at mRNA level):
RNA elongation, processing, transport, alternative
splicing, stability, efficiency of translation
Post-translational regulation (at protein level):

protein translation, processing, transport, stability,
targeting
Modified or
degraded
proteins __
In eukaryotes gene expression
also involves posttranscriptional
and posttranslational processes
(Ferrier7_F33.1_p465) C opyright 2008 W ohers K hiwerHeaItli| Lippincott W iliams & Wilkins
II. Strategies of Gene Regulation in E. coli
A. Alternative sigma factors recognize distinct sets of gene promoters
E. c o li sigma factors recognize promoters with different consensus sequences
Factor Use -35 Sequence Separation -10 Sequence
a 70 general TTGACA 16-18 bp TATAAT

a 32 heat shock CNCTTGAA 13-15 bp CCCCATNT
60
a nitrogen CTGGNA 6 bp TTGCA
B. Operon model --- bacterial genes are organized along DNA in a way that allows for
COORDINATED expression of related genes
One regulatory region will be responsible for the regulation of many related genes
Lac operon:
Promoter Operator
Repressor
gene
\ / Structural genes
_L_i___ CAP site | p | o z y a _ l_
----------------->
mRNA

III. Regulation of Gene Expression in Eukaryotes
A. Gene organization
1. No operons in eukaryotes (a) Cannot make polygenic mRNA because of the scanning
model of ribosome initiation of translation
(b) Will not find multiple ORFs in eukaryotic mRNA
2. Gene clusters: Related genes near each other on the same chromosome
Similar idea to an operon but NOT exactly the same (each gene has its own promoter)
Nevertheless, close spatial localization in a subregion of the nucleus allows for

(a) sequential unpacking of chromatin; and
(b) unpacking of a small region of chromatin opens up several related genes
B. Levels of transcriptional regulation
There are two levels of transcriptional regulation:
1. at DNA level
2. at chromatin level
(Ferrier7_F30.27_p426)
1. Transcriptional regulation at the DNA level:
(a) Eukaryotic promoter elements RNA pol II

core promoter: TATA box, initiator site
regulatory DNA sequences for specific genes (responsive elements): enhancers and
silencers.
-445 -114 -40 -30 +1

Initiation
-------------1------------- site
Responsive elements L J
--------- i--------
Core promoter
(R. Ritchie, MSU)
promoter-proximal regulatory sites (hundreds of bases from +1)
I ------
I I I I
-1000 7/ -200 -100 +1
distant regulatory sites (thousands or tens of thousands of bases from +1)
(b) Transcriptional regulatory proteins

basal (general) transcription factors: TFII-A, TFII-B, TFII-D, etc
specific transcriptional factors - OVER and ABOVE the general (basal) transcription
factors (TFII-A, B, D, etc.)
Basal transcription
Specific transcription factors RNA Polymerase II
TF II D
jpL A TAIA DOX
-445 -114
(R. Ritchie, MSIJ)
Bind to DNA at specific sites Group of proteinsthat
called enhancers/silencers. are essential for transcription
Interacts with the transcription from an eukaryotic promoter.
complex to increase/decrease Are involved in the
rate of transcription. formation of the pre-initiation
Specific for certain genes (set complex and the recruitment
of genes). of RNA polymerase II.
Same for every gene.
(b) Transcriptional regulatory proteins (<continued)
RNA Polymerase II
(R. Ritchie, MSU)

Core promoter
Regulatory proteins often have two distinct domains
Transcription
Variable
regulatory
linker
domain
(R. Ritchie, MSU)
Recognizes and b in d s to Site o f p ro te in -p ro te in in teractio n

specific DNA sequences Interacts w ith the tra scrip tio na l
("responsive elem ents") m achinery (basal complex)
DNA-binding domain (DBD)
DNA-binding domains/proteins usually use one of four structural motifs to interact

with DNA:
(a) helix:-turn-helix; (b) zinc finger; (c) leucine-zipper; and (d) helix-loop-helix
A. Zinc fingers B. Leucine zipper C. Helix-turn-helix D. Helix-loop-helix
A TRD could possibly either activate or repress depending on specific conditions

(e.g. presence or absence of co-activators, etc.)
What might TRD do?
(1) direct interactions with basal transcription factors
(2) recruit co-activators that modify chromatin
ATP-dependent remodeling enzymes that induce sliding of nucleosomes

along DNA, increasing the gaps for RNA pol II, TFII-D, etc. to gain access to
DNA
Target gene
( Nudeosome-
\ remodelling complex
( Nudeosome-
(Modified from: V. M. Weake and J. L. Workman, Nature Reviews Genetics 11 (2010) 426-437)
(3) covalent modification of histone proteins
(acetylation; methylation; phosphorylation, etc.)
(c) What turns on transcriptional activators?

a. steroid hormone receptors Carrier
Steroid hormones are hydrophobic and can cross the

plasma membrane to bind to specific receptors in cytosol.
The receptor has: (a) hormone-binding domain;

(b) DNA-binding domain; and
(c) transcription regulatory domain.
Upon binding to the steroid hormone, the hormone-

receptor complex translocates into the nucleus.
Using its DBD, the hormone-receptor complex binds to
hormone-responsive elements on the DNA.
Via its TRD, the hormone-receptor complex recruits the
BMB 515
Gene Expression_______________________________________Dr. Ritchie: rritchie@msu.edu
machinery to activate hormone-responsive genes.
Examples of the receptor as a transcription factor pathway of signal transduction:
H o rm o n e R ecep to r D N A sequences G enes a c tiv a te d Biological

Response
Cortisol Glucocorticoid Glucocorticoid PEP Carboxy Kinase Gluconeogenesis
Receptor Responsive
Element (GRE)
Estradiol Estrogen Receptor Estrogen Ovalbumin Oviduct function
Responsive (ovarian cycle)
Element (ERE)
(Tamoxifen is an antagonist (competitive inhibitor) that targets the estrogen receptor; it is used against
breast cancer because some breast tumors rely on estrogen-mediated pathways for proliferation.)
b. cholesterol metabolism: SREBP
Sterol Responsive Element-binding Protein is a trans

membrane protein at the plasma membrane. Low
cholesterol concentration signals a protease, which cleaves
the cytoplasmic domain , releasing it to be translocated into
the nucleus.
This cytoplasmic domain has: - DBD: binds to sterol regulatory element

- TRD: activates genes in cholesterol metabolism
c. NF-k B
Nuclear Factor (K-chain) is anchored in the cytoplasm by

I k-B. Bacterial lipopolysaccharide (LPS) or viral infection
(induction of a cytokine such as interleukin 1P), results in Triezenberg andJ. Wang MSU)
ubiquitin-tagging and degradation of I k -B. This releases NF-k B to be translocated into the nucleus and
activate genes for inflammatory response.
2. Transcriptional regulation at the chromatin level
Epigenetic modifications: chemical modifications to the chromatin that regulate gene

expression, without changing the primary DNA sequence, and are heritable. Includes DNA
methylation and covalent histone modification
DNA inaccessible due to

methylation (gene inactive)
o
Methyl groups attached
to the CpG islands
regulate gene activity
Low/no methylation, DNA
accessible (gene active)
O
Groups attached to histone tails
determine the activity of the DNA
wrapped around them
Chromatin remodelling makes certain

regions of DNA available fortranscnption
C hrom atin remodeller
(S. Rajender et al, Mutation Research 727 (2011) 62-71)

(a) DNA methylation:
[Note: DNA methylation is discussed here in the context of gene regulation]
( l) What gets methylated?
(Ferrier7_F30.2_p412)
DNA methylation, in the context of gene regulation, is the covalent attachment of a methyl
group to the C5 position of the cytosine pyrimidine ring of a CpG dinucleotide in the DNA.
CpG dinucleotides consist of a cytosine bound to a guanine by a phosphodiester bond, rather
than CG base-pairing.
DNA methylation is important for normal embryonic development and for normal functioning
of adult organism. DNA methylation covers most of the genome (with the exception of CpG
islands), but it is found predominantly in repetitive regions of the genome, such as satellite
DNA and transposable elements (including long interspersed elements (LINES) and short
interspersed elements (SINES)). DNA methylation is also found, to a lesser extent, at
imprinted genes.
Transcriptionally Silenl
Heterochromalin C lo s e d " H e te ro c h ro m a tin
V ersus "Open"E u ch ro m a tin
Heterochromatin (not expressed) is

heavily methylated, resulting in tight
packing.
Euchromatin (expressed) contains

much less methylation and is more Environmental
Cues
loosely packed. Transcriptionally Active
Euchromatin
Transcriptionally Repressed
Euchromatin
TF
s /
(O.G. McDonald and G.K. Owens, Circulation Research 100 (2007) 1428-1441)
_ ft* 11/11
CpG islands - DNA sequences of more than 200 base pairs which are GC rich, with a greater
than expected number of CpG dinucleotides. CpG islands are usually found in and/or near the
promoter region of many genes and are typically unmethylated.
(2) Functions of DNA methylation:
i. Methylation that happens on ALL chromosomes for regulation of gene expression:
Transcriptional gene silencing
o Directly by inhibiting binding of specific transcription factors
o Indirectly by recruiting proteins with associated repressive chromatin
remodeling activities
Regulation of chromatin structure euchromatin (transcriptionally active - less
methylated) vs heterochromatin (transcriptionally inactive - heavily methylated)
Genome stability suppression of repetitive elements
o silencing of repetitive and centromeric DNA
o suppression of homologous recombination between repeats
o transposon silencing
ii. Methylation that happens only on selected chromosomes (e.g. chromosome 15) for
genomic imprinting:
Genomic imprinting
iii. Methylation that happens only on the X-chromosomes of females (inactivation of the
X-chromosome to maintain gene dosage):
X-chromosome inactivation (females)
Clinical Relevance: Epigenetic alterations contribute to cancer development.

Hypermelhylated,
Hypomethylation repetitive
heterochromatin
o Genomic instability
o Loss of imprinting (LOI)
o Activation of oncogenes
Hyperm ethylation
o CpG islands may be
aberrantly methylated in
cancer cells leading to gene
silencing (usually of a tumor
suppressor gene).
Hypomethylated Tumor suppressor gene

CpG region
Turnpenny: Emery's Elements o f Medical Genetics, 14e
Copyright 2011 by Churchill Livingstone, an imprint of Elsevier Ltd. All rights reserved.
(T umpenny 14_F 14.8_p219)

(3) D onor o f methyl group: S-adenosyl-m ethionine (SA M )
Unmethylated Methylated
cytosi cytosine
DNMT
5'
------ C H Z
Adenine
5'-CpG-3'
h 1 r h
OH OH
S-Adenosyl methycmine S-Adenosyl homocysteine
(SAM) (SAH)
(R. Ritchie, MSU)
(4) D N A methyltransferases (DNM T): class o f enzym es that carry out the D N A methylation
reaction. D N A methyltransferases transfer methyl groups from SAM to the cytosine in the
CpG dinucleotides, and they establish and maintain D N A methylation patterns in the genom e.
D N M T 1: this enzym e is responsible for maintenance o f D N A methylation patterns.

After D N A replication, DNM T1 adds methyl groups to the new ly synthesized D N A
strand based on the presence o f methylation in the CpG dinucleotide in the
complementary template D N A strand.
D N M T 3A and DNM T3B: these enzym es catalyze the de novo D N A methylation. De
novo D N A methylation patterns are established early in em bryogenesis, around the
time o f implantation.
DNM T2: does not methylate D N A , it methylates a cytosine base in the anticodon loop
o f the aspartic acid transfer RNA.
(5) G enom ic imprinting: although you inherit duplicate copies o f your genes, the maternally
derived copy o f som e genes (or the paternally derived copy o f other genes) may be
required/utilized in unique manners during specific stages o f development. Imprinting refers
to m odification o f a gene (e.g. methylation pattern) as it is transmitted through the father or
the mother.
M ethylation patterns change from generation to generation. The imprinting pattern o f an

individual is established prior to fertilization and remains during life. The imprints are
erased in germ cells during gam etogenesis and new patterns are established for the next
generation prior to fertilization.
Norm ally, for imprinted genes, only one copy o f the gene is active. Improper imprinting can
result in tw o active copies or tw o inactive copies, w hich can lead to abnormal developmental
processes.
HI
I
Chromosome 15
(R. Ritchie, MSU)
(6) Epigenetic reprogramming: process through w hich most genom ic D N A patterns are erased
(usually by demethylation) and reestablished. There are tw o epigenetic reprogramming events
during embryonic developm ent, one that occurs during pre-implantation developm ent and
affect all cells, and a second one that w ill only occur on primordial germ cell (embryonic cell
that w ill form germ cells).
Epigenetic reprogramming in the zygote: most D N A methylation is rem oved or erased

follow ing fertilization during pre-implantation developm ent, when extensive
demethylation o f the genom e takes place (does N O T affect genom ic imprints). This
process functions to reestablish totipotency.
Sperm cell
PGC j Spermatocyte
precursors
Spermatogoniim
p Prosperiratogo mun
Zygote
Zygote j
o PG
Csit-f
3
PGCs settled in gonad
Sex differentiation
*A. Onset o f
merosis
Non-growing
oocyte
Fully grown oocyte

Mil oocyte/egg
,-

, /
/
Oocyte Maturation
growth : Ovulation
(Modified from: H. Sasaki and Y. Matsui; Nature Reviews Genetics 9 (2008) 129-140)
Epigenetic reprogramming in primordial germ cells: happens during post-implantation
developm ent and erases the methylation marks o f imprinted genes in the primordial
germ cell genom es. This erasure is important to make sure that the epigenetic marks in
the primordial germ cells can be reset and reflect the sex o f the developing embryo,
o Rem em ber that germ cells need to be imprinted w ith proper methylation
patterns according to the sex o f the developing embryo,
o Somatic cells do not undergo this second epigenetic reprogramming. During
post-implantation developm ent imprinted marks m ust be maintained in som atic
cells because correct expression o f the imprinted genes is essential for normal
som atic development.
Prader-Willi syndrome and Angelm an syndrome are tw o different syndromes, w hich are both
linked to the sam e imprinted region o f chrom osom e 15. In this imprinted region som e o f the
genes are silenced in the maternal chrom osom e and som e are silenced in the paternal
chrom osom e. Therefore, a defect on chrom osom e 15 w ill lead to loss o f different gene activities,
depending on whether the chrom osom e cam e from the mother or the father. In Prader-Willi
syndrome, gene activity that normally com es from the father is m issing. On the other hand, on
Angelm an syndrome gene activity that normally com es from the mother is m issing.
Prader-W illi Syndrome Angelman Syndrome
(R. Ritchie, MSU)

X = deleted or non-functional
Clinical manifestations to be described by Dr. Amalfitano.
E x e r c is e to c o n f ir m o u r u n d e r s t a n d in g :
Paternal r~ * i- * I""* I- *
chromosome 15: zz ZNF127 NDN SNRPN IP W UBE3A
Maternal
chromosome 15: zz ZNF127 NDN SNRPN IP W UBE3A
(R. Ritchie, MSU)
1. A man has a daughter and a son. If you look at the daughters chrom osom e 15 what w ould be
the status o f the follow ing genes in her paternally derived chrom osom e 15 in her som atic cells?
H ow about in the girls egg cells?
Somatic cells: E gg cells:

GENE Methylated? Expressed? GENE Methylated? Expressed?
ZN127 ZN127
NDN NDN
SNRPN SNRPN
IPW IPW
UBE3A UBE3A
2. A man has a daughter and a son. If you look at the sons chrom osom e 15 what w ould be the
status o f the follow ing genes in his paternally derived chrom osom e 15 in his som atic cells? H ow
about in the b oys sperm cells?
Som atic cells: Sperm cells:

GENE Methylated? Expressed? GENE Methylated? Expressed?
ZN127 ZN127
NDN NDN
SNRPN SNRPN
IPW IPW
UBE3A UBE3A
(b) Covalent histone modification:
H istones are globular proteins with an N-terminal tail. A m ino acid residues in the histone N -
terminal tails can be m odified in a variety o f ways, w hich results in changes in the chromatin
structure.
N ucleosom e
R ecall nucleosom e structure
(H 2 A , H 2 B , H 3, n 4 )o
N-terminal tails o f histones can be
post-translationally m odified (see below ) w hich w ill
change chromatin structure.
Functions o f covalent histone modification:

Establishment o f chromatin structure (usually by affecting the interaction o f histones
with D N A or by affecting the contact between different histones in adjacent
nucleosom es)
Regulating the binding o f non-histone proteins (which may carry enzym atic activities,
such as rem odeling A TPases that further m odify chromatin).
o m odified histones can function as a binding site
o histone m odification can function to disrupt an interaction betw een a histone
and a binding factor.
(Modified from: A. J. Keung et at, Nature Reviews Genetics 16 (2015) 159-171)
Post-translational m odification o f the core histone N-terminal tails include: acetylation,

methylation, phosphorylation, ubiquitylation and sumoylation.
(1) Acetylation:
a. Residue: addition o f acetyl groups to lysine residues (K-ac) in H3 and H4
b. Enzyme: histone acetyl transferases (H A Ts)
c. Functions:
- transcriptional activation
- Opens up chromatin by reducing positive charges o f histones , therefore
w eakening interaction with PO4' on D N A backbone and reducing the packing
o f nucleosom es
- Function as a binding site for ATP-dependent rem odeling enzym e, w hich
induces sliding o f nucleosom es to increase gaps for the basic transcriptional
machinery (R N A pol II, TFII-D, etc) to gain access to D N A sequence
(Activator I (Activator
H istone deacetylases (H D A C s) rem ove acetyl groups from histone tails causing chromatin to
be more com pact and leading to transcriptional repression.
(2) Methylation:
a. Residue: Both lysine (K -m e) and arginine (R -m e) residues in histone tails can be
methylated
b. Enzyme: histone methyltransferases
c. Function: D epending on w hich lysine or arginine residue is methylated it can lead to
transcriptional activation (e.g. H 3K 4m e) or repression (e.g. H 3K 9m e)
(3) Phosphorylation:
a. Residues: serines (S-ph), threonine (T-ph), and tyrosine (Y-ph) residues in the N -
terminal tails
b. Enzymes: kinases that phosphorylates histone tails and phosphatases that rem oves the
phosphate group
c. Function: It generally leads to gene activation
(4) Ubiquitylation:
a. Residue: Ubiquitin is attached to lysine residues (K-ub)
b. Enzymes: sequential action o f three enzymes: E l-activating, E2-conjugating, and E-
3-ligating enzym es
c. Function: Can lead to both gene activation (e.g. ubiquitylation o f H 2B ) and gene
silencing (e.g. ubiquitylation o f H 2A )
(5) Sumoylation:
a. Residue: Covalent attachment o f small ubiquitin-related m odifier (SU M O ) m olecules
to lysine residues (K-su) in all four core histones
b. Enzymes: E l, E2, and E3 enzym es
c. Functions:
- gene silencing
- It antagonizes acetylation and ubiquitylation that w ould otherwise occur on
the same lysine residue
T h u s , D N A m e t h y la t io n , a lo n g w it h h is t o n e m o d if ic a t io n s , p la y a r o le in p a c k in g a n d
u n p a c k in g o f c h r o m a t i n ------ > r e g u la t io n o f g e n e e x p r e s s io n
Start o f transcription
(R. Ritchie, MSU)
R E Q U IR E D R E A D IN G
IV . S p e c ia liz e d T y p e s o f R e c o m b in a tio n
A . Transposons: jum ping genes (a.k.a. m obile genetic elem ents)
Transposon
Recipient DNA
Transposon
1. Transposons are the dispersed repetitive D N A sequences (SINES and LINES) that are
capable o f m oving from one location to another in the genom e, w hich can occur through non-
hom ologous recombination events.
a. Recall these transposable elem ents make up ~ 45% o f the genom e.
b. Som e o f these transposons (~ 3%) actually carry protein-coding genes.
2. Potential effects o f transposon insertion into a new location in the genome:

a. Insertional inactivation (disruption! o f a gene: occurs when a transposon gets inserted
into a gene.
Example: A transposon inserts into the insulin gene. Result: N o insulin can be made in
this cell ( if this was a cell that was supposed to make insulin).
+ Insulin gene Insul in gene
b. Insertional activation o f a gene: som e transposons carry transcription signals w hich take
over the regulation (on or off) o f the gene w hen they get inserted nearby. The result is
inappropriate expression o f the gene.
Example: A transposon carrying a transcription signal inserts into the regulatory region
upstream o f the insulin gene. Result: In this cell, the insulin gene is expressed according
to the transposon signals (w hich may cause too m uch or too little insulin to be made).
Insulin gene Insulin gene

c. D eletion o f a gene: occurs by hom ologous recombination o f tw o transposons on the
same chrom osom e causing the D N A in betw een to be deleted.
Example: Recom bination betw een 2 transposons flanking the insulin gene causes
the deletion o f the entire insulin gene. Result: N o insulin can be made in this cell
abed wxyz
3. Other transposon biological impacts:

a. Bacterial transposons often carry antibiotic resistance genes
b. Effect o f viruses: W hile viruses are N O T transposons viruses that integrate into the
host genom e have similar effects as transposons
B. Site-specific recom bination: Imm unoglobulin genes

The im m unoglobulins are made o f heavy and light chains. The vast number o f possible
recombination events that make the im m unoglobins lead to the m illions o f antibodies w e need
to fight infections.
The Basis of Molecular Techniques
In this lecture we describe many of the principles and basic techniques that
are used for the screening, diagnosis and treatment of disease. Molecular
diagnostics and recombinant DNA technology is a rapidly advancing field in
medicine; therefore, having a strong foundation in the basis of the
techniques will allow you to learn and adapt as the field moves forward.
The applications of this technology to specific clinical scenarios will follow
in the subsequent two lectures.
Suggested Reading:
Ferrier: Chapter 34: Biotechnology and Human Disease - from Southern Blotting
through Polymerase Chain Reaction
Turnpenny: Chapter 3: Chromosome and Cell Division - Molecular Cytogenetics (p.

34-37); Chapter 4: DNA Technology and Applications-from Restriction Fragment
Length Polymorphism through Amplification-Refractory Mutation System (ARMS)
PCR (p. 59-60)
Objectives:
1. Understand the techniques that can be used to obtain fragments of DNA or copies of
genes (restriction endonuclease, cDNA, reverse transcriptase).
2. Understand the techniques that can be used to identify DNA sequences (gel
electrophoresis, hybridization assay, probe, Southern blot, northern blot (RNA), western
blot (protein), Sanger (dideoxy) sequencing).
3. Understand the techniques that can be used to amplify DNA sequences (recombinant
DNA, DNA ligase, cloning vector, genomic library, cDNA library, polymerase chain
reaction (PCR)).
1. Describe the features of DNA sequences recognized by restriction endonucleases.

Given a DNA sequence and a list of recognition and cleavage sites for restriction
enzymes, identify the positions and number of DNA fragments that would be produced.
(Obj. 1)
2. Describe the synthesis of cDNA, including the role of reverse transcriptase. Be able
to distinguish between cDNA and genomic DNA based on the components present in
the sequence. (Obj. 1)
3. Explain the main limitation and the uses for chemically synthesized DNA. (Obj. 1)
4. Describe the process of gel electrophoresis and what information is obtained. (Obj. 2)
5. Explain the physical basis of hybridization assays. Recall the composition of probes
and the factors that determine their stringency. (Obj. 2)
6. Identify the kinds of information revealed by Southern, Northern, and Western blots.
Summarize these procedures, identifying the molecules being detected and the probes
used. (Obj. 2)
7. Explain the basis of the Sanger (dideoxy) sequencing method. Outline the steps
involved. Note the limitations of the method. (Obj. 2)
8. Describe in words or diagrams the general concept of recombinant DNA. Describe
the role of DNA ligase. Describe the essential features of a cloning vector. List five
types of cloning vectors and their distinguishing features. (Obj. 3)
9. Recall the types of foreign DNA that can be used as an insert. Compare the
informational content of genomic and cDNA and synthetic DNA clones. (Obj. 3)
10. Outline procedures for selecting and screening colonies for which have taken up the
vector and which of those are recombinant, respectively. (Obj. 3)
11. Describe the concept of clone libraries and how such libraries are constructed.
Compare the information content of genomic libraries and cDNA libraries. (Obj. 3)
12. Describe the process and products of the polymerase chain reaction (PCR). (Obj. 3)
The Basis of Molecular Techniques

a. Obtaining Fragments of DNA and Copies of Genes
i. Restriction Enzymes
1. The discovery of restriction enzymes in the late 1960s was the

start of the biotechnology age.
2. When they were first discovered, it was not as a biotechnology

tool but for their natural biological role in bacterial viral defense.
a. Bacteria contain restriction enzymes that cut double stranded

DNA at specific sequences (restriction sites). When a bacterial
virus (bacteriophage) injects its DNA into a host bacterium, the
hosts restriction enzymes cut the invading DNA thereby
preventing infection. The host protects its own DNA by marking
the restriction sites with methyl groups (- CH3) that prevent the
restriction enzyme from being able to cleave the site.
M ethylated
Recognition
Site
B acterial Genom e
(M. Faner, MSU)
T h e R estriction M od ification S ystem in B a cteria
3. Not long after the initial discovery of restriction enzymes their

utility as a molecular tool was recognized. Two major
applications of restriction enzymes are cutting DNA into smaller
specific fragments to...
a. facilitate its study, and specifically, in the identification and

characterization of genes.
b. be recombined Coined) with DNA from different genomes with

the intent of studying a genes function, expression and
regulation. Or to create recombinant gene products that can be
used in the treatment of disease (i.e. insulin, human growth
hormone).
BMB515
Danna and Nathans. 1971. Proc N at A cad Sci. 68: 2913
T h e F irst u se o f a R estrictio n E n z y m e as a M o lecu la r T ool. Products derived from digestion o f the

DNA o f the oncogenic Simian Virus 40 by the restriction enzyme Hin&W. The bands correspond to
DNA fragments o f different sizes separated by gel electrophoresis.
4. The nature of restriction enzymes

a. Scientists have isolated restriction enzymes that recognize and
cut more than 100 unique restriction sites.
b. Restriction sites are typically 4-6 bases long and the sequence
is an inverted repeat.
c. Produces DNA fragments with sticky or blunt ends. These
ends can be resealed with the enzyme DNA ligase.
----------------
----------------
EcoR\ SmM
Cleavage Site
Cleavage Site
5. . . C - C - C - G - G - G . . . 3
1
-------- 1
-------- 1
Cn
1
1
1
1
Q -----
o -----
o
----- O
----- >
----- >
0
o
1
1
1
1
cn
w
0
l
3... C T T A - A - G - 5
t Cleavage Site ^ Cleavage Site
5 \..G -O H -A -A -T -T -C ...3 ' 5 ... C - C - C - OH 0 - G -G -G ...3

1 . 1 I I I
1 . 1 I I I
3 \.,C -T -T -A -A -(p ) / HO -G ...5 3 '. . . G - G - G - ( p) H O -C -C -C ...5
\ \ /
Blunt ends
Sticky ends
(M. Faner, MSU)
P ro d u ctio n o f S tick y an d B lu n t E n d s b y T w o D ifferen t R estrictio n E n zym es
E x erc ise 1. Using the AcoRI recognition sequence from the previous page, find the AcoRI sites in the
DNA sequence below and predict how many fragments would result after its digestion.
5 ... G A C G C G T C C T A G G T G A C C G G A T C C A T G G A A T T C G C G G C C A C T G G T T A A C
CTG CG CAG G AT CCACTGG CCT AG G TACCTTAAG CACCG G TG ACCAATT G
ii. Reverse Transcription (cDNA)
1. It is possible to obtain DNA w ithout introns by using an enzym e

o f retroviral origin, reverse transcriptase.
2. G iven the m RN A of a gene and a com plem entary prim er the

enzym e will synthesize DNA that is com plem entary (cDNA) to
the mRNA.
3. Because the tem plate is spliced m RN A the product contains the

open reading frame, 5 and 3 untranslated regions and polyA tail
but not introns, prom oters, enhancers o r intergenic DNA.
4. A n advantage of this approach is the ability to express a

eukaryotic gene in a prokaryotic host even though the host does
not have splicing machinery.
S y n th esis o f cD N A
iii. Chem ical Synthesis
1. DNA o f up to ~ 100 nucleotides can be chem ically synthesized.
2. Prim ary uses for these oligonucleotides are as prim ers fo r in vitro
DNA synthesis using polym erases and as probes in hybridization
assays fo r identifying DNA sequences.
b. Id e n tify in g D N A S e q u e n c e s
i. G el E lectrophoresis
1. DNA and RNA can be separated by size and shape using gel
electrophoresis.
2. The negative charge o f nucleic acid backbones allo w them to

move through a porous gel m atrix when an electric charge is
applied. The m olecules will m ove aw ay from a negative electrode
at one end of the gel tow ards a positive electrode at the other
end.
3. S m aller m olecules are able to move through the gel more easily
than large fragm ents and therefore appe ar m ore tow ards the
positive electrode.
4. Fragm ents of nucleic acids of known size are run in an adjacent

lane to serve as a m olecular w eight m arker/ladder.
5. The gel is then treated with a stain to visualize the nucleic acid
fragm ents.
A. Electrophoresis B. Image after staining
Hepatitis C Virus
Sample
DNA Digest
wells
_______ 1 MW (-----------------*-----------------)
Gel 1 1 (bp) BstUI Hinfl Rsal Mval
\n iT iJru rijry ri Direction

of
Larger
molecules
migration
't
Smaller
molecules
(M. Faner, MSU)

A d ap ted from Filippo et at. 2012. Virol J. 9:242
Gel Electrophoresis. A) Schematic o f gel electrophoresis. B ) Visualization o f patterns obtained from

the restriction digest o f the hepatitis C virus genome.
ii. Probes/Hybridization Assays
1. A large number of molecular diagnostics techniques are based

on the ability of two complementary single stranded nucleic acid
oligomers to form a double stranded structure. The process of
base pair formation between a probe and its target is called
annealing or hybridization. Their ability to do this is based on the
same principles that govern genomic DNAs double helix
structure.
2. In clinical diagnostics probes, are most often synthetic DNA or

RNA but can also be composed of cDNA or genomic DNA
fragments.
3. The stringency, or the degree to which a probe will tolerate
mismatches and still anneal, can be manipulated by probe length
and experimental conditions (temperature and salt). Conditions
can be such that a single mismatch will prevent annealing.
4. Probes are labeled in order to detect the degree to which

hybridization has occurred. Common labels include radioactive
isotopes, fluorescent tags, or affinity tags.
a. Southern Blot
i. The first application of hybridization as means to identify a

specific DNA sequence was the Southern blot. It allowed
scientists to identify a single gene and determine its size
among the thousands of fragments created using newly
discovered restriction enzymes. Southern blotting is still an
important technique used in research and clinical laboratories.
ii. The method consists of these general steps:
1. Separate DNA (ex. fragments of genomic DNA after

restriction digest) molecules using gel electrophoresis.
2. Blot (transfer) the separated molecules to a filter paper.
3. Add the sequence specific probe and allow annealing to

occur.
4. Image the filter paper to observe hybridized fragments.
b. Northern Blot
i. A similar technique as the Southern but it determines the size

and abundance of an RNA species rather than DNA.
c. Western Blot
i. Another technique based on similar principles as the Southern

blot but it detects the size and abundance of proteins. Look
forward to more extensive coverage of this technique in MMG
531 pg.
d. Some other techniques that employ probe hybridization are

allele specific oligonucleotides, fluorescence in situ
hybridization (FISH), and microarrays.
S o u th e rn N o rth e rn W e s te rn
(D N A ) (R N A ) (P r o te in )
-i_n_n_n_n_r T_n_n_n_n_r i_n_n_n_n_r
E le c tro p h o re s is
T r a n s fe r to p a p e r ^ I I
B ands not
a c tu a lly v is ib le -"1-I =_";I
= - i " -
P ro b e a n n e a lin g
ÎD N A /R N A
P ro b e
I 1
D N A /R N A
P ro b e ^ A n tib o d y
P ro b e
Im a g e to
v is u a liz e b a n d s - -
(au to ra d io g ra m o r flu o rescen ce)
(M . Faner, M S U )
S o u th e rn , N o rth ern and W estern B lo ttin g P ro ced u res

iii. S equencing
1. S anger (dideoxy) M ethod
a. This m ethod is based on DNA polym erases need fo r a 3 -OH

group fo r synthesis to occur. The addition o f a small am ount of
dideoxynucleoside triphosphates (ddNTPs) to a reaction
containing DNA polym erase, a tem plate, a primer, and
deoxynucleoside triphosphates (dNTPs) results in the synthesis
of fragm ents of DNA. The lengths of these fragm ents
correspond to positions in the DNA sequence w here a certain
base occurs. The read length fo r one sequencing reaction is
400-600 nucleotides and the throughput is low.
b. How it works:
i. The sequencing prim er binds to the tem plate of unknown

sequence and prim es synthesis of a com plem entary DNA
strand (5 '^ 3 ') .
ii. Sanger's trick is to include the fou r dN TPs (as substrates)

A N D fou r ddNTPs, each labeled with a different dye (red for T;
green fo r A ; blue for C ; and "b lack" fo r G).
iii. dd stands fo r dideoxy (no OH on either 2' or 3'-positon of

ribose of nucleotide) and thus that chain cannot be extended
(DN A polym erase requires free 3'-O H....)
iv. Thus, the sequencing reaction results in a collection of

fragm ents w hose lengths differ by one nucleotide; these are
separated by capillary electrophoresis to generate a pattern of
colored peaks.
Prim er
Prim er
-OH
T em plate of f l
unknow n sequ e n c e -C TA A G C TC G A C T
r
T em plate of
DNA Polym erase unknown sequ e n c e
dNTPs, ddNTPs
DNA Polym erase,

Dye labeled dNTPs, ddNTPs
I
segm ents of DNA
S e p a ra te fragm ents
by capillary electrop ho resis j
G A TTCG A G C TC ^^ GATTCGAGfjE|*
G A T T C G ^^ -G A rrfjj^
G ^^
G A T T C G A G C '^ ^ ^ -G A T T C G A G C Ê Î
G A T T C G /^ ^ l-GAT
gattc^ E E
-G A ^ ^
'2 0 '2 5 ' 30
M
G A T T
l k Mill
C G A G C T G A
C o m p u te r g en e ra te d result a fte r bands (M. Faner, MSL
m igrate p ast d e te c to r
S a n g er (d id eo x y ) S eq u en cin g M eth o d
c. Amplification
i. Cloning
1. The process of cloning involves combining a specific gene or

DNA segment (insert) with a small carrier DNA (vector) and then
replicating it. Replication of the recombinant DNA occurs both by
increasing the cell number and by creating multiple copies of the
DNA per cell.
2. The steps involved in the process are as follows:
a. The foreign DNA and the vector DNA are digested by the
appropriate restriction enzyme (ex. Pst\).
i. The foreign DNA can be genomic DNA, cDNA or synthetic

DNA.
ii. The vector we will most refer to is a bacterial plasmid but

there are several other types of vectors as shown in the table
below. Essential features of a vector include:
1. An insertion site where a specific restriction enzyme

cleaves to open the vector.
2. An origin of replication that allows the vector to replicated

independently from the host genome.
3. A selectable marker that allows the identification of cells

that have taken up the vector (usually an antibiotic
resistance gene).
H ost In sert
V ector T ype O rganism Size R ange S p ecial F eatures
E . c o li , up to 10 - double-stranded circular
Plasmids yeast kbp - antibiotic resistance
- double-stranded linear
X phage E . c o li 10-15 kbp - efficient cloning of larger fragments
Cosmids E. c o li 25-40 kbp - even larger fragments

Bacterial Artificial
Chromosomes - very large fragments for gene
(BACs) E. c o li 50-200 kb mapping, genome organization
- very large fragments for gene
Yeast Artificial mapping, genome organization
Chromosomes 100-1,000 - replication origin, centromere,
(YACs) yeast kbp telomeres
iii. The insert DNA is then ligated into the vector using the
enzyme DNA ligase. The matching sticky ends of the digested
DNA allow this process to occur. In this example a successful
ligation disrupts the ampicillin resistance gene.
iv. E. coli cells are then forced to take up the recombinant

plasmid in a process called transformation. Some bacteria are
able to take up plasmids naturally but in the cloning process
the cells are treated in a way that forces them to do so.
v. The cells are then grown on agar containing the appropriate

antibiotic (ex. Tetracycline) to select for bacteria that have
taken up the plasmid.
vi. In order to select the colonies for which have taken up the
plasmid and to screen for which of those is recombinant (not
just an empty vector) individual colonies are spotted in
identical positions on a plate with tetracycline (of the cells that
grow some contain empty vector and some contain
recombinant vector) and a plate with tetracycline and
ampicillin (cells that grow contain empty vector).
pBR322
plasmids
0)
p8R322 is cleaved at the ampicillin
Pstl restriction
resistance element by Pitt.
I endonuclease
b
o v o Foreign DNA
Foreign ONA is ligated to cleaved

pBR322. Where ligation is successful,
the ampicillin-resistance element is
disrupted. The tetracycline-resistance
element remains intact.
<D
f. toll cells are transformed, then
grown on agar plates containing transformation
tetracycline to select for those that of t. toll cells
1
have taken up plasmid.
selection of
transformed cells
All colonies Agar

Q have plasmids containing
tetracycline
Individual colonics are transferred
to matching positions on additional
plates. One plate contains tetracycline, colonies transferred
the other tetracycline and ampicillin. for testing
A
Colonies with
recombinant
plasmids
Agar containing Agar containing

tetracycline (control) ampicillin + tetracycline
Cells that grow on tetracycline but not on tetracycline *

am picillin contain recombinant plasmids with disrupted
am picillin resistance, hence the foreign ONA. Cells with pBR322
without foreign ONA retain am picillin resistance and grow on
both plate*. Nelson4_F9-5_p312
P la sm id C lon in g
vii. An additional method that is
used to screen colonies is hybridization
with a molecular probe. This method is
conducted in the following manner:
1. An agar plate with bacterial

colonies is transferred to a filter paper.
2. The cells are treated with alkali

to lyse the cells and denature the DNA
within.
3. A labeled DNA/RNA probe is

allowed to hybridize to the cellular DNA on
the filter paper.
4. Excess (unbound) probe is

washed away and the paper is imaged to
identify the colonies that contain the DNA
of interest.
ii. Clone Libraries
1. Clone libraries are a collection of clones made simultaneously

from a given sample. The types of libraries are as follows:
a. Genomic libraries are a set of clones that together contain all of

the DNA sequences of a given genome.
b. A cDNA library is set of clones that together contain all the DNA
sequences produced by reverse transcription of the mRNA
isolated from a tissue or cell. This library represents all of the
genes being transcribed at a given time.
iii. P olym erase Chain Reaction
1. The polym erase chain reaction (PCR) is the in vitro am plification

of a specific fragm ent of DNA w ithout an origin of replication. The
technique has revolutionized m olecular biology by allowing
scientists to obtain large am ounts (enough to m anipulate in the
lab) of DNA o f interest.
2. The process is as follows:
a. Tw o synthetic DNA prim ers are designed to be com plem entary

to DNA sequences flanking the target region.
b. Step 1 of a PCR cycle involves denaturing (by heating to

~95C) the double stranded target DNA.
c. Step 2 involves a decrease in tem perature (~55C) to allow the

prim ers to anneal to the target DNA.
Cycle Q d. In step 3 the
UnampMiod DNA
f Targeted sequence temperature is increased to the
Cycle * ideal temperature (~70C) for
Otntlufe and
Pnmar anneal pnm ttt extension of the primer by a special
DNA potymerase
heat stable DNA polymerase (Taq
Primer extension DNA polymerase can withstand the
95C denaturing temperature in
Cycle 2
step 1)
Denatureand
anneal pnrrwi
e. These steps are cycled
25-30 times.
Primer extension
3. Each cycle doubles the
amount of target DNA in the
Cycle 3 previous which results in an
exponential increase in copy
Denature and
number of the fragment of interest.
anneal prtmarj
Each cycle takes about 1.5 minutes
so in 1 hour there are billions of
copies of the target DNA.
4. PCR is extremely
Primer extension
sensitive. In theory you only need a
single molecule of the target DNA
to start with.
5. It is quantitative. The
(Turnpenny14_F4.6_p57) more DNA you start with the
greater the yield.
T h e P o ly m er a se C h ain R ea ction
6. The product can be

used for many things including sequencing, cloning, digestion
with restriction enzymes, or as a probe.
The Application of Molecular Techniques in the Screening, Diagnosis,
and Treatment of Disease (Parts 1 and 2)
The previous lecture provided you with a foundation in many of the

important techniques that form the basis of modern molecular diagnostics.
We will now discuss how those basic techniques have been adapted and
combined to address the needs of the clinical world. Learning about
specific examples of molecular techniques used in the screening, diagnosis
and treatment of disease in current clinical settings will provide you with the
background necessary to learn about more techniques as they become
relevant in your medical education and future practice. The importance of
molecular techniques in clinical settings continues to grow and the
complexity of the methods as well as how to interpret the information
obtained is increasing. As a future physician it is essential that you have a
sound scientific background so that you can effectively use the technology
available and be able to explain to your patients what the tests are and
what they mean.
Suggested Reading:
Turnpenny: Chapter 4: DNA Technology and Applications - from Sanger Sequencing
through Variable Number Tandem Repeat (p. 61-67); Chapter 4: DNA Technology and
Applications - Diagnosis in Non-Genetic Disease (p. 71)
Objectives:
1. Understand DNA polymorphisms and their importance in disease (single nucleotide
polymorphism, variable number tandem repeat, copy number variation).
2. Understand how the basic molecular techniques have been adapted and combined to
address the needs of the clinical world (Southern blot, multiplex PCR, allele specific
oligonucleotide (ASO) probes, restriction fragment length polymorphism (RFLP), Sanger
sequencing, comparative genomic hybridization (CGH), microarray, fluorescent in situ
hybridization (FISH), PCR, transcriptomics, reverse transcription - polymerase chain
reaction (RT-PCR), next generation sequencing, Iliumina sequencing, whole exome
sequencing, human growth hormone).
3. Understand what information each technique provides and which technique is most
appropriate for a given clinical scenario (cystic fibrosis, fragile X syndrome, lung
adenocarcinoma, breast cancer, unexplained developmental delay/intellectual disability
(DD/ID), autism spectrum disorders (ASD), multiple congenital anomalies (MCA),
hepatitis C, human growth hormone, Miller syndrome).
1. Explain what DNA polymorphisms are and describe the main types. Explain the
importance of DNA polymorphisms in disease. (Obj. 1)
2. Categorize techniques as being useful for the screening/diagnosis of disease when

the gene or specific mutation is known or being useful when the genetic basis of the
disease is unknown. (Obj. 2 and Obj. 3)
3. Describe the use of Southern blotting and variable number tandem repeat (VNTR)
analysis in the diagnosis of disease. (Obj. 1, Obj. 2 and 3)
4. Discuss the utility of multiplex PCR. (Obj. 2 and 3)
5. Demonstrate the application of allele specific oligonucleotide (ASO) probes or

restriction fragment length polymorphism (RFLP) analysis to reveal polymorphisms.
Provide examples of how these techniques can be used in the diagnosis and/or
treatment of disease. (Obj. 1, 2 and 3)
6. Rationalize the basis for using DNA sequencing methods to analyze a genetic
condition. (Obj. 2 and 3)
7. Describe comparative genomic hybridization (CGH), providing examples of the kind

of information that can be derived, and explain its limitations in terms of the mutations
being analyzed. (Obj. 1,2 and 3)
8. Explain the basis of chromosomal microarray analysis and how array based
techniques can be used to globally genotype individuals at numerous genetic loci. (Obj.
1,2 and 3)
9. Explain how PCR can be used to detect small genetic mutations and give an example
of how the results can be used to determine the treatment course for a disease. (Obj. 1,
2 and 3)
10. Describe the fundamental basis for the technique of fluorescent in situ hybridization
(FISH) including the types of probes used and the information that each can reveal.
Interpret the result of the test given the LSI/CEP (locus specific identifier/centromere
enumeration probe) ratio. (Obj. 1, 2 and 3)
11. Describe transcriptomics and explain its utility. Rationalize how surveying thousands
of genes at once allows for greater diagnostic precision, as well potential for improved
therapy of diseases such as cancer. (Obj. 1,2 and 3)
12. Summarize the use of reverse transcription - polymerase chain reaction (RT-PCR)
in the diagnosis of infectious disease. (Obj. 1, 2 and 3)
13. Explain what is meant by next generation sequencing and the motivation behind its
development. (Obj. 2 and 3)
14. Summarize the steps utilized by the lllumina sequencing platform. (Obj. 2)
15. Provide examples of the clinical utility of next generation sequencing. (Obj. 3)
16. Outline the steps involved in producing recombinant human growth hormone.
Explain the uniqueness of the approach that they took and why it was necessary. (Obj.
2)
17. Given a case be able to design a viable approach (using the techniques discussed
in the last three sessions) for the screening, diagnosis or treatment of the condition
described. (Obj. 1, 2 and 3)
I. The use of Molecular Techniques in Diagnosis and Screening of

Disease
a. Recall the cookbook analogy introduced by Dr. Ritchie in BMB 515 in

the DNA and Chromosome Structure lecture. In the kitchen, there are
two sets of cookbooks: one set passed down from Moms family and
another from Dads.
i. Each set has 23 books where one book = one chromosome.
1. Each book is themed by country of origin. Book 1 (both Moms

set and Dads set) contains recipes from Afghanistan, etc.
2. The recipes in the cookbooks from Mom and Dad are for the
same dishes but have slight variations in ingredients. In the book
containing Mexican recipes, for example, Moms family cookbook
has beef tamales while the corresponding one from Dads has
pork tamales.
ii. Each recipe in a book corresponds to a gene.
iii. Each word in a recipe corresponds to a triplet of

nucleotides.
iv. Each letter in a word corresponds to one nucleotide.
v. Nucleotide - a single letter in the words of a recipe
Dad's Mom's
Cookbooks Cookbooks
b. DNA Polymorphisms
i. It has been estimated that there are approximately 30 million base

pairs of variation between individual human genomes. When a
particular variation (at a given locus) is present in more than 1% of
the population it is called a polymorphism. The polymorphism may
be a change that is harmless; lead to an increased susceptibility to
a disease; or, infrequently, cause a disease. Polymorphisms are
often used to diagnose disease. There are three major types of
polymorphisms:
D N A M olecule 1. Single nucleotide
polymorphisms (SNPs;
V ersio n 1 GG CTTAC
snips) consist of a
V ersio n 2 GGCATAC
single base change (one
V ersio n 3 G G CC TAC
t letter change in a word of
SNP
D N A M olecule a particular recipe).
Versio n 1 (C G G )s
Versio n 2 (C G G )160
2. Tandem repeats
Versio n 3 (C G G )2eo (variable number tandem
f repeats, VNTR) are short
VNTR
sequences of DNA at
various locations in the
D eletion D u p lication
-
genome that are
1 co py o f B 3 co p ies o f B J
repeated one after
another (one word of a
recipe repeated many
(M. Faner, MSU) times). The number of
repeats varies among
Three Types of Polymorphisms A. Single nucleotide
polymorphism (SNP) B. Variable number tandem repeats individuals.
(VNTR) C. Copy number variation
3. Copy number
variation (deletions and duplications) is when there are different
numbers of copies of a specific genetic sequence (deletion or
repetition of a particular recipe).
c. Inherited genetic disease
i. When the gene or the specific mutation is known.
1. Southern blotting is used to detect VNTRs in Fragile X

syndrome.
a. Fragile X syndrome (BMB 527 genetic anticipation lecture) is

the most predominant cause of heritable intellectual disability.
The syndrome is associated with a variable number of tandem
repeats of CGG (a word in the recipe is repeated over and
over) in the 5 UTR of the fragile X mental retardation gene 1
(FMR1) gene. FMR1 is located on the X -chrom osom e and the
syndrom e dem onstrates X -linked inheritance.
b. The greater num ber of CGG repeats the m ore affected the
individual will be. The unstable nature of the repeats can lead to
repeat expansion during m eiosis in a carrier fem ale; because of
this each successive generation is at a higher risk of expressing
the syndrom e. Southern blotting is the gold standard for
diagnosing fragile X syndrom e.
c. The procedure is as follow s:
i. Extract patient DNA and digest with EcoRI to obtain fragm ents
containing the FMR1 gene.
ii. Perform gel electrophoresis and blotting.
iii. H ybridize with a probe that is com plem entary to a region

adjacent to the mutation.
iv. Im age the blot to visualize the size of the repeat.
d. The num ber o f repeats and the ir associated phenotype are as

follow s:
i. R epeats from 0-44 are normal
ii. R epeats from 45-54 are interm ediate
iii. R epeats from 55-199 are perm utation
iv. R epeats greater than 200 have the full m utation
Probe
(CGG)n FMR1 ORF
5 UTR 11 i w
A E c o Rl
EccR \
| = affected
^ = mildly affected
} = appeared normal/
not tested
Adapted from Rousseau eta/. 1991. N E ng !J M ed. 325:1673
VNTR A n a ly sis in F ra g ile X S y n d ro m e A. Schematic of the

S o u th ern B lo t a n d
FMR1 gene showing the location of the VNTR and probe B . Detection of fragile X
syndrome in a family
2. Multiplex polymerase chain reaction followed by allele specific

oligonucleotide probes for cystic fibrosis screening.
a. Cystic fibrosis (CF) is a common autosomal recessive disorder

that manifests as chronic pulmonary disease and exocrine
pancreatic insufficiency. The disorder is caused by mutation of
the cystic fibrosis transmembrane regulator (CFTR) gene (PSL
536 epithelial transport lecture). More than 1,800 mutations in
the gene have been reported. Mutations are most often SNPs,
a deletion, addition, substitution of a single letter in the recipe.
The 23 mutations with a frequency of greater than 0.1% are
most often tested for on screening panels. An example that
tests for 8 mutations is discussed below.
i. Multiplex PCR involves performing more than one PCR

reaction in the same tube. In the case of this CF screening
example 7 primer pairs were designed to amplify regions of
the patients CFTR gene that have a potential mutation. The 7
primer pairs will be sufficient to test 8 mutations because one
of the regions has 2 possible mutations. The product of the
multiplex PCR will be billions of copies of each of the 7 CTFR
regions. The amplified DNA is then probed using ASOs.
ii. Allele specific oligonucleotides (ASOs) are probes that are

designed to be sensitive enough to detect a single nucleotide
change (a single letter in the recipe). The probe will hybridize
to the target DNA and produce a signal if and only if it is a
perfect match. In the CF screening example ASOs were used
to probe the multiplex PCR reaction described above. The
general procedure is as follows:
1. Apply the multiplex PCR reaction products to a membrane.
2. Hybridize with probes for wild type and mutant alleles

designed for each region of interest (8 regions in this case
for a total of 16 probes).
3. Image the membrane to determine the genotype of the

patient.
a. Example: The Patient 1sample produces a signal
for AF508 but not for F508(wt) indicating that the
genotype is homozygous for the AF508 mutation.
The patient does not have any of the other
mutations because there are only signals for the wt
alleles.
b. Now interpret patient 8.... will post answer on D2L.

Patient
1 2 3 4 5 6 7 8
F508{W T>
AF508 m
G 5 4 2 {W T ) m
G 542X m
1 7 1 7 -1 G {W T ) m m m m m m m
1 7 1 7 -1 G >A m
2 1 8 3 A A (W T ) m m m m m m m
m 2 1 8 3 A A i-G m
R 553(W T) m m m m m m m
R553X
W 1 2 82 (W T ) m m m m m m m
W 1282X
N 1 3 03 (W T ) m m m m m m m m
N 1303K
M 48 (W T ) m m m m m m m m
I1 4 8 T m
J M ed Genet. 33:475
A S O D o t B lo t Im a g e from A n a ly sis o f E ig h t C ystic F ib ro sis P atien ts
3. Sanger sequencing for whole gene analysis in cystic fibrosis

diagnosis.
a. In the USA it is standard for all newborns that screen positive

for CF to undergo genotyping. A positive screen is indicated by
a combination of lab tests (sweat chloride and immunoreactive
trypsinogen) and a screening panel (like the ASO dot blot
above). Genotyping is important because it can indicate
potential molecular therapies as well as predict variant
presentations like late onset, pancreatitis, or vas deferens
anomaly.
b. Genotyping is straightforward if the patient has 2 common

mutations that are identified using a screening panel but in
some cases only one or neither of the mutations is detected. In
3862 order to identify both mutations that patient
I
C T C T T G N G A AG A. would be referred for whole gene analysis.
5 -> 3 (forw ard strand)

c. The method of choice is Sanger
(dideoxy) sequencing. The size of the
CFTR gene (-189 kb) necessitates the use
of many sequencing reactions that together
cover the regions with clinical significance
(exons, splice junctions, regulatory regions
and a few intronic regions).
d. The example shown is a patient who

Lucarelli etat. 2006. Ana! Biochem. 353: 226
is heterozygous for the mutation 3862 G
S eq u e n c in g D a ta fro m a P a tien t A (a change in a single letter of the recipe).
w h o is H ete ro zy g o u s fo r th e So on Sanger sequencing, both G and A
M u ta tio n 3 8 6 2 G -> A in th e C F T R
G en e
show up on the colored trace for position
3862.
ii. When the genetic cause is unknown.
1. Comparative genomic hybridization (CGH) for unexplained

developmental delay/intellectual disability (DD/ID), autism
spectrum disorders (ASD) and multiple congenital anomalies
(MCA).
a. CGH is a technique that maps complete or partial chromosome

gains and losses (resolution 5-10 Mb, one half to a whole cookbook)
in a genome wide and objective way. It is useful when there is no
prior information about the chromosomal aberration.
b. CGH compares the hybridization of genomic DNA from cells of

interest (test DNA) and genomic DNA from karyotypically normal cells
(reference DNA) to a normal metaphase (when chromosomes are
condensed and aligned at the equator of the cell) spread. A typical
analysis would include the following:
i. Test DNA and reference DNA are labeled with a green and a
red fluorochrome, respectively.
ii. The labeled DNA samples are mixed in equal amounts and
cohybridized to a normal metaphase spread.
iii. The fluorescent image is captured and the green to red signal
ratio is quantified digitally.
iv. The signal ratio reveals copy number changes. An elevated

green to red ratio indicates a gain of material and a reduced
green to red ration indicates a loss.
Test DNA Control DNA
w i / - V . **!
i
Digital image processing
Fluorescence
microscopy 1
Fluorescence Ratio
Modified from Oostlander eta/. 2004. C/in Genet. 66:488 and Riegel. 2014. Genet Mo/Bio/. 37: 194
C o m p a ra tiv e G en o m ic H yb rid iza tio n P ro ced u re

Comparative Genomic Hybridization A. Fluorescent image of a whole genome CGH B. Close up
chromosome 13 shows a red region indicating a loss of genetic material C. Quantitation of the
fluorescent signal from chromosome 13 verifies a loss of genetic material.
2. Array comparative genomic hybridization (array CGH) for

unexplained developmental delay/intellectual disability (DD/ID),
autism spectrum disorders (ASD) and multiple congenital
anomalies (MCA).
a. Array CGH is based on the same principles as traditional CGH

but instead of hybridizing to whole chromosomes (metaphase
spread) the test and reference DNA is hybridized to fragments
of chromosomal DNA of a known location. The origin of this
known DNA is bacterial artificial chromosomes (BAC) clones or
synthetic oligonucleotides. The fragments are spotted on a grid
of precise location (an array).
b. A rray CGH w as a significant im provem ent in technology
(resolution down to 50 kb o r about a single recipe) from
traditional CGH and is now the first tier diagnostic fo r these
patients.
Test DNA Control DNA
D ig ita l image processing

Fluorescence
microscopy
Hybridization Fluorescence Ratio
Oostlander e ta l. 2004. Clin G e n e t 66: 488
A r r a y C o m p a ra tiv e G en om ic H y b rid iza tio n P ro ced u re
c. M any poorly understood syndrom es have now been classified

using array CGH. The exam ple show s three individuals with
unexplained mental retardation w ho when the ir genom es w ere
analyzed w ere found to have 17q21.31 m icrodeletions.
Array CGH A. Three individuals with unexplained developmental
delay and the same distinct clinical phenotype B. Array CGH analysis
identified a 17q21.31 microdeletion in the three patients.
d. Cancer
i. PCR for epidermal growth factor receptor 1 (EGFR1) testing in lung
adenocarcinoma.
1. With a case mortality of - 85%, lung cancer is the most lethal
cancer in the USA. In the following example a specific type of
lung cancer, non-small cell lung adenocarcinomas with an
EGFR1 mutation have a mean survival of 8 months (without
treatment).
2. EGFR1 is a tyrosine kinase (BMB 515 Receptor to nucleus
signaling cascades lecture) that when mutated can lead to
cancer because of dysregulated cell growth and proliferation.
EGFR1 mutations that have been observed to cause cancer are
small deletions and point mutations (changes to a single letter in
the recipe).
3. EGFR1 mutant adenocarcinoma can be treated with a tyrosine

kinase inhibitor (erlotinib, gefitinib, afatinib) to prolong survival
from 8 months to 2 years. Tyrosine kinase inhibitors are not
effective against any other type of lung cancer therefore it is
essential to identify individuals who will respond before
administering the drug.
4. A PCR based technique called allele refractory mutation system

(ARMS) can be used to determine which lung cancer patients are
suitable for treatment with a tyrosine kinase inhibitor.
5. The technique depends on a property of Taq polymerase that

prevents it from extending from a primer that has a 3 mismatch
with its target. In an ARMS analysis a primer is designed so that
it will be complementary to the mutant allele and will only extend
from mutant DNA. Therefore, if a product is obtained, that patient
has an EGFR mutation and is eligible for treatment with a
tyrosine kinase inhibitor.
Mutant specific prim er extends on mutant template
Mutation of interest Taq

/
Mutant specific primer
I I I I I I II I I I I II
Mutant template I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Mutant specific primer does not extend on wild type template
Taq
Mutant specific primer

Wildtype template I I I I I I I I I I I I I I I I I I I I I I I I I I I I
(M. Faner, MSU)
Allele Refractory Mutation System (ARMS) PCR

6. This assay is available as an FDA approved com panion
diagnostic from Q iagen under the trade nam e therascreen
EG FR RGQ PCR kit . The kit screens fo r 21 different m utations,
15 of w hich indicate use of afatinib. The other 6 indicate
resistance to the drug.
ii. Fluorescence in situ hybridization (FISH) fo r EG FR2 gene

am plification (one recipe appears m ultiple tim es) in breast cancer.
1. FISH uses fluorescently labeled probes to detect chrom osom al

abnorm alities in tissues, blood and cytology sam ples. M any
types of cancer contain chrom osom al abnorm alities including a
type of breast cancer that is caused by EG FR2 (a m em ber of the
sam e fam ily of tyrosine kinases as EGFR1) gene am plification.
The am plification of the EG FR2 gene leads to an overexpression
o f the encoded tyrosine kinase w hich leads to cancer.
2. To detect EGFR2 gene am plification using FISH tw o types of

probes (labeled with different colors) are used.
a. A centrom ere enum eration probe (CEP) targets repetitive

sequences near the centrom ere of a specific chrom osom e. The
purpose of the probe is to identify the num ber of copies of a
specific chrom osom e in the cell.
b. A locus specific identifier (LSI) probe targets a specific region of

interest on a chrom osom e to detect chrom osom al gains, losses
o r translocations.
3. The probes are hybridized to a fixed tissue sam ple and annealing
is visualized and quantified using a fluorescent m icroscope and
associated com puter software. The copy num ber o f the EGFR2
gene is assessed using the LSI/CEP signal. A ratio >2 is positive
fo r gene am plification and a ratio <2 is negative.
4. A positive result fo r EG FR2 gene am plification indicates the
patient is likely to respond to a m onoclonal antibody drug
(trastuzum ab, trade nam e Herceptin) that targets EGFR2.
5. A n FDA approved com panion diagnostic from Dako called

HER2 IQFISH pharm D x (FYI, EGFR2 is aka HER2) uses the
approach described above.
Fluorescence in situ Hybridization Analysis of EGFR2. EGFR is a.k.a HER2

iii. Microarray gene expression profiling to determine breast cancer
prognosis.
1. Breast cancer patients with poor prognostic features benefit the

most from adjuvant chemotherapy. Chemotherapy treatment of
patients with good prognostic features often does more harm
than good. One way to determine the prognosis of breast cancer
patients and therefore the appropriate treatment is by
determining the gene expression signature (transcriptomics) of
the tumor using a microarray platform.
2. This method follows the same principle as array CGH but instead
cDNA is used both for the target sequences on the chip and for
test and reference samples. By monitoring cDNA levels the
amount of RNA being transcribed from a given gene is measured
(how many times the chef made a given recipe).
3. The example shown demonstrates how the expression of a

group of 70 specific genes in tumor tissue correlates with the risk
of metastasis and death. A microarray platform that analyzes the
expression of these 70 genes is now an FDA approved in vitro
diagnostic offered by Agendia under the trade name
MammaPrint.
=
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Reporter Senes
van de V ijver. 20 02. N E n g iJ Med. 25: 1999
G en e E x p ressio n P ro file o f 295 P a tien ts w ith B rea st C an cer
e. Infectious Disease
i. Hepatitis C is a serious form o f viral hepatitis caused by the

hepatitis C virus (HCV). In the USA, it is estim ated that 3.2 million
people are chronically infected with HCV and hepatitis C is the
leading cause of death from liver disease.
1. HCV has a single stranded RNA genom e that harbors one ORF
bordered by a 5UTR and a 3UTR. Further discussion to com e
in MMG 532.
ii. Reverse transcription - polym erase chain reaction (RT-PCR) to

detect active HCV infection.
1. It is necessary to perform a laboratory test to diagnose HCV

infection because the sym ptom s are scarce and nonspecific. RT-
PCR is a sensitive test com m only used to diagnose HCV
infection. The general protocol is as follows:
a. The patient serum or plasm a is separated from a blood sam ple.
b. The HCV RNA genom e is converted to cD N A using a prim er

that is com plem entary to the 5 UTR and the reverse
transcriptase enzyme.
c. The cD N A is then am plified by PCR.
d. A probe that is com plem entary to the HCV PCR product is

allowed to hybridize.
e. The hybridization m ixture is analyzed using gel electrophoresis

and im aged according to the nature of the label on the probe.
HCV in blood
HCV RNA
cDNA
PCR primers
PCR products
Probe hybridization
(M. Faner, MSU)
RT-PCR Procedure to Diagnose Active HCV Infection
f. An FDA approved in vitro diagnostic that uses this approach is

COBAS AMPLICOR HCV Test by Roche Molecular
Diagnostics.
iii. Restriction fragment length polymorphism (RFLP) for HCV

genotyping.
1. There are six major genotypes of HCV. The treatment that a

patient will benefit most from is specific to the HCV genotype
infecting the patient. The treatment is expensive and has severe
side effects therefore it is essential for physicians to test for the
genotype to determine an appropriate course of treatment.
2. RFLP is one method that can be used to determine the genotype

of HCV. This technique is founded on the idea that genotype
specific polymorphisms result in genotype specific restriction
sites. The procedure is as follows:
a. The patient serum or plasma is separated from a blood sample.

b. The HCV RNA genome is converted to cDNA using a primer
that is complementary to the 5UTR and the reverse
transcriptase enzyme.
c. The cDNA is then amplified by PCR.
d. The PCR product is amplified by a second round of PCR in

order to obtain quantities sufficient for visualization using a
stain.
e. The PCR product is digested with a series of enzymes that will

produce a characteristic pattern for each of the genotypes.
f. The restriction fragments are analyzed using gel

electrophoresis and stained for visualization.
g. A patient whose restriction pattern matched that of genotype 1a

would receive a different treatment regimen than if it had
matched genotype 2. Examples of the restriction patterns for
these genotypes are shown in the image below.
HCV COL_175 HCVCOL_120

Genotype 1a Genotype 2
MW , 7 MW
(bp) B stU I H in fl R sal M v a l' (bp) B s tU I H in fl R sal M val
330
330
100
100
10
10
Filippo e ta t. 2012. Virol J. 9:242
Genotyping HCV using RFLP

f. When to use Which Technique
i. It is the clinical information that should dictate the answer...then do

the cheap stuff first.
1. Does the clinical presentation and manifestation suggest a

known condition?
2. Is the condition associated with a chromosomal disorder that

would be observable on karyotype analysis?
3. Is the etiology of the condition associated with a specific gene?
4. Are there common mutations known for this candidate gene

such that appropriate probes/panels are already available?
ii. In certain cases a genetic cause is suspected for the clinical

observations but not ascribable to any known genetic abnormality
(e.g. unexplained developmental delay or intellectual disability). In
these cases you need to go to high throughput techniques such as
array CGH or whole exome sequencing (see required reading).
iii. The decision tree below can help you decide an appropriate
technique when faced with a clinical scenario. As you can see more
than more technique will allow you to arrive at similar information.
Is the causative gene known?
No Yes
Karyotype
CGH (5-10 Mb)
Array CGH (50 Kb) I
Whole exome sequencing (single nt) Is the causative mutation known?
Single nucleotide Larg e^

polymorphism amplification/deletion
RFLP FISH RFLP
ARMS-PCR Array CGH Southern Blot
Southern Blot Sanger Sequencing
ASO
Further investigation
Northern Blot
Western Blot
iv. Ex. Which of the following is the most appropriate test for a
newborn suspected to have pyruvate dehydrogenase deficiency
(high blood levels of pyruvate, lactate, and alanine;
unresponsive to thiamine, lipoic acid, and biotin administration)?
A) Routine banded karyotype

B) Array CGH
C) Sanger sequencing of genes coding for subunits
D) Whole exome sequencing
E) Targeted mutation analysis such as ASO
v. The only way to learn how and when to use the technology
discussed in these lectures is to practice using the problems in
the problem sets and practice exams in both BMB 515 and BMB
527.
II. Use of Molecular Techniques in the Treatment of Disease
a. Production of therapeutic proteins

i. Therapeutic proteins such as insulin and human growth hormone
can be produced in bacteria using recombinant DNA technology.
ii. Human growth hormone (HGH) is a 191 amino acid protein that
when deficient causes dwarfism. Growth can be restored in deficient
individuals by administering exogenous HGH. Prior to the early
1980s the only source of exogenous HGH was a limited supply
from the pituitary glands of human cadavers. The potential for using
bacteria to produce HGH in this case is clear.
iii. The decision to use E. coli as the host and a bacterial plasmid as
the vector was straight forward. The design of the insert DNA
required more creativity. The length of the protein is such that it
would not be efficient to create a fully synthetic version. They also
could not use a natural cDNA version since it contains the sequence
of a signal peptide that must be cleaved in order to create the
mature protein (prokaryotes do not have the machinery required for
this eukaryotic process). These considerations led them to develop
a unique combination of the two approaches which is described
below.
1. The cDNA for HGH was created and purified.
2. A natural EcoRI site was used to remove the mammalian signal

sequence. Because of the position of the restriction site the
codons for the first 24 amino acids were also removed.
3. To restore the first 24 codons a synthetic oligonucleotide coding

for amino acids 1-24 was created and ligated to the cDNA for
amino acids 24-191 using matching EcoRI sticky ends.
4. The insert DNA and the expression vector were digested with
HindiII and then ligated. The insert was joined with the vector
such that it is adjacent to a bacterial promoter sequence which
allows the mammalian DNA to be expressed in E. coli.
5. The recombinant plasmid was transformed into E.coli and now
we have the capability to express and purify the protein for use
as therapy in individuals deficient in human growth hormone.
iv. Recombinant DNA techniques can also be used to produce more

complex proteins in mammalian cell culture. Examples include
tissue plasminogen activator and hematopoeitic growth factor.
Required Reading
a. Future Direction: Next Generation Sequencing
v. The first draft of the human genome cost about 3 billion dollars and
took several years to generate. The primary technology used was
bacterial cloning (as discussed in the Basis of Molecular techniques
lecture, using bacterial artificial chromosome (BAC) vectors)
followed by Sanger sequencing. Each capillary electrophoresis
instrument (the instrument required to semi-automate Sanger
sequencing) is capable of generating 96 concurrent sequences in
about 3 hours (low through put).
vi. For whole genome sequencing to be a useful clinical tool the cost
and time need to be substantially less than Sanger sequencing.
During the same time as the first human genome draft was being
created new sequencing technology was being developed in
response to that demand. Several different platforms have been
developed, all of which are referred to as next generation
sequencing. The key to the efficiency of all of the platforms is the
ability to perform massively parallel (billions of sequencing reads at
once) in a non-relational format. The non-relational aspect of this
type of sequencing is made possible because a draft sequence was
created using Sanger sequencing previously. One of the next
generation platforms is described in detail below.
vii. Illumina sequencing technology
1. The first phase of the protocol involves amplifying the DNA to

achieve a quantity that will produce a signal large enough to be
detected by the sequencing instrument. The Illumina platform
uses bridge amplification for this purpose.
a. Randomly fragmented genomic DNA is ligated to synthetic DNA

adapter sequences.
b. Bind/anneal the single stranded fragm ents to the surface o f a
flow cell. The surface of the flow cell is coated w ith a dense
lawn o f prim ers that are com plem entary to the adapter
sequences.
c. Add dN TPs and polym erase to initiate am plification. Extension

o f flow cell prim ers using the fragm ented genom ic DNA as a
tem plate results in copies of the genom ic DNA being tethered
to the flow cell surface.
d. The tethered genom ic DNA copies can then anneal with

adjacent prim ers on the flow cell surface to create a bridge.
e. The prim er is extended using the bridge DNA as a tem plate.
f. The double stranded bridges are denatured leaving another

tethered copy of genom ic DNA.
Adapters
DNA
Fragments ||JI m b *
r V T
DNA
Polymerase
M. Faner, MSU C usters
A m p lifica tio n P h a se o f Illu m in a S eq u en cin g. Letters in the figure correspond to the text
discussion above.
g. Cycles of denaturing, annealing and extension (just like
conventional PCR) are repeated to create several million dense
clusters of genomic DNA copies.
2. After amplification the genomic DNA copies tethered to the flow

cell are sequenced using a technique called sequencing by
synthesis.
a. To prepare for sequencing the reverse strands of DNA are

cleaved and washed away.
b. To initiate sequencing four fluorescently tagged nucleotides,

primers, and DNA polymerase is added. A key feature of this
technology is the fluorescently tagged nucleotides. They are
nucleotides that have been modified by adding a fluorescent
tag at the 3 position. The tag not only allows the monitoring of
which nucleotide was incorporated but also blocks the 3
position so that no more nucleotides are allowed to be added.
c. The four fluorescently tagged nucleotides compete for addition

to the growing chain of DNA being synthesized by DNA
polymerase. Only the nucleotide that is complementary to the
template strand will be incorporated.
d. After the addition of the nucleotide the fluorescent signal is

read. The signal determines the base call.
e. The blocking tag is chemically removed from the nucleotide.
f. The next four fluorescently tagged nucleotides are added and

the cycle is repeated until the read is completed.
g. This process is occurring simultaneously for all strands in a

cluster and all clusters on a flow cell making it massively
parallel.
h. The data are then aligned to a reference genom e.
i. Please w atch the Y outube video on this process at

http://w w w .youtube.com /w atch?v=w om K fikW lxM .
S eq u e n c in g b y S y n th esis P h a se o f Illu m in a S eq u en cin g. Letters in the figure correspond to

the text discussion above.
viii. The application of next generation sequencing in rare M endelian

disorders
1. In general this technique is useful to determ ine the cause o f a

disorder when one is unknown. I t can detect S N P s and small
duplications and am plifications anyw here in the genom e
(changes in a single letter of a recipe and repeated o r deleted
sections of recipes).
2. The current Illum ina platform can sequence a w hole human

genom e fo r $1,000 (cost o f reagents, depreciated cost of the
instrum ent, and cost of paying technicians to operate it) but the
instrum ent costs at least $10 m illion dollars. V ery fe w custom ers
have the volum e of sam ples that would m ake the investm ent
worth it. T here are also a significant am ount of oth er overhead
costs (i.e. electricity) that m ake it prohibitive fo r m ost institutions.
Until w hole genom e sequencing becom es feasible fo r everyone
scientists have devised w ays to use next generation sequencing
in m ore targeted and therefore m ore affordable w ays for the
clinical setting.
3. G enom e com plexity reduction techniques allow a clinical test to

target m ultiple genes as part o f a sequencing panel o r even
w hole exom es (every exon in the genom e). O ne technique uses
hybridization w ith oligonucleotide probes to select for the
sequences of interest.
4. O ne exam ple in w hich w hole exom e sequencing has been

particularly useful is in the identification o f the genetic basis of
rare M endelian (m onogenic) diseases. Factors such as the
infrequency o f the disease, locus heterogeneity, and reduced
reproductive fitness have resulted in less than half o f m onogenic
diseases having a known underlying genetic basis.
5. W hole exom e sequencing has been used to identify m utations in

the gene fo r the enzym e dihydroorotate dehydrogenase
(pyrim idine biosynthesis) as the genetic basis o f M iller syndrom e.
Whole Exome Sequencing in the Identification of Rare Mendelian Disorders.
A. Phenotypic features of an individual with Miller syndrome. B. Locations
of mutations found in the dihydroorotate dehydrogenase gene of individuals
with Miller Syndrome.
v. The application of next generation sequencing in reproductive

health
1. Next generation sequencing also holds tremendous promise in

the context of reproductive health. During pregnancy an
appreciable amount of cell-free DNA in maternal blood is derived
from the fetus. Sequencing of the cell-free fetal DNA is being
rapidly adopted to detect aneuploidies. In the future, similar
methods may be able to diagnose most Mendelian disorders
prenatal ly.
2. Look forward to more discussion on this in BMB 527.

DNA Repair
We have now covered the mechanism by which DNA is replicated for the
purpose of passing on the genetic information to its progeny. We will next
consider how uncorrected errors in replication and reactions that damage
DNA occur to alter the information coded for by DNA. We will distinguish
between the physical changes in the DNA to the corresponding informational
changes that occur.
As the repository of genetic information it is essential that DNA damage is
repaired. While damage is infrequent it is significant due to the low biological
tolerance for DNA changes therefore we will discuss the mechanisms by
which these changes are repaired. Finally, we will discuss how changes in
DNA sequence influence genetic diversity.
Suggested Reading:
Ferrier: Chapter 30: DNA Structure, Replication, and Repair - DNA R e p a ir Chapter 32:
Protein Synthesis - Consequences of Altering the Nucleotide Sequence
Turnpenny: Chapter 2: The Cellular and Molecular Basis of Inheritance - from Mutations
through Mutations and Mutagenesis (p. 22-28)
Objectives:
1. Understand the different sources of DNA damage and the effect that they have on the
DNA structure and function (mutation, deamination, depurination, intercalating dyes,
radiation damage transitions, transversions, insertions, deletions, as well as silent,
missense, nonsense, frameshift mutations, cell-cycle, apoptosis).
2. Know the various mechanisms of repair used to correct DNA damage (direct repair, base
excision repair, nucleotide excision repair, mismatch repair).
3. Understand the impact of change in the DNA sequence on genetic diversity (allele,
locus, homozygous, heterozygous, wild type allele, mutant allele, loss of function mutation,
gain of function mutation).
1. Describe the process by which physical damage to DNA becomes fixed as a

permanent mutation. (Obj. 1)
2. Describe how deamination, depurination, intercalating dyes and other chemicals, and
radiation damage results in mutations. (Obj. 1)
3. Recognize and provide examples of transitions, transversions, insertions, deletions, as

well as silent, missense, nonsense, and frameshift mutations. (Obj. 1)
4. Distinguish between the effects of mutations in protein-coding regions and in

regulatory regions of DNA. (Obj. 1)
5. Distinguish between cell-cycle arrest and apoptosis as cellular responses to DNA

damage. (Obj. 1 and Obj. 2)
6. Describe the general features of the following DNA repair mechanisms, and human
genetic diseases associated with defects in these mechanisms: (Obj. 2)
a. Direct repair of alkylation and UV photoproducts.
b. Base excision repair and DNA N-glycosylases.
c. Nucleotide excision repair pathway
d. Mismatch repair
7. Define and apply the following terms: allele, locus; homozygous and heterozygous;
wild type and mutant alleles. (Obj. 3)
8. Describe and distinguish the biochemical and genetic effects of loss of function and gain
of function mutations. (Obj. 3)
I. DNA Mutations (Damage)

A. Key Principle: DNA damage yields permanent mutations ONLY when
'sealed in' by replication
If damage is repaired properly, nothing happensnot a mutation
B. Causes and effects of mutations

1. Spontaneous mutations
a. replication errors: base mismatches
(i.e. a DNA polymerase error that is not caught by the proofreading
function)

b. changes to bases: affecting base-pairing
Example: deamination
(i.e. cytosine can spontaneously deaminate and form uracil, which is
NOT a DNA base)
0(3
loss of bases from nucleotides: produces gaps in template

d
Example: depurinationclips off a purine base (breaks N-

glycosidic bond such that the base is gone, but phospho-
deoxyribose backbone is still intact)
< i-
2. Chemically induced mutations

a. oxidative damage
(1) Bases are modified
(2) Addition of hydroxyl groups
(3) Causes shape changes in how fits in
helixresults in mispairing
b. alkylation (carbon/hydrogen groups)

(1) Bases are modified
(2) Addition of methyl (-CH3)
or ethyl (-CH2CH3) groups
(3) Now cannot make proper hydrogen thymine
bonds between basesresults in mispairing of bases.
c. nucleotide (or nucleoside) analogs

(1) Commonly used in chemotherapy
(2) Extensive mispairing>-many mutations>oancer cell
dies 0
5-fluorouracil
d. intercalating dyes
(1) Inserts between base pairs
in helix and pushes bases in a single
DNA strand apart
(2) Result: new base is inserted
across from where dye sits to fill in gap frameshift mutation
3. Radiation damage to DNAtwo types
a. ultraviolet (UV) irradiation: (sun, tanning
beds)
(1) Pyrimidine dimers and 6-4
photoproducts
(2) So DNA structure is skewed and
cannot base pair correctlycausing
mutations.
b. ionizing radiation: (X-rays, radioactivity)
(1) Causes DNA double stranded breaks
(2) Very hard to repairDNA backbones broken; get many DNA
fragments
(3) Chromosome translocations result (due to chromosome
fragments)
Translocationscell tries to put the DNA pieces back together to form the 46
chromosomes, but usually connects the broken pieces in the wrong order.
C. Informational Impact of Mutations

(Key theme: distinguish physical changes from informational changes)
1. Mutations in protein-encoding regions of DNAthe exons

a. nucleotide substitutions (physical change)2 types:
(1) transitions one purine substituted for another purine

(i.e. A^G);or one pyrimidine substituted for another pyrimidine
(i.e. C>T)
(2) transversions a purine substituted for a
pyrimidine (or vice versa) (i.e. A^T)
possible informational changes:
(1) silent mutations - no effect
(i.e. CAG CAA, both code for Gin)
(2) missense mutations - results in change of
a single amino acid (i.e. CAG CGG;
result Gin Pro)
(3) nonsense mutations - change from amino
acid codon to stop codon (i.e. CAG
(Ferrier7 F32.3)
UAG; result Gin stop)
b. Deletions or insertions (physical change)
possible informational changes: (recall codon is a triplet set of
nucleotides) Addition of base
mRNA
(1) frameshift mutationsif add/delete CAfflCCUAUCGCU
nucleotides NOT in a multiple of 3 Ser
OCI -Ser
Tyr Gly
(2) disruptionsvery large insertion or A d d itio n o f U
deletion of nucleotides
(3) Generally result in non-functional proteins '\ z \ y \ , u c a C c u a u g g c u
Ser Pro M et Ala

(4) NOTE: If insertion/deletion DOES occur 5-E
-E nd | 3 '-E n d -
is sets of 3, ultimately results in D eletion o f C
insertions/deletions of one or
more amino acidsbut is NOT a ' \ A A A , U C A C U jA U G G C U \ / \ / \ / \ ,
Ser Leu Trp

frameshift mutation)
Deletion of base
(Ferrier7_F32.5)
c. Duplications or amplifications (physical change)
[Often occur in meiosis when homologs are paired up; or in DNA
replication with the sister chromatids]
possible informational changes:
(1) gene dosage effectsextra copies of gene t [protein]
problems
(2) evolutionary impactkeep one copy intact, try to improve
other copies or have them take on new functions
d. RNA splicing mutations
2. M utations in re g u la to ry re g io n s o f DNA (physical change)
possible inform ational changes:
a. increased o r decreased expression o f the gene
b. incorrect response to expression signals
c. transcriptional regulatory signals m ake m ore/less of RNA
d. translational regulatory signals m ake m ore/less of protein
E x e rc is e to c o n firm o u r u n d e rs ta n d in g
M u ta tio n s
In s tr u c tio n s : W ith a partner, w o rk through the first exam ple to g e th e r. Then,
take turns on the next exam ples. Be sure that both partners understand the
concepts and how to approach these problem s!
************************************************************************************
E x e rc is e 1 . In each o f the follow ing pairs of DNA sequences, the normal
sequence is shown on the left and a m utated sequence on the
right. Identify the types o f physical and inform ational changes
represented by the m utations
P hysical: transition, transversion, insertion, deletion
Inform ational: silent, m issense, nonsense, fram eshift/disruptions
Normal Mutation
A. 5'... GCT GTG GGG ...3' 5'... GCT GAG GGG ...3'
CGA CAC CCC DNA CGA CTC CCC
i
Physical: 5'... GCU GUG GGG ...3' RNA 5'... GCU GAG GGG ...3'
i
Info: Ala Protein Ala
B. 5' TTT TGG GGA 3' 5' TTT TGA GGA 3'
AAA ACC CCT AAA ACT CCT
Physical: 5'... UUU UGG GGG ...3' 5'... UUU UGA GGG ...3'
Info:
Phe Phe
C. 5'... GGC CGA TGG ...3' 5'... GGC GAT GGG ...3'
CCG GCT ACC CCG CTA CCC
Physical: 5'... GGC CGA UGG ...3' 5'... GGC GAU GGG ...3'
Info:
Gly Gly
**********************************************************************'*************'*
Exercise 2. Identify the types of physical and information changes that result
in the altered amino acid sequences shown below.
A. ... Pro- Thr- Asp B. ... Leu- Gly- Tyr ... C. Val- Ala- Asp- Ala
... Pro- lie- Asp ... Leu- Gly- Stop ... Val- Pro- Met- Gin
Physical:
Informational:
II. Cellular responses to DNA damage

A. Cell growth control
1. cell cycle checkpoints: G1 arrest

a. During assess DNA damage
b. If too much damage, arrest cell in G1 stage for
repair
(S. Triezenberg and
2. programmed cell death (apoptosis) j.wang,Msu)
a. Severe/widespread damage, cell cannot repair
b. Cell commits suicide, called apoptosisso damage is not
passed on
B. Bypass strategy: trans-lesion synthesis
1. In S phase during DNA replication, if damage encountered
2. specialized DNA polymerases just put in nucleotides to keep going,

finish replication (N means any deoxynucleotide)
--------
________ XX__________
C. Repair mechanisms: actual correction of damageseveral types to

discuss
III. DNA Repair Mechanisms
A. Direct repair of DNA damage

1. dealkylation enzymesremove the methyl, ethyl groups
2. photoreactivation of pyrimidine dimers

Some organisms can use visible light to fix these dimers
This type of direct repair CANNOT be done in humans
(Humans use nucleotide excision repair for pyrimidine dimers)
B. Base excision repairacts on single

bases that are damaged Base Excision Repair
5'
1. DNA N-glycosylase A C T A G G C T C G A
T G A i C C G A G C T
a. removes damaged base by
cleaving N-glycosidic bond Base i* damaged
b. creates AP siteapurinic or I
apyrimidic site apurinic A C T A G G U T C G A
TG A T C C G A G C T
missing purine base;
apyrimidicmissing a Uracil DNA gtycosylase
pyrimidine base
1
c. Note: backbone intact, just 5'
A C T A G G TCG A
base is missing T G A T C C G A G C T
AP endonuclease
2. AP endonuclease (endonucleases .hboM'P
\
cut in the middle of strands)
5' A C T A G G 'C T LC G A
T G A T C C G A G C T
Recognize that base is missing
Cleaves the backbone just to 5- DNA polymerase.
side of AP site DNA ligase \
A C T A G G C T C G A
T G A T C C G A G C T
Deoxyribose phosphate lyase
removes deoxyribose-P
3. DNA polymerase, DNA ligasefills the gap; seals nick

C. Nucleotide excision repairrepairs larger 5' i i i i i i i i i i i i
regions of damage [including pyrimidine dimers 11 1 1 1 1 1 1 1 1 1 1
in humans] 1 Bases are damaged
1. general mechanism 5' i i i i i i i i i
i i i i i i ............
a. Endonucleases + Exonucleases patrol
Endonuclease cuts
DNA remove a short stretch of DNA damaged strand
surrounding damage 5J'T. r r
i
I NI
I I I I I I I I I I I I
b. DNA polymerase fills in gap
I Exonuclease removes
f DNA, including damage
c. DNA ligase seals nick
5'T T~T
2. Clinical note: xeroderma pigmentosum I I I I I I I .........
a. Defect in any of the several genes that Polymerase
makes new DNA
encode for the XP proteins required for
5' I I I I I I TT
nucleotide excision repair in humans - I I I I I I I .........
Patients cannot do nucleotide excision DNA ligase^
repair. seals nick
I II I I I I I I I I I
I II I I I I I I I I I
b. They are VERY sensitive to UV light
because they cannot repair the DNA damage
D. Mismatch repair (e.g. replication errors)
1. Which strand to fix?
a. Both nucleotides OK, but Replication:
mismatched base J *
is incorporated T
mismatched (due to DNA *
polymerase error during replication) CTAG c

Repair of mismatch
in non-methylated 1
b. Parent strandalways correct daughter strand
*
c. Parent strand is methylated in CTAG
various locations Mettiylat on of
So mismatch repair enzymes scan I d a jc n te ' strand
GATC
the DNA for methyl groups to CTAG
identify which strand is the parent
(S. Triezenberg and
strand, then fixes the error on the opposite strand. J. Wang, MSU)
d. Lag in time before daughters strands get methylated to allow

for this proofreading stage
2. Clinical note: hereditary nonpolyposis colorectal cancer (HNPCC):
genetic defects in m ism atch repair
m akes a person m ore susceptible to colon cancer
E. Double-stranded break repair _________________________
1. N on-hom ologous end joining

a. Repair m echanism fo r double-stranded
DNA breaks (i.e. from ionizing ____
radiation)
b. V ery erro r prone and m utagenic
c. Result: chrom osom e translocations
Cell trying to put back pieces to form ------- -
46 chrom osom es ^ ...
However, no tem plate fo r end joining w hen (s. Triezenberg andj. wang, msu>
both strands are broken
Fragm ents of chrom osom es put back together
in wrong order.
2. R ecom bination repair:

a. R ecom bination between hom ologous chrom osom es (or sister
chrom atids) to patch double-stranded breaks or replication
blockage on one chrom osom e
b. Especially during replication w hen one undam aged double

stranded DNA is nearby (i.e., sister chrom atid). Thus, more
prevalent during S/G 2 phase.
c. BRCA1 and BRCA2: key hum an genes that participate in

recom bination repair
W om en w ith defects in either BRCA1 or BRCA2 have a > 80%

chance o f developing breast cancer.
For an online anim ation o f recom bination repair, please visit the you tube
video below: http://w w w .youtube.com /w atch?v=9P E dqB uD M H M
IV. G e n e tic D iv e rs ity a n d D ise a se
A. G enetic Diversity: G enotype Dictates Phenotype
1. G enes encode proteins (or RNAs) that perform specific functions

fo r the cell/organism .
Nucleotide Amino acid Protein folding Protein

sequence sequence ^ & modification activity
Genotype Phenotype
2. I
im plications fo r individuality / adaptation / evolution
B. T erm inology
1. A lleles different form s a gene m ay have in a population

a. DNA sequence differences reflect m utations acquired during
history/ancestry of each chrom osom e.
b. N ote: Diploid organism s (i.e. hum ans) have two alleles of each
gene (one from mom, one from dad)
2. hom ozygous two identical alleles o f a gene
3. heterozygous tw o different alleles o f a gene
4. polym orphic locus a gene in w hich m any different alleles occur at

great frequency w ithin a population.
5. wild type allele the norm al allele, o r the version m ost often seen
in a population
6. m utant alleles are different alleles than norm ally seen

(N ote: m utant alleles are not necessarily defective som e are
im provem ents)
7. locus w here a gene is found on a chrom osom e (i.e. a ge nes

location on a chrom osom e)
8. genotype(1) the genetic constitution of an individual;
(2) More specifically, an individuals alleles present at a
locus
9. phenotypethe observed biochemical, physiological, and

morphological characteristics of an individual as
determined by his/her genotype and the environment
in which it is expressed.
V. Principles of Biochemical Genetics

[How informational changes may lead to diversity OR disease]
A. Central concept:
1. Physiology (traits that are expressed) depends on biochemistry

of cell/organism
2. Biochemistry is accomplished by enzymes (AND structural,

signaling, transport, and regulatory proteins)
3. Enzymes are encoded by genes (as are other proteins and RNA
components)
So: mutations in genes changes in enzyme (or other type of protein)

changes in metabolism
changes in physiology/health
B. Inborn errors of metabolisminherited physiological disorders,

which arise from defect(s) in biochemical pathway(s).
1. How many inherited metabolic diseases are known?

> 1000 defined genetic disorders
2. How frequently do they occur?
a. each distinct disorder: 1/10,000 births

b. in aggregate: textbook indicates 1/2500 live births
m ore likely 1/300 live births will have som e sort
of genetic disorder
3. The biochem istry helps:
a. recognize the disease: recognizable phenotype ^ specific

diagnostic te st
b. understand th e disease
c. identify targ ets fo r therapy:
(1) W ork around the problem (pathway)
(2) Provide the end product by another m eans

O r avoid toxic interm ediate
(3) G ene therapy to restore function
VI. B io c h e m ic a l G e n e tic s : D is tin g u is h b e tw e e n tw o ty p e s o f m u ta n t

a lle le s
[M ain focus here is on m utations leading to disease (as opposed to diversity)]
A. Loss o f function alleles (LO F) m ost com m on (because lots of w ays

to break th e gene)
1. Typically recessive: requires 2 defective copies of the gene to

express the disease
a. gene product (protein, enzym e) is not expressed from m utant
allele
OR
b. m utant allele expresses defective gene product (dim inished
activity)
c. in hom ozygote, pathology arises from:

lack of biochem ical product O R accum ulation of interm ediate
d. in heterozvaote. loss of activity (from one allele) is often
compensated by allosteric regulation of enzyme from wild type
allele
thus, NO pathologybut are carriers
2. Occasionally dominant (are exceptions): only one defective gene

required to express disease
Due to either:
a. haplo-insufficiency - half as much is not enough
(1) when cell depends on a certain amount of a protein; half the

amount is not enough
(2) Examples: structural proteins (collagen); signaling

molecules; receptors
b. dominant negative alleles: mutant protein interferes with

function of normal protein
(1) Proteins that work in pairs (or complexes)when one is

defective, other(s) cannot work either
(2) Examples: leucine-zipper proteins work in pairs; PDH

complex
B. Gain of function alleles (GOF): enzyme (or other protein) does

something new
1. Typically dominant (dominant inheritance)

a. Do the wrong job
OR
b. Do the job, but at wrong time or place
2. Phenotype is (almost always) allele-specific (i.e. only one enzyme

responsible)
a. are very few ways (types of mutations) that will give a new
function
b. Thus GOF alleles are much rarer than LOF alleles
3. gain of function often m eans loss of regulation
a. loss of regulation of expression: gene is O N/O FF w hen it

should not be on/off
b. loss of regulation of activity: a receptor responding w ithout the

signal present
Receptor to Nucleus Signaling Cascades
If you will recall, you were introduced to signaling as one of the major themes of Cell
Biology and Pathophysiology at the beginning of PSL 539 and we noted that we were going
to be returning to the signaling theme throughout PSL 539 and BMB 515. There are six major
receptor-mediated cell-to-cell signal transduction pathways that will be discussed in various
parts of our curriculum as follows:
1. Receptor, itself, is an ion channel (e.g. nicotinic cholinergic receptor).

PSL 539
2. Receptor is coupled via a G-protein to adenylate cyclase and the cAMP cascade
(e.g. (31 , |32 , or J33 adrenergic receptors).
PSL 539
3. Receptor is coupled via a G-protein to phospholipase C and the phosphoinositide
cascade (e.g., a1-adrenergic receptor or M3 muscarinic cholinergic receptor).
PSL 539
4. Receptor as a guanylate cyclase pathway.
(with emphasis on ANF and NO)
PSL 539
5. Receptor as a transcription factor pathway.

BMB 515, Sessions 22-23
6. Receptor as a tyrosine kinase pathway.
BMB 515, Session 28
In Sessions 22-23 we discussed the receptor as a transcription factor signaling

pathway and in this lecture we will discuss the last pathway: receptor as a tyrosine kinase.
The receptor as a tyrosine kinase pathway utilizes receptors which have intrinsic
enzyme activities themselves. We will discuss the general theme of the receptor as a tyrosine
kinase pathway in some depth. We will illustrate the branches of the pathway using the
insulin signaling system. Finally, we will relate the receptor as tyrosine kinase pathway to
oncogenes and cancer.
Discovery of tyrosine kinase activity in components of the hormone and growth factor
signaling pathways and in products of tumor cells provided a major unifying theme between
scientists working on cell growth and their counterparts working on cancer.
Suggested Reading:
Ferrier: Chapter 23: Metabolic Effects of Insulin and Glucagon - Insulin - Mechanism only
Rhoades and Bell: Chapter 1: Flomeostasis and Cellular Signaling - Molecular Basis of
Cellular Signaling - through Plasma membrane receptors activate signal transduction
pathways and Tyrosine kinase receptors signal through adapter proteins to activate the
mitogen-activated protein kinase pathway (p. 9-14); Second Messenger roles - Lipids have
important second messenger regulatory functions, including immune response mediation (p.
18) and Mitogenic Signaling Pathways (p. 21-23)
Required Reading:
Rhoades and Bell: Chapter 1: Homeostasis and Cellular Signaling - Clinical Focus 1.2.
Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia (p. 10)
Objectives:
1. Delineate the domain structure and activities of a tyrosine kinase (TK) receptor and
describe the initial events upon hormone/ligand binding.
2. Distinguish the mitogen-activated protein kinase (MAP kinase) and the phosphatidyl
inositol 3-OH kinase (PI3 kinase) pathways in terms of distinctive intermediates, kinases,
and the types of biological response (SH2 domain, GTPase, Ras, neufibromin, insulin,
insulin-like growth factor).
3. Define oncogenes and proto-oncogenes.
4. Explain how some oncogenes are related to growth factors or growth factor receptors
(epidermal growth factor receptor 1 and 2, chronic myeloid leukemia, lung
adenocarcinoma, breast cancer).
1. Delineate, in terms of structure, binding properties, and enzymatic activity, the domains
of a tyrosine kinase (TK) receptor. (Obj. 1)
2. Explain (or schematically diagram) the events following the binding of hormone/ligand to
a TK receptor: what happens and what is its purpose? (Obj. 1 and Obj. 2)
3. Define the structural and binding properties of a protein containing an SH2 domain. Cite
at least three examples of such a protein. (Obj. 1 and Obj. 2)
4. List, in sequence, the classification of components in the mitogen-activated protein
kinase (MAP kinase) cascade. (Obj. 1 and Obj. 2)
5. Distinguish the three branches of MAP kinase cascade, in terms of the key distinctive
kinase and the general type of biological response. (Obj. 2)
6. Explain how phosphatidyl inositol 3-kinase (PI3K) binds to an activated TK receptor and
how this signal transduction pathway can affect metabolic changes via its effects on
proliferation. (Obj. 2)
7. Using insulin as an example, illustrate signal transduction from a TK receptor via the
MAP kinase cascade and PI3K cascade. (Obj. 1 and Obj. 2)
8. Explain how oncogenes are related to growth factors/growth factor receptors and
provide an example to illustrate the relationship. (Obj. 3 and Obj. 4)
9. Explain how the understanding of oncogenes can be used in the treatment of chronic
myeloid leukemia and breast cancer. (Obj. 3 and Obj. 4)
The Receptor as a Tyrosine Kinase Pathway
I) The receptor is a transmembrane protein
extracellular domain: ligand binding domain (binds
hormone), e.g. epidermal growth factor (EGF)
transmembrane domain: anchors in membrane
intracellular domain: tyrosine kinase (TK)
-----NH - C H - C O i
J* ---------------------^ |
Cep -o-p-o-
Tyr residues oH J
on proteins
W.K k i _ . m., ir | k
A) Hormone binding leads to activation of TK activity (Rhoades4_Fl.ll_pl4)
One consequence of TK activation is autophosphorylation

B) Phosphorylated tyrosines on the TK receptors serves as a tag (flag)
C) These tags are bound (recognized) by proteins with
SH2 domains (Src homology 2) --- domains in certain proteins with amino
acid sequence identity/similarity to the Src oncogene (see below)
chromo domain methylated Lys
bromo domain acetylated Lys
[certain protein modules recognize specific post-translational modifications]
II) Proteins with SH2 domains serve as the coupler between activated TK
receptors and the downstream signaling system
Proteins with SH2 domains bind to the activated TK receptor.
They, in turn, bind other proteins (exchange factors, other kinases),
thereby recruiting these latter proteins to the activated TK receptor
to be phosphorylated at specific Tyr residues by the activated TK.
Two major signaling pathways, each one of which has multiple branches:
A) The Mitogen Activated Protein Kinase Pathway
(uses exchange GTP-GDP factors and GTPases)
B) The Phosphatidyl Inositol 3-OH Kinase Pathway
(uses lipids as signaling intermediates)
III) The MAP Kinase Pathway and Its Branches
(Rhoades4_F1.12_p15)
Basic Elem ents of MAP Kinase Pathway:
Stimulus
Coupler/Adapter
Initial Response
Receptor
Exchange Factor
GTPase
MAPKKK
MAPKK
MAPK
Transcription
Factor
BIOLOGICAI
RESPONSE
(R. Ritchie, MSU)
III) The MAP Kinase Pathway and Its Branches (continued)
Stimulus: EGF Growth Factors
Mitogens
Receptor: EGFR Extracellular domain: ligand binding

Cytoplasmic domain: tyrosine kinase activity
Initial Response: Autophosphorylation of Tyr residues on the

receptor
Coupler/Adaptor: Grb2 SH2-bearing proteins recognize P04-Tyr

Recruit other proteins to the activated receptor
(to be phosphorylated and activated)
Exchange factor: SOS Stimulate GTP-GDP exchange of GTPase
GTPase: Ras
Activated Ras stimulates three branches

of Ser/Thr kinases
I
I 1
MAPKKK: B-Raf MEKK3 M FKK1.4
TAK MLKs
MAPKK:
Y
M E K l/2 MKK3/6
i I
M KK4/7
V J v
SAP/
MAPK: <extracellular signal- (p38 MAP (Jun N'-terminal
regulated kinase) kinase) kinase)
cytoplasm
nucleus
c j a
Transcription c-fos C-jllll
Factor
Biological proliferation differentiation, inflammation

Response apoptosis (programmed cell death)
Clinical Relevance:
Growth factor
Growth factor receptor
Farnesyl
membrane anchor
Activation
Phosphorylation
Bridging | Activates
protein V
Q|rp Inactivation by.
hydrolysis o( GTP
(Modified from K um ar9_Fl.ll)
Failure to inactivate Ras causes cancer due to continuous proliferation. I am

going to show you two examples:
Loss of GTPase activity: Mutations in Ras such that GTP is not hydrolyzed
causes Ras to be permanently activated -> continuous growth signal (e.g.
colon adenocarcinoma).
Neurofibromin (NF-1 protein produced by the NF-1 gene) binds to Ras in

neuronal tissues and inactivates Ras (by activating the GTPase domain).
The inherited disease neurofibromatosis type I is caused by a mutation in
NF-1 prolonged GTP binding and activation of Ras increased cell
proliferation.
Active
Inactive
(Lieberman4_F18.11)
IV) The PI3 Kinase Pathway and Its Branches
Stimulus: insulin Growth Factor
Receptor: Insulin R Tyrosine Kinase
Initial Response: Autophosphorylation of Receptor
P04-Tyr on the receptors are

recognized by insulin receptor
substrate-1 (IRS-1) and IRS-1
becomes phosphorylated
Coupler/Adaptor: PI3K Phosphatidylinositol 3-OH Kinase
(p85 regulatory subunit of PI3 Kinase
00ÔCOOOCCC Plasma
I membra
has SH2 domain to bind to P04-Tyr)
Phosphatidylinositol
_<>
PI 4,5-bis-phosphate
(PI-4,5-bisP)
PI 3,4,5-tris-phosphate
(PI-3,4,5-trisP)
Docking site for
pleckstrin homology (Lieberman4_F11.12)
I
domains
cycIins/Cdks
Other PI3K signaling molecules and their function:

transcription factors
I
1) PIP2: Phosphatidylinositol 4',5'-bisphosphate (PI-4,5-bisP) in nucleus (pRb/E2F)
- membrane phospholipid. PIP2 is phosphorylated at 3
position of inositol by PI3K to become PIP3. I
proliferation
2) PIP3: phosphatidylinositol 3',4',5'-trisphosphate (PI-3,4,5-
trisP) - membrane docking sites for pleckstrin homology (PH) domain.
3) PKB: protein kinase B (also called Akt) - has PH domain and is a serine-
threonine kinase. Will be recruited to the membrane by PH domain.
4) PDK1: phosphoinositide-dependent kinase-1 - has PH domain. Recruited
to membrane by PH domain to phosphorylate and activate PKB.
V) The complexity of the insulin signaling system
Hormones: Insulin
Insulin-like growth factors: IGF-1, IGF-2
Receptors: insulin R, IGF-1 R, IGF-2R
Three receptors cross-reactive (e.g. insulin R will

bind insulin best but will bind IGF-1 and IGF-2)
All tyrosine kinases autophosphorylation
P04-Tyr on the receptors are recognized by
insulin receptor substrate-1 (IRS-1)
(R hoades4_Fl.ll_pl4)
which, in turn, is phosphorylated to yield P04-Tyr on IRS-1
IRS-1 couples to both the MAP kinase and PI3 kinase pathways
APC
CD4/
C D 8-
Adapter
proteins
T cell Z A E -70........ . __ PI 3-Kinase
Initiation of
TCR-mediated , GDP/GTP I
signals PLCyl / exchange /
factor /
PLCyl GTP/GDP Activation of

,--------------- y activation x exchange on PI3-Kinase
Biochemical / \ Ras, Rac
intermediates I
Increased Diacylglycerol
cytosolic Ca2+ (DAG) RasGTP, PIP3
Rac*GTP
I
Active Akt, t Protein
enzymes Calcineurin PKC mTOR synthesis
Transcription
I
[n f a t )
\
[N F - k-B|
i
I AP-1 I
factors (Abbas5_F5.10_pll5)
VI) Oncogenes related to hormone/growth factor mechanisms

A) Definitions and nomenclature
Oncogene: genes of tumor-causing viruses that are responsible for the
transformation of cells (from normal to tumor phenotype)
protein products of oncogenes: pMWnc
pp(phosphoprotein); gp (glycoprotein)
Proto-oncogene: genes in normal cells that are similar/identical to viral

oncogenes (viruses probably stole these cellular
genes during passage)
B) Discovery and revelations

Observation of TK activity
1) receptors for hormones------ insulin R
2) receptors for growth factors EGFR
3) protein products of certain oncogenes/proto-oncogenes
avian sarcoma virus oncogene src
avian erythroblastosis virus oncogene --- erb B
C) Certain oncogenes are structurally related to growth factors or growth
factor receptors
normal cell product viral oncogene
growth factor PDGF B-chain p28v'sis
growth factor receptor EGFR gp65v'erbB2
EXTRACELLULAR Cysteine-
rich
SIDE domain
Transmembrane Transmembrane
helix helix
I Tyrosine Tyrosine
CYTOSOLIC kinase kinase
SIDE domain domain
COO- COO-
(R. Ritchie, MSU)
Genes in control of cell growth altered--------- > tumor cell

Growth factor
Growth factor receptor
Famesyl membrane anchor
(Kumar9_F7.25)
D) Clinical Relevance'.
CHRONIC
NORMAL MYELOGENOUS
CHROMOSOMES LEUKEMIA
1) Chronic Myeloid Leukemia (CML): 9 22 9 22
o Patient inherit Philadelphia chromosome
(fusion of bcr gene with part of cellular abl (c-
abl) gene). .B C R
locus A B L -B C R
o The c-abl gene codes for a protein tyrosine
kinase with unknown substrates, - ABL
oncogene
> hybrid gene
o The abnormal Bcr-Abl fusion protein has

unregulated TK activity (always ON), Tyrosine
kinase - _I Tyrosine
' kinase
o Gleevec (imatinib mesylate) is a drug that inhitor
functions as a TK inhibitor (specifically bind I
Activation of
to and inhibit only the active site of the Bcr- growth factor
signaling
Abl fusion protein) and it is used to treat CML pathways
(targeted therapeutic strategy).
(Kumar9_F7.26)
BCR (chromosome 22) ABL (chromosome 9)
M
V-, DNA breakage
v Translocation \ Translocation
DNA breakage
B C R -A B L (der chromosome 22)
Q
1
RAS STAT
i
AKT
1 1
Growth factor independent proliferation and survival
Normal differentiation
(Kumar9_F13.32)
2) Oncogenic mutations involving growth factor receptors:

o Epidermal growth factor receptor 1 (EGFR1 - aka EGFR, HER1, ErbB1)
- Several different point mutations are found in a subset of lung adenocarcinomas.
- Mechanism: mutations lead to constitutive activation of EGFR 1 tyrosine kinase
(always ON).
- Tyrosine kinase inhibitor can be used to treat lung adenocarcinomas with
EGFR1 mutations.
o Epidermal growth factor receptor 2 (EGFR2 - aka HER2, Neu, ErbB2) -

overexpression of growth factor receptors
- Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine
kinase receptor member of the epidermal growth factor receptor (EGFR)
family.
- Mechanism: EGFR2 gene is amplified leading to overexpression of HER2
receptor and constitutive tyrosine kinase activity (always ON).
- HER2 is overexpressed in 10-20% of breast cancers.
- Patient with breast cancer overexpressing HER2/neu --- Poor prognosis.
- Transtuzumab (Herceptin) is a monoclonal antibody that targets the
extracellular domain of HER2 blocks growth proliferating signal of HER2/neo.
LIP ID M E T A B O LIS M
O U TLIN E OF LIP ID M E T A B O LIS M LE C TU R E TO PICS
I. O v e rv ie w o f L ip id M e ta b o lis m
II. F a tty A c id (FA) O x id a tio n (a.k.a. p -o x id a tio n o f F a tty A c id s )
III. K e to n e B o d y S y n th e s is
IV. F a tty A c id S y n th e s is
V. C o m p a ris o n o f FA O x id a tio n a n d FA S y n th e s is
VI. In te g ra tio n o f C a rb o h y d ra te a n d L ip id M e ta b o lis m
VII. R e v ie w a n d In te g ra tio n o f M e ta b o lic P a th w a ys
I. OVERVIEW OF LIPID METABOLISM ( Wilkins, pgs. 132-134)
A. Importance of Lipids
1. Storage of fuel:
Fat accounts for most of our stored energy, BUT not our
Fuel Reserves of a Normal 70-kg Man
___________________________ (kg)___________
Tissues
Fat (adipose triacylglycerols) 15 141,000
Protein (mainly muscle) 6 24,000
Glycogen (muscle) 0.150 600
Glycogen (liver) 0.075 300
Circulating Fuels
Glucose (extracellular fluid) 0.020 80
Free fatty acids (plasma) 0.0003 3
Triacylglycerols (plasma) 0.003 30
Total______________________________________166,000
a1 (dieters) Calorie = 1 kcal = 4.184 kJ.
2. Structural role > membranes

a. hydrophobic barrier
b. compartmentation
c. communication
3. Hormonal role
a. steroids
prostaglandins, thromboxanes,
b. eicosanoids (includes prost
leukotrienes, and resolvins)
c. newly discovered lipid hormones: sphingosine 1-phosphate and
platelet activating factor (acetyl-glyceryl-ether-phosphorylcholine)
4. Vitaminslipid-soluble: Vitamins A, D, E, and K

I. O V E R V IE W OF LIPID M E T A B O LIS M (continued)
B. S tru c tu re s /N o m e n c la tu re o f F a tty A c id s a n d L ip id s
1. Fatty acid structures and nom enclature

a. N um bering from the carboxylic acid (-CO O H) end the carbon of
the carboxylic acid group is carbon 1 (C 1 )
b. N um bering from the methyl group carbon (C H 3-) the carbon of
the methyl group is called the om ega (w) carbon
W ilk in s , T a b le 6.1, p. 134: F a tty a c id s o f im p o rta n c e to h u m a n s
2. C om m on Lipid S tructures (see W ilkins, F igure 6.1, p. 133)
a. P h o s p h o lip id s (m em brane lipids)
b. T ria c y lg ly c e ro l (a.k.a. triglyceride) non-m em brane lipids

(energy store)
c. Sphingolipids and Glycolipids (also membrane lipids)
(C. Wilkins, MSU)
RCOOH
HO-CHCH=CH(CH2)12CH3 (fatty acid) HO-CHCH=CH(CH2)12CH3
0
HC-NH,
I H C -N -C -R
ch 2oh
H
sphingosine ch 22
01
oh
phosphorylcholine sugar
ceramide
HO-CHCH=CH(CH2)12CH3
0
HO-CHCH=CH(CH2)12CH3
0 H C -N -C -R
H C -N -C -R
CH2OPOCH2CH2N+(CH3)3
O'
sphingomyelin
(a phosphosphingolipid)
d. Cholesterol (a membrane lipid)

C. O v e rv ie w ( R oad M a p ) o f L ip id M e ta b o lic P a th w a y s
D ietary lipids are digested and a b sorb ed; Blood transports dietary and
endogenous lipids (via lipoprotein particles); tissues/cells s to re , utilize,
and m etabolize the lipids.
(C. Wilkins, MSU)

Lipid Metabolism
FATTY ACID OXIDATION
Required reading:
Wilkins: p. 135-145 Chapter 6, Section: (3-oxidation of fatty acids
Coursepack: Dietary Issues and Clinical Problems regarding Impaired FAO
Suggested reading:
Ferrier: Chapter 16, I. Overview through II. Fatty Acid Structure; Chapter 16, IV. Fat
Mobilization and Fatty Acid Oxidation
Objectives:
1. Define the process of fatty acid oxidation.
2. Explain the purpose of the pathway of fatty acid oxidation.
3. Identify where fatty acid oxidation takes place in the cell, and what tissues can carry
out this pathway.
4. Explain how fatty acid oxidation is carried out in the cell.
5. Explain when fatty acid oxidation takes place.
6. Identify the clinical results of deficiencies of particular dehydrogenases in fatty acid
oxidation.
Measurable Outcomes: You should be able to ...

A. Identify the sources of free fatty acids and how they are transported in the blood.
(Obj. 1 and 3)
B. Identify the starting and ending products of the pathway, including their structures.
(Obj. 1)
C. Identify all the products of the pathway, the enzymes that produce them, and for
what the products can be used. (Obj. 2)
D. Explain how acyl CoA is shuttled into the mitochondria, the role of carnitine in the
process, and the major clinical consequences of defects in this system. (Obj. 3)
E. Describe the four repeated steps of fatty acid oxidation, and identify the enzymes and
cofactors involved in these steps. (Obj. 1 and 4)
F. Calculate the number of cycles needed to oxidize fatty acids of even- and odd-
numbered chain lengths, and the total number of products produced (i.e. the number
of acetyl CoA, propionyl CoA, FADH2 , and NADH produced for the complete
oxidation of the fatty acid in question). (Obj. 2 and 4)
G. Diagram how propionyl CoA is converted to succinyl CoA, and which enzyme in this
conversion requires Vitamin B12. (Obj. 3 and4)
H. Identify the regulatory enzymes of fatty acid oxidation; identify what is the primary
regulator of CAT I and how hormones regulate fatty acid oxidation. (Obj. 5)
I. Identify the clinical results of deficiencies in particular dehydrogenases of fatty acid
oxidation. (Obj. 2, 3, 4, and 6)
II. FA TTY A C ID O X ID A TIO N (a.k.a. p-oxidation or FA degradation)
A. W hat?
B. W hy?
C. W here?
D. How?
F ig u re 6.2, p. 136: O verview o f p-oxidation of fatty acids

E. Generation of Free Fatty Acids (free FAs or FFAs)
1. Various lipases (depending on the source) yield free fatty acids
Sources of free fatty acids:
a. Dietary Fat (triacylglycerides=triglycerides=triglycerol=TAGs)
(1) Transported by lipoproteins (chylomicrons) in the blood
(2) FFAs released b y______________________
(3) FFAs are then absorbed by various tissues
b. Stored Fat (also TAGs)
1) In adipose tissue
(2) FFAs released b y_____________________and other lipases
(3) FFAs bound to ____________for transport in blood to tissues.
c. Phospholipids
(1) Membrane components
(2) FFAs released b y______________________
(3) FFAs are bound to FA binding proteins in the cell (many
different specific kinds)
(4) FFAs can be used for oxidation [or they can be messengers,
i.e. DAG and IP3, or have other functions]
2. NOTE: Free Fatty Acids are not lying around FREEdangerous!

a. Free FAs act like detergents
b. Could disrupt membranes if left free
F. Activation of the Free Fatty Acids

1. Before the FAs can be oxidized, they must be primed for reaction
in an ATP-dependent acylation reaction to form _______________.
2. This activation occurs on th e _________________________and is

catalyzed by a n __________________________________
3. Reaction: Figure 6.3, p. 137: Activation of a free fatty acid

G. Transport of Activated FAs to the Mitochondrial Matrix (across inner
mitochondrial membrane)
1. Recall:
a. Activation to acyl-CoA occurred o n _______________________
b. Inner mito. membrane is very______________
2. Enzymes involved in the transport steps:

a . ______________________________ , which is ____________
b. Translocase
as it transports acyl-carnitine into the matrix, it simultaneously
transports free carnitine back out to the intermembrane space.
c . ______________________________ , which is not_________
3. Sources of carnitine
Figure 6.4, p. 138: The carnitine shuttle

H. The FO U R repeated steps of p-oxidation. (F ig u re 6.5, p. 140)
1. Step 1:
a. R eaction:
b. R eaction type:
c. Enzyme:
d. C ofactor or additional reactant:
2. Step 2:
a. Reaction:
b. Reaction type:
c. Enzyme:
3. Step 3:
a. Reaction:
b. Reaction type:
c. Enzyme:
4. Step 4:
a. Reaction:
b. Reaction type:
c. Enzyme:
So a C 16 acyl CoA (palm itoyl CoA) w ould re q u ire ___ cycles of p-oxidation
and w ould yield: ___ acetyl C o A ,____FADH 2 , a n d ____ (NADH + H+)
I. Special Cases in (3-oxidation of various FAs
1. Unsaturation in wrong place AND/OR in wrong configuration (is cis-
but need trans-)the double bond can be moved into correct position
and configuration
2. Odd number of carbons in acyl chain

a. Example: C 15 6 acetyl CoA + 1 propionyl CoA (3C)
See Wilkins text, Figure 6.6, p.143: Conversion of propionyl CoA,

from (3-oxidation of odd-numbered carbon fatty acids, to succinyl
CoA for complete oxidation by the TCA cycle.
b. The first step requires___________________

c. The third step requires_____________used by the enzyme
When?
J. Regulation of (3-oxidation
1. (3-oxidation of FAs and FA synthesis are______________
2. In starvation: adipose cell Hormone sensitive lipase is:
a. stimulated by hormones such as_________________

b. In the fed state, lipases are inhibited by the hormone_
3. Entry of FAs into the mitochondrial matrix is also regulated

a. CAT I:
4. Two enzymes of the (3-oxidation pathway are also regulated:

a.
b.
K. Energy (ATP) yields for acyl CoAs
(NOTE: Problems like these will NOT be on the exam.)
1. Table 6.2, p. 142: Net energy (ATP) yield from the complete
catabolism of palmitoyl CoA (Cie)
2. How about a C m FA?
Products of 3-oxidation From TCA cycle ATP yield

7 acetyl CoA (7 x) 3 NADH (x 3) 63
(7 x) 1 FADH2 (x 2) 14
(7 x) 1 GTP (x1) 7
7 FADH2 (x 2) 14
7 NADH (x 3) 21
1 propionyl CoA -> - 1 Succinyl CoA
1 GTP (x1) 1
1 FADH2 (x 2) 2
1 NADH (x 3) 3
125
*2 high energy P O 4 2' bonds broken during -3
activation stage & 1 more for prop. CoA to succ.
CoA conversion
122 ATP (Net yield)
L. Dietary Issues Regarding Fatty Acid Oxidation
1. Dietary intake of polyunsaturated partially hydrogenated lipids (i.e.
margarine)
a. Partial hydrogenation is chemical reduction
b. Results in removal of some double bonds AND rearranges other
c/s-double bonds to trans- double bonds >trans-fats
c. These accumulate in membranes
d. Body responds with increased levels of______ , in blood (LDLs)
e. Ultimately, same as eating_________
2. Excess oxidation
a. Occurs when fat is the main energy sourceexamples:
starvation, diabetic conditions, certain diets that restrict
carbohydrate intake
b. RESULT: Production of
c. Normally KB production is not a problem
M. Clinical Problems associated with Impaired Fatty Acid Oxidation

Overall: Complex symptoms, difficult to diagnose
1. Carnitine deficiencies
Results:
a. Decreased ability of tissues to utilize long-chain fatty acids as a
fuel
b. Accumulation of toxic amounts of fatty acids (straight and
branched-chains) in cells
2. Secondary carnitine deficiencies also occur
a. Liver disease
b. Malnutrition
c. Strict vegetarian diets
d. Increased requirements due to pregnancy, burns, trauma, etc.
e. Hemodialysis
3. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency
a. Four specific fatty acyl CoA dehydrogenases
b. A very common inborn error of metabolism
c. Causes decrease in FA oxidation, severe hypoglycemia
d. Treatment: carbohydrate-rich diet
Lipid Metabolism
KETONE BODY SYNTHESIS
Required reading:
Wilkins: p. 145-150, Chapter 6, Section: Ketone Body Synthesis
Coursepack: CASE STUDY Systemic Carnitine Deficiency: A Treatable Disorder
Suggested reading:
Ferrier: Chapter 16, V. Ketone Bodies: Alternative Fuel for Cells through end of chapter
Objectives:
1. Define the process of ketone body synthesis.
2. Explain the purpose of the pathway of ketone body synthesis.
3. Identify where ketone body synthesis takes place in the cell, and what tissue(s) can
carry out this pathway.
4. Explain how fatty acid oxidation is carried out in the cell, and how ketone bodies are
utilized by other tissues.
5. Explain when ketone body synthesis takes place (i.e. under what conditions are
ketone bodies produced).

A. Identify the starting and ending products of the pathway, including their structures.
(Obj. 1)
B. Identify all the products of the pathway, the enzymes that produce them, and for
what the products can be used. (Obj. 2)
C. Describe the steps of ketone body synthesis, and explain the fates of the 3 ketone
bodies produced. (Obj. 3 and4)
D. Explain the regulation by acetyl CoA concentrations that coordinate fatty acid
oxidation, ketone body synthesis, and gluconeogenesis in the liver. (Obj. 5)
E. Explain why starvation increases ketone body production. (Obj. 2 and 5)

III. K E TO N E B O D Y SYN TH ES IS
A. W hat?
B. W hy?
C. W here?
D. How?
Pathway of ketone body synthesis (3 to 4 steps). (F ig u re 6.7, p. 146)
1. Step 1:
a. R eaction:
b. R eaction type:
c. Enzyme:
d. C ofactor or additional reactant o r product:
2. Step 2:
a. Reaction:
b. Reaction type:
c. Enzyme:
3. Step 3:
a. Reaction:
b. Reaction type:
c. Enzyme:
e. How is acetone form ed?
4. Step 4:
a. Reaction:
b. Reaction type:
c. Enzyme:
e. Purpose of reaction:
E. C onditions favorable for Ketone Bodies S ynthesis ( W ilkins, p. 148-149)
1. Entry of acetyl CoA into T C A cycle depends o n __________________

availability.
2. If O AA concentration is low, may mean carbohydrate (glucose) is

unavailable, thus O AA is consum ed to fo r m _______________ .
[OAA] could be low under the follow ing conditions:

a.
b.
c.
3. U nder these 3 c o n d itio n s ,________________ predom inates to form

________ , the m ajor fuel source fo r the brain.
4. p-oxidation of fatty acids then occurs to form e x c e s s __________ , but

since O AA is not available, it is diverted to form
F. Fates of ketone bodies (again, are water soluble) Wilkins, p. 149-
ISO.
1. acetoacetate:
2. (3-hydroxybutyrate:
3. acetone:
(Ferrier7_F16.23)
G. Excess ketone body productionketosis

1. High levels of ketone bodies in the blood is termed________ and
in urine is termed_____________
2. Leads to________________ .
3. Usually seen in uncontrolled, type 1 diabetes

REQUIRED READING
CASE STUDY
Systemic Carnitine Deficiency: A Treatable Disorder
The 3 % year-old boy was born to non-consanguineous parents from Chihuahua,

Mexico after an uncomplicated pregnancy and delivery. A brother had died at 3 months
of age of a "liver problem after an unexplained coma. The parents and three siblings
are well.
At 3 months of age the boy was admitted in a coma to a community hospital and
suffered a cardiac arrest. Examination revealed hepatomegaly and cardiomegaly and a
serum glucose of 15 mg/dl (normal, 60-100 mg/dl). The patient eventually recovered
and the hepatomegaly resolved. At 6 months of age he developed congestive heart
failure after an upper respiratory tract infection and was brought to the medical center of
the University of California, Los Angeles. Hepatomegaly and hypotonia were noted on
admission. On day 3 he became lethargic and developed generalized seizure activity
and cardiac arrest, but he was successfully resuscitated. Laboratory studies at the time
of the arrest revealed a blood glucose level of 15 mg/dl without associated acidosis or
ketosis; mild elevation of serum aspartate aminotransferase (SGOT) (337 I.U./liter;
normal, 6-36 I.U./liter) and alanine aminotransferase (SGPT) (179 I.U./liter; normal, 10
45 I.U./liter)l and hyperammonemia (300 :g/dl; normal < 69 :g/dl). Delayed milestones
(developmental quotient 66; normal 100), proximal-muscle weakness, and growth
retardation (weight and height below the 3rd percentile) were noted after recovery.
Metabolic studies showed normal glucose, galactose, and fructose tolerance, and a
normal 10 hour fasting blood glucose, with no increase in lactate, pyruvate, or ketone
bodies. The electroencephalogram, brain scan, and chromosomal studies were
unremarkable. Also normal were the plasma levels of electrolytes, calcium,
phosphorus, magnesium, bilirubin, throxine, thyroid-stimulating hormone, and growth
hormone. Results of total serum protein determination, serum electrophoresis,
cerebrospinal fluid studies, and studies of immune function were also normal. Between
acute episodes, serum levels of glucose, ammonia, SGOT, SGPT, and creatine
phosphokinase were all normal.
The patient was again admitted with cardiorespiratory arrest after upper respiratory tract
infections at ages of 20, 24, and 33 months. The episodes were associated with liver
enlargement, elevations of transaminases to >2000 I.U./liter, and of creatine
phosphokinase from 1500 to 33, 000 I.U./liter. Maintenance glucose requirements
varied from normal (3 mg/kg of body weight per/min).
A muscle biopsy specimen contained large amounts of neutral lipids. Liver biopsy also
showed severe but nonspecific fatty changes. Abnormal mitochondrial structure, many
electron-dense lysosome-like bodies, and dense, laminate, rounded lipofuscin-like
particles were evident.
REQUIRED READING (cont.)
A thirty-two hour fasting study was performed. During the fasting period the patient had
no nausea, cramps, or pigmenturia. However, at thirty-two hours he suddenly had a
cardiorespiratory arrest characterized by an absence of cardiac electrical activity. He
was successfully resuscitated and intravenous glucose was administered.
At the start of the fast his blood glucose levels was 91 mg/ml, but by twenty-four hours it
had fallen to 66 mg/dl. Plasma triglyceride levels rose from 66 to 126 mg/dl and free
fatty acids increased from 0.1 to 2.0 mEq/liter. Serum SGOT rose from 36 to 1450
I.U./liter, with no increase in creatine phosphokinase or aldolase. Ammonia increased
from 40 to 134 :g/dl. The most significant finding, however, was the lack of production
of measurable ketone bodies.
During the 11th admission, the diagnosis of carnitine deficiency was considered because
of fatty changes in the liver and the lack of production of ketone bodies after twenty-four
hours of fasting. At the time the patients height and weight were normal but his
developmental quotient was 40, and he had a variety of neuromuscular abnormalities.
A computerized axial tomography (CAT) scan of the brain revealed marked
enlargement of both lateral ventricles and of the sulci between the cerebral gyri.
adapted from: H.S. Paul, "Clinical Studies in Medical

Biochemistry, Oxford University Press (1987)
Questions:
1. Why does lipid accumulate in the liver in carnitine deficiency?
2. Why was the patient unable to produce ketone bodies after a 24 hour fast?
3. Would the symptoms be different if the disease state were due to:
a. deficient synthesis of carnitine
b. deficient activity of carnitine- acyl transferase I
c. deficient activity of carnitine - acyl transferase II
If so, in what respects?

REQUIRED READING ( c o n t.)
Answers to questions on Carnitine Case Study

Systemic Carnitine Deficiency: A treatable disorder
Q1. Lipid will accumulate in the liver in carnitine deficiency because fatty acyl CoA
cannot be transported into mitochondria and therefore it will be available in the
cytoplasm to be incorporated into triglycerides.
Q2. Ketone bodies will not be produced because fatty acids that are mobilized during
fasting cannot enter the mitochondria and thus cannot make acetyl CoA and
ketone bodies.
Q3. The symptoms would be very similar if the deficiency were in synthesis of
carnitine or in the activity of carnitine acyl transferase I (CAT I).
You might expect something different from a carnitine acyl transferase II (CAT II)
deficiency: in theory, the fatty acid would get into the mitochondria but not
reconverted to fatty acyl CoA and therefore not be metabolized. But there would
be no release of carnitine, so inward transfer would also be inhibited and the
result would be the same. (Lipid accumulation in cytoplasm)
Lipid Metabolism
FATTY ACID SYNTHESIS
Required Reading:
Wilkins: p. 151-167, Chapter 7, entire chapter
Coursepack: Integration of Metabolism
Suggested Reading:
Ferrier: Chapter 16, III. Fatty Acid De Novo Synthesis
Objectives:
1. Define the process of fatty acid synthesis.
2. Explain the purpose of the pathway of fatty acid synthesis, and why linoleic and
linolenic acids are essential fatty acids.
3. Identify where fatty acid synthesis takes place in the cell.
4. Explain how fatty acid synthesis is carried out in the cell.
5. Explain when fatty acid synthesis takes place.
6. Compare and contrast the overall similarities and differences of the reciprocal
pathways of fatty oxidation and fatty acid synthesis
A. Explain how acetyl CoA, which is produced in the mitochondrial matrix, is shuttled
out to the cytosol, and why a special export mechanism is required.
{Obj. 1, 2, 3, and 4)
B. Identify the committed step of fatty acid synthesis, the enzyme that catalyzes this
step and its regulatory effectors. {Obj. 2, 4, and 5)
C. Describe the four main repeated steps of fatty acid synthesis, and identify the
enzymes and cofactor(s) involved. {Obj. 1, 2, and 4)
D. Explain the advantages of having all the enzymes involved in fatty acid synthesis
(except acetyl CoA carboxylase) bound together in one large complex, and the
role the phosphopantetheine group plays in the synthesis process. {Obj. 1, 2, and 4)
E. Calculate how many cycles of the four repeated steps of fatty acid synthesis are
necessary to generate the major productpalmitate (Ci6:o fatty acid).
{Obj. 1,2 and 4)
F. Identify the regulatory enzymes of fatty acid synthesis, what regulates them, and how
hormones regulate fatty acid synthesis. {Obj. 2, 4, and 5)
G. Explain how longer-chain fatty acids (Cis and higher) are synthesized and how
double bonds are introduced into the acyl chain. {Obj. 2 and 4)
H. Explain why linoleic and linolenic acids are dietary requirements. {Obj. 2)
I. Compare and contrast the overall similarities and differences of the reciprocal
pathways of fatty oxidation and fatty acid synthesisincluding substrates,
products, cofactors, enzymes and regulation of the pathways. {Obj. 6)
IV. FATTY ACID SYNTHESIS
A. What?
B. Why?
C. Where?
D. The net reaction of palmitate (C1 6 ) synthesis and malonyl CoA

synthesis. (Figure 7.1, p. 152)
E. How?
Overview of fatty acid synthesis. (Figure 7.2, p. 153)
F. General information
1. Sources and uses of acetyl CoA. (Figure 7.3, p. 154)
2. Excess food intake in any form -> fatty acids
3. FA synthesis regulated by: (food intake - energy use) along with

Hormones:
a.
b.
c.
4. Fat is NOT RAPIDLY MOBILIZEDduration of storage:

a. Fat 150 days
b. Protein 10 days
c. CHO 1 day
5. First step to fat storage: transport acetyl CoA to the cytosol

G. M alate/C itrate/P yruvate shuttle (See F ig u re 7.4, p. 155)
1. P roblem : acetyl CoA is form ed inside t h e __________________ , but

Fatty acid synthesis takes place in t h e ___________________ .
2. Solution: m alate/citrate/pyruvate shuttle g e ts ____________ to the

____________ A N D generates s o m e ________________ as well.
H. Form ation of Malonyl CoA. (F ig u re 7.5, p. 156)

1. Reaction:
2. This reaction i s ________________ , and is t h e _________________ in

FA synthesis.
3. Enzym e i s ______________________________
a. R e q u ire s ________________
b. The tw o steps o f this reaction are sim ilar to the pyruvate

carboxylase reaction steps (s h o w n in F ig u re 5.5, p. 104)
I. The Fatty A cid S ynthase C om plex (FAS com plex)

1. A large m ultienzym e com plex (240,000 mol. wt.) that contains:
The enzym atic com ponents of the fatty acid synthase com plex. (T a b le 7.1,
p. 157)
2. The interm ediates of FA synthesis are attached to an

, w hich is a part o f the FAS com plex.
J. The fou r key repeated steps of the fatty acid synthase com plex.
(F ig u re 7.6, p. 159)
1. Step 1:
a. R eaction:
b. R eaction type:
c. Enzym e:
d. C ofactor or additional product:
2. Step 2:
a. Reaction:
b. Reaction type:
c. Enzyme:
3. Step 3:
a. Reaction:
b. Reaction type:
c. Enzyme:
4. Step 4:
a. Reaction:
b. Reaction type:
c. Enzyme:
R E P E A T Steps 1-4 (+2 tran sfe r steps), usually 7 tim es to produce

J. The Reactions of the FAS complex continued
(Ferrier7_16.9)
Fatty Acid Synthase figure (above) key:
Steps 1&2: Acetyl CoA-ACP transacylase Loads an acetyl unit to the sulfhydryl group of the acyl
carrier protein (ACP) and then transfers it to the cysteine sulfhydryl group of the the p-
ketoacyl-ACP synthase (and transfers the growing acyl chain in subsequent rounds in the
same manner step #2).
Step 3: Malonyl CoA-ACP transacylase Loads malonyl units onto the sulfhydryl group of the ACP
(the long arm that can reach all of the enzyme active sites).
Step 4: p-ketoacyl-ACP synthase (a.k.a. the condensing enzyme) Condenses the acetyl unit (or
growing acyl chains in subsequent steps) onto carbon 2 of the malonyl units on the ACP,
with loss of CO 2 .
Step 5: p-ketoacyl-ACP reductase Reduces the keto group on the p-carbon to a hydroxy group,
with simultaneous oxidation of NADPH + H+ to NADP+.
Step 6: p-hydroxyacyl-ACP dehydratase Dehydrates (removes H2 O) and forms a frans-alkene
between carbons 2 and 3.
Step 7: Enoyl reductase Reduces the alkene to an alkane, with simultaneous oxidation of NADPH
+ H+ to NADP+.
REPEAT: Steps 2-7 (usually 6 more times)
Step 8: Palmitoyl thioesterase Cleaves off final product, usually palmitate (a C 16 0 fatty acid)
which quickly gets attached to CoA to form palmitoyl CoA
K. Further Processing of newly synthesized fatty acids Wilkins, p. 161
1. The major product of the FA synthase complex is____________
2. In eukaryotes, further processing occurs in the________________
3. Elongation of fatty acids
a . ____________ is the donor of 2 -carbon units for elongation.
b. it is sequentially added to the__________________ of both
saturated and unsaturated fatty acids.
4. Desaturation of fatty acids
a . ___________________can be introduced at various positions
b. The reactions to make double bonds are carried out by several
enzymes (termed mixed-function oxidases) including a reductase
and a desaturase.
c. However,________________ desaturate beyond C9
(J. Wang, MSU)
L. Essential FAs = certain FAs cannot
be made by the human body w \= /w v w y s'CoA
1 . ________ : omega 6 (w-6 ) It
0
Cl8:2A9, 12 Linoleyl CoA ( C 10, a 9, 12)
2. ________ : omega 3 (w-3) to
Cl8:3A9, 12, 15
DESATURASE - ZH
3. From these can make:
a .___________________ Y 0
n
(C20:4A5, 8 , 11, 14) V W y w W W c-s' CoA
b. <C18.A6, 9,12)
^ O O C -C H ^ C O -S-C o A *
c.
W . COA-SH
V 2
* o
Importance of
co-3 co-6
(fish oils) 1) In seed oils ( C^. AS. l l , 14)
1) Inhibit PG and LT [vegetable, olive,
synthesis [are poor soybean oils, etc.]
DESATURASE - 2.H
substrates for
PGHS/COX-1] 2) Preferred substrates
for PG and LT
2) Protects against synthesis. v v \^ y \= /\= /\= y v \ _s_c0A
inflammation,
0
allergies, and heart Arachidonyl CoA (C^g. a 5 . 8, 11, 14 )
disease due to plaque
BMB 515
V.-VI. Comparison of FAO/FAS; Integration Carb/Lipid Met. Dr. Wilkins: mindockc@msu.edu
W hen?
M. Regulation o f Fatty A cid S ynthesis W ilkins, p. 162-163
1. In FA synthesis, the com m itted step is:
a. Reaction:
b. Enzyme:
c. R egulated in several ways:
^G lobally by hormones:
________________ ( )
________________ ( )
-^ C e llu la rly by:
ii.
iii.
3. NOTE: FA synthesis is m axim al when carbohydrate and energy are

plentiful, and when fatty acids are scarce.
V. C O M P A R IS O N OF FA O X ID A TIO N A N D F A S YN TH ES IS
A. Features distinguishing p-oxidation (FA oxidation) and FA synthesis
(T a b le 7.3, p. 163)
B. Com parison of the fou r repeated steps of FA oxidation and FA

synthesis pathways. (F ig u re 7.7, p. 164)
C. Reciprocal Regulation o f FA oxidation and FA synthesis pathways

(T a b le 7.2, p. 162)
VI. In te g ra tio n o f C a rb o h y d ra te a n d L ip id M e ta b o lis m
W ilkins, p. 164-167
S ee F ig u re 7.8, p. 166: W e ll-fe d s ta te ; a n d F ig u re 7.9, p. 167: S ta rv a tio n

o r in s u lin -d e fic ie n c y s ta te
REVIEW AND INTEGRATION OF METABOLIC PATHWAYS
SELF-REVIEW AND SELF-ASSESSMENT ASSIGNMENT
The major metabolic pathways are reviewed and integrated on the next four pages.
First is a review the control sites of the pathways, emphasizing that biosynthetic and
degradative pathways are invariably distinct. To help you review: the regulatory
enzymes and biochemical components at key junctions of carbohydrate, lipid, and
amino acid metabolism are presented as an assignment for you to fill in the tables and
lists.
Objectives:
1. Explain how metabolism can be regulated effectively by distinct biosynthetic and
degradative pathways, as well as how these pathways are compartmentalized both
within cells and/or specific tissues.
2. Identify the key enzymes that control carbohydrate and lipid metabolic pathways.
3. Identify key molecules that serve as branch points between several metabolic
pathways, and the specific enzymes/pathways that use these molecules or produce
them.
4. Identify, and explain why, which pathways of lipid and carbohydrate metabolism
would operate together under high or low insulin/glucagon ratio conditions.

I. Generalizations on Metabolism
A. Explain how metabolism can be effectively regulated by distinct biosynthetic and
degradative pathways. (Obj. 1)
B. Describe how metabolic pathways can be governed by activity of committed step

enzymes and by compartmentation. (Obj. 1 and 2)
C. Explain why different organs can respond to the same hormone/stimulus in

opposite directions. (Obj.4, and PSL 539 objectives)
II. Control Sites of Major Metabolic Pathways

A. Explain the enzymes and the molecular basis of coordinate control of glycolysis
versus gluconeogenesis, glycogenolysis versus glycogenesis, fatty acid synthesis
versus (3-oxidation. (Obj. 2, 3, and 4)
B. Describe the regulation of the citric acid (TCA) cycle, pentose phosphate pathway,
and lipolysis of triacylglycerol. (Obj. 2)
III. Key Junctions of Metabolic Pathways
A. Recall that glucose-6-phosphate can choose alternative fates: glycolysis,
gluconeogenesis, pentose phosphate shunt, and glycogenesis. (Obj. 3)
B. List the sources of pyruvate and its metabolic fates. Recognize pyruvate as a
major link between amino acid metabolism and carbohydrate metabolism. It is
also the substrate in a decisive reaction in metabolism, committing the carbon
atoms of carbohydrates and amino acids to oxidation by the TCA cycle or to lipid
synthesis. (Obj. 3)
C. List the sources of acetyl CoA and its metabolic fates. Use this information to
account for the fate of acetyl CoA under particular metabolic conditions. (Obj. 3)
IV. Integration of Lipid/Carbohydrate Metabolism

A. Explain which pathways in the liver would be active under excess nutrient
conditions. (Obj. 3 and 4)
B. Explain which pathways in the liver would be active under starvation or insulin
deficiency conditions. (Obj. 3 and 4)
VII. REVIEW AND INTEGRATION OF METABOLIC PATHWAYS
A. Generalizations on Metabolism
1. Biosynthesis and biodegradation pathways usually distinct;
Permitting more effective, reciprocal regulation
glycogenolysis glycolysis
e.g., G L Y C O G E N G LU C O SE PYRUVATE
glycogenesis
gluconeogenesis
2. Rates of metabolic pathways are governed by key enzymes not just substrate
concentration. Key enzymes usually:
a Catalyze the committed step and/or essentially irreversible steps
b. Are regulated by cellular effectors and by hormones

This control affects enzyme activity
enzyme level (rate of syn./degrade.)
3. Metabolic patterns can also be determined by compartmentation

Example: FATTY ACIDS
in cytosol FAs go to TAGs, phospholipids, etc.
in mitochondriaFAs go to beta-oxidation; ketone body synthesis (liver)
4. Tissue-specific enzymes [isozymes] and receptors allow DIFFERENT tissues

to respond differently to SAME stimulus.
Examples:
a. Glucagon receptors in liver, not muscle: thus liver does GNG, not muscle, in
response to low blood glucose.
b. Epinephrine different effects in liver versus muscle (receptors in both)

(1) in liver: [F-2,6-BP] | , glycolysis | > promotes high blood sugar (i.e. liver
does GNG to release glucose into the blood.
(2) in muscle: [F-2,6-BP] | , glycolysis | > to make lots of ATP quickly to

fight or flight
(3) Due to different isozymes of PFK 2/phosphatase (FBP 2 )which respond

differently to phosphorylation.
B. Control Sites of Major Metabolic Pathways
1. Major Metabolic Pathways KEY ENZYMES
(S. Ferguson-Miller, MSU)
GLYCOGENOLYSIS GLYCOGENESIS GLYCOLYSIS
glycogen glucose glucose-6-P
1 ,
t2 f'
t
glucose
r
glycogen
r
pyruvate
LIPOLYSIS/GENESIS AMINO ACID OXIDATION FATTY ACID OXIDATION
triglyceride amino acids fatty acyl CoA

7\
4
ItT * > glutamate t 17
fatty acylcarnitine
<> It,
'f NH3 + a Kg (mitochondria)
glycerol + fa tty acid i n i
acetyl CoA
UREA
key: 1) g lyco g en phosphorylase 2) hexoklnase 3) glycogen synthase 4) PFK 5) PK 6) Pyruvate carboxylase

7) PEPCK 8) F-l ,6-blsphosphatase 9) Gluc-6-phosphatase 10) PDH 11) isocitrate DH
12) a KgDH 13) succinyl CoA syn. 14) acetyl CoA carboxylase 15) hormone-sensitive lipase
16) acyltransferase 17) g lu tam ate DH 18) CPS 19) CPTI
2. Reciprocal Regulatory MechanismsSummary: Fill in the tables below as
to whether the effectors, etc. activate or inhibit the enzymes or pathways
indicated.
a. Glycolysis versus Gluconeogenesis
EFFECTO RS ENERGY HORM ONES

(c e llu la r ) A T P /A D P (or A M P ) (sy ste m )
(c e llu la r )
F r u c to se 2 ,6 -B P t In su lin G lu c a g o n
G ly c o ly sis
(P F K 1 )
GNG
(F B P 1 )
b. Glycogenolysis (breakdown) versus Glycogenesis (synthesis)
EFFECTO RS HORM ONES

(c e llu la r ) (sy ste m )
G lu c o se -6 -P E p in e p h r in e /G lu c a g o n In su lin
G ly c o g e n o ly sis
(g ly c o g e n p h o sp h o r y la se )
G ly c o g e n e sis
(g ly c o g e n sy n th a se )
c. (3-Oxidation of Fatty Acids versus Fatty Acid Synthesis
EFFE CTORS ENERGY HORM ONES

(cel u la r) (c e llu la r ) (sy ste m )
P a lm ito y l M a lo n y l C o A t In su lin G lu c a g o n
C oA
fi-o x id a tio n (substrate of
(C A T I/C P T I) CAT I)
F A sy n th e sis (substrate of
(a c e ty l C o A c a r b o x y la se ) FA synthase)
C. Key Junctions of Metabolic Pathways (Sources/Fates of Key Intermediates)
1. Glucose-6-P
a. Sources (what enzymes/pathways lead to glucose-6-P production):
(1) Hexokinase (Glucokinase)
(2) Gluconeogenesis
(3) Glycogenolysis (glycogen breakdown)
b. Fates (what enzymes/pathways use glucose-6-P):
(1) Glycolysis
(2) Pentose Phosphate Pathway
(3) Glycogenesis (glycogen synthesis)
(4) Free glucose
2. Pyruvate Fill in the sources and fates

a. Sources:
( 1)
(2 )
(3)
b. Fates:
( 1)
(2 )
(3)
(4)
3. Acetyl CoA Fill in the sources and fates

a. Sources:
( 1)
(2 )
(3)
(4)
b. Fates:
( 1)
(2 )
(3)
(4)
D. Integration of Lipid/Carbohydrate Metabolism

See Wilkins: pgs. 164-167 and Figure 7.8, p. 166; and Figure 7.9, p. 167)
Clinical Applications: Clinical Case
Abnormalities in Hormonal Regulation and Its Effects on
Metabolism
This case illustrates how the presenting complaint and symptoms can be traced back to
a deficiency in a single hormone. These are revealed at the cellular and biochemical
levels, whose laboratory data need to be interpreted in light of the past medical history
as well as the results of the physical examination. You will need to draw upon
information learned throughout the BMB 515 course. This case will be used to show:
(a) inter-connectedness between metabolism of carbohydrates and fats; (b) effects on
blood pH and compensation mechanisms; and (c) protein structures and molecular
techniques used in developing replacement insulin.
Suggested reading:
Ferrier: Chapter 25, entire chapter
Objectives: Active participation in this case session will serve to ...

1. Review how the pathways of carbohydrates and lipids are integrated.
2. Explain how the lack of insulin in type 1 diabetes affects specific metabolic pathways.
3. Describe the factors that contribute to the shift in fuel usage in diabetics and the
metabolic consequences.
4. Relate how the shift in fuel usage impacts blood pH regulation and, in turn, how this
is manifested in the laboratory data on blood gases and in the presenting symptoms
of an individual.
5. Outline an approach to produce insulin for diabetes therapy using recombinant DNA
technology.
I. Case Study
There is a video clip introducing the case scenario, which you can access at the link
http://streaming.msu.edu/storemedia/download/koernerg/BMB514/BMBCCA.mov
(To play this video clip, you will need to download Quick Time Player 1 from Apple.)
Presenting patient: Sally Spartan

30-year old Caucasian female
Height: 5 4
Weight: 130 lbs
Temperature: 97 Fahrenheit (normal range: 97.8 - 99.1)
Blood Pressure: 100/50 mm Hg (90/60 to 120/80)
Pulse: 120 beats/min (60-100/min)
Respiration: 40 breaths/min (12-18/min)
Chief complaint:
I have been extremely tired lately and I have been vomiting for three days. I have
also been going to the bathroom all the time. I also noticed that I have been
forgetting things.
History of present illness:
Ms. Spartan checked into the emergency department (ED) complaining of
lethargy, vomiting, and polyuria of three-day duration. She had started vomiting 3
days ago with no known cause. She is extremely tired despite 8 hours of sleep per
night. Ms. Spartan was so confused that she actually came to the wrong hospital
and was admitted here.
Past medical history:

(a) diabetes mellitus of three year duration, controlled by NPH and regular insulin;
(b) in the past two months, she had been admitted to ER twice for diabetic
ketoacidosis and had noted persistent acetone in her urine since discharge, for
which she had gradually increased her regular insulin to 20 units;
(c) other regular medications included amitriptyline, diazepam, and propranolol,
which had been started in an investigative study of (3-blockers in brittle diabetes
Social history:
Divorced, with many emotional problems.
Physical exam revealed:

(a) Kussmaul respiration; (b) severe dehydration; (c) elevated pulse rate.
Laboratory data (blood gas chemistry and other pertinent data):
Admission 1/2 hr 2 1/2 hr 6 hr Normal Units Usual Name

Plasma
Na 138 142 143 139 136-145 meg/l plasma sodium
K 5.0 3.7 4.2 4.8 3.5-4.5 meg/l plasma potassium
Cl 103 104 114 114 100-106 meg/l plasma chloride
plasma (Total)
C02 4 7 8 12 26-27 meq/l
CO2 (content)
pH 7.03 7.37 7.28 7.35-7.45 pH units pH
PCO2 8.6 11.5 13.8 18 35-45 mm Hg CO2 partial pressure
HCOs Undetectable 6 8 25-26 meq/l plasma bicarbonate
BUN 18 5 10-20 mg/dl blood urea nitrogen
fasting blood sugar
FBS 400 300 175 90 60-100 mg/dl (blood glucose)
Potentially relevant data left out of above case study:
Normal range Units Usual Name

M 14-18 g/dl
Hb F 12-16 g/dl hemoglobin
Child 11-17 g/dl
arterial oxygen
PO2 (arterial) 95-100 mm Hg partial pressure
urine glucose 0 mg/dl urine sugar
urine pH ca. 6 pH units (4.5-8.0 often measured)
Na+ + K+- (CL + HCO3 ) 14-15 meq/l anion gap
II. Clinical Case: Worksheet
A. Pre-class Reading: Read Chapter 25, Ferriertext
B. Read the case study and lab report.

1. What laboratory data are outside of the normal ranges?
2. What does the past medical and family history tell you about the patient?
3. What do the pH, low bicarbonate, and elevated blood glucose indicate to you
regarding changes in the patients metabolic pathways?
4. What are Kussmaul respirations and what do they indicate?
C. Case study questions

1. What would be your likely diagnosis for this patient?
2. Using the diagram on the next page, draw in the arrows to indicate what
pathways would be going on during insulin deficiency conditions.
3. How does the lack of insulin cause the liver to shift to ketone body production?
4. What are ketone bodies?
5. Which specific ketone bodies cause a decrease in bicarbonate? Explain why.
6. Based on the patients pH at the time of admission to the hospital, what is the
patients condition? Explain your rationale.
7. What information did you gather to support your order for an arterial blood gas
(ABG) for this patient?
8. List all observations (physical exam and laboratory data) that suggest the
patient is trying to compensate for the low blood pH.
9. Calculate the HCO3 and pH of the patient at 2.5 hours post-admission. What
does the pH tell you about the patient?
10. Using your knowledge of recombinant DNA technology and the structure of
insulin, design an approach to produce insulin for therapy in diabetic patients.
a. What organism would you use as a host?
b. What important structural feature of insulin should be considered in the

design?
11. Why does insulin have to be injected rather than taken orally?
12. Using you knowledge of the signal transduction pathway of insulin, what might
be a (cheap) laboratory test for the biological activity of recombinant insulin
(without resorting to expensive bioassays involving experimental animals)?
INTEGRATION OF LIPID/CARBOHYDRATE METABOLISM
(During Starvation or Insulin Deficiency Conditions)
In the Liver
.........regulation TRIACYLGLYCEROLS
Plasma ------- reaction
GLUCOSE fatty acvl hormone
(ketosis)
o 0 Increased rate in
starvation/insulin
deficiency_____
transferase fa t
m o b iliz a t io n
sensitive
lipase
O KETONE
0 BODIES Fatty acyl CoA Fatty acid
1(/>' acvl CoA
|Pyruvate] T synthetase fatty acid
svnthase
Gluconeogenesis
KETONE complex
BODIES Carnitine
CAT I Malonyl CoA
acetyl CoA
carboxylase
Acetyl CoA
citrate
Ivase
CHOLESTEROL
Citrate
lOAAl NADH
-P
^
CD
|Qxaloacetate|
00
OPPORTUNITY TO EVALUATE THE COURSE
AND THE INSTRUCTORS
BMB 515 has now been completed. Students will have an opportunity to complete an
evaluation of the course: BMB 515 and its instructors: Dr. M. Faner, Dr. J. He, Dr. R
Ritchie, and Dr. C. Wilkins.
The evaluation page is available at: http://kobiliak.msu.edu/. Down the right-hand

column, hover over Evaluation/hospital Forms and select Pre-Clerkship Evaluation.
On the next screen, click on Begin Login. On the next screen, click on Student Login.
After you enter your first and last names and PID, click on Verify Student. Choose the
semester being evaluated (FS17) and click on Evaluate Selected Semester-Year.
Proceed with the Course and Instructor evaluation.
Although a list of names of students who have logged in is kept, this is for the purpose of
letting students know which forms they have completed and for checking who has not yet
logged into the system. The evaluation data provided by each student is completely
dissociated from the log-in name in the databasethere is absolutely no way to link data
entered with log-in name.
It is an expectation of the College of Osteopathic Medicine, as part of professional

behavior, that all students will provide evaluation of faculty and courses. Feedback via
these evaluations will provide important information regarding student perspectives on
the quality of the course. The information gained from these evaluations will be used to
develop future offerings of BMB 515.
Bibliography
BMB 5 15 F 1 7 -4 0 1
1. Ferrier, Denise R. "Fig 7.9" from Biochemistry, 6th ed. Ed.(W olters Kluwer
H ealth/Lippincott W illiam s & W ilkins, 2014) pp. 1 [1 pages] ISBN: 9781451175622
4. Ferrier, Denise R. "30.27" from Biochemistry, 6th ed. Ed.(W olters Kluwer
5. Turnpenny P, El lard S "Fig 3.15 Stages of meiosis" from Emery's Elements o f Medical
Genetics 14th Ed., 14 Ed.(Elsevier Ltd. (Global), 2012) pp. 40 [1 pages] ISBN:
9780702040436
6. Turnpenny P, Ellard S "Fig 3.17 Stages of m itosis" from Emery's Elements o f Medical
9780702040436
7. Turnpenny P, Ellard S "Fig 3.16 Stages of oogenesis and sperm atogenesis" from
Emery's Elements o f Medical Genetics 14th Ed., 14 Ed.(Elsevier Ltd. (Global), 2012) pp.
42 [1 pages] ISBN: 9780702040436
8. T urnpenny P, Ellard S "Fig 3.27 G eneration of som atic m osaicism " from Emery's
Elements o f Medical Genetics 14th Ed., 14 Ed.(Elsevier Ltd. (Global), 2012) ISBN:
9780702040436
9. T urnpenny P, Ellard S "Fig 3.13 Stages of m itosis" from Emery's Elements o f Medical
9780702040436
10. Ross MH, Pawlina W "Fig 2.24 Proteasom e-m ediated degradation" from Histology: A
Text and Atlas 6th ed., 6 Ed.(Lippincott W illiam s & W ilkins, 2011) pp. 45 [1 pages] ISBN:
9780781772006
11. Ross MH, Pawlina W "Fig. 3.12" from Histology: A Text and Atlas 6th ed., 6 Ed.
(Lippincott W illiam s & W ilkins, 2011) pp. 87 [1 pages] ISBN: 9780781772006
12. Ross MH, Pawlina W "Fig 3.10 Cell cycle and checkpoints" from Histology: A Text
and Atlas 6th ed., 6 Ed.(Lippincott W illiam s & W ilkins, 2011) pp. 86 [1 pages] ISBN:
9780781772006
13. Ross MH, Pawlina W "Fig 2.21 Pathways of delivery of m aterials fo r digestion in
lysosomes" from Histology: A Text and Atlas 6th ed., 6 Ed.(Lippincott W illiam s &
W ilkins, 2011) pp. 41 [1 pages] ISBN: 9780781772006
14. Ross M, Paulina W "Fig 3.3" from Histology: A Text and Atlas 7th, 1 Ed.(Lippincott
W illiam s & W ilkins, 2016) pp. 77 [1 pages] ISBN: 9781451187427
15. Ross M, Paulina W "Fig 3.11" from Histology: A Text and Atlas 7th, 7 Ed.(Lippincott
16. Mescher, A nthony "Fig 3.8" from Junqueira's Basic Histology: Text and Atlas,
Thirteenth Edition, 13; 13th Revised ed. Ed.(MCGRAW-HILL EDUCATION, 2013) pp. 1 [1
pages] ISBN: 9780071780339
17. Ferrier D, Harvey R "Fig 5.22" from Lippincott's Illustrated Reviews: Biochemistry
(5th ed.), 5 Ed.(Lippincott W illiam s & W ilkins, 2010) pp. 66 [1 pages] ISBN:
9781608314126
18. Ferrier D, Harvey R "Fig 11.4 The structure of UPD-glucose" from Lippincott's
Illustrated Reviews: Biochemistry (5th ed.), 5 Ed.(Lippincott W illiam s & W ilkins, 2010)
pp. 127 [1 pages] ISBN: 9781608314126
19. Ferrier D, Harvey R "Fig 23.3B Form ation of human insulin from preproinsulin" from
Lippincott's Illustrated Reviews: Biochemistry (5th ed.), 5 Ed.(Lippincott W illiam s &
20. Ferrier D, Harvey R "Fig 2.6 Peptide helix stru ctu re " from Lippincott's Illustrated
Reviews: Biochemistry (5th ed.), 5 Ed.(Lippincott W illiam s & W ilkins, 2010) pp. 16 [1
pages] ISBN: 9781608314126
21. Ferrier D, Harvey R "Fig 31.1" from Lippincott's Illustrated Reviews: Biochemistry
(5th ed.), 5 Ed.(Lippincott W illiam s & W ilkins, 2010) pp. 431 [1 pages] ISBN:
9781608314126
22. Ferrier D, Harvey R "Fig 2.7 S tructure o f a B-sheet" from Lippincott's Illustrated
Reviews: Biochemistry (5th ed.), 5 Ed.(Lippincott W illiam s & W ilkins, 2010) pp. 17 [1
pages] ISBN: 9781608314126
23. Ferrier D, Harvey R "Fig 3.8 Effect of pH on th e oxygen a ffin ity of hem oglobin" from
Lippincott's Illustrated Reviews: Biochemistry (5th ed.), 5 Ed.(Lippincott W illiam s &
24. Chandar N, Vicelli S "Fig. 21.5" from Lippincott's Illustrated Reviews: Cell and
Molecular Biology, 1 Ed.(LIPPINCOTT WILLIAMS & WILKINS INC., 2010) pp. 400 [1 pages]
IS B N :9780781792103
IS B N :9780781792103
IS B N :9780781792103
27. Lieberman M, Marks AD "Fig 11.12" from Marks' basic medical biochem istry: a
clinical approach, 4 Ed.(Lippincott W illiam s & W ilkins, 2013) ISBN: 9781451100037
clinical approach, 4 Ed.(Lippincott W illiam s & W ilkins, 2013) pp. 1 [1 pages] ISBN:
9781451100037
clinical approach, 4 Ed.(Lippincott W illiam s & W ilkins, 2013) ISBN: 9781451100037
30. Rhoades RA, Bell DR "Fig 1.12 A signaling pathw ay fo r tyrosin e kinase receptors"
from Medical Physiology 4th ed., 4 E d.(Lippincott W illiam s & W ilkins, 2013) pp. 15 [1
pages] ISBN: 9781609134273
31. Rhoades RA, Bell DR "Fig 1.11 General structures of the tyrosin e kinase receptor
fa m ily " from Medical Physiology 4th ed., 4 Ed.(Lippincott W illiam s & W ilkins, 2013) pp.
14 [1 pages] ISBN: 9781609134273
32. Rhoades RA, Bell DR "Fig 1.13" from Medical Physiology 4th ed., 4 Ed.(Lippincott
33. Amon, Angelika ; et al "Fig 13-11" from Molecular Cell Biology, 8 Ed.(W. FI.
FREEMAN, 2016) pp. 588 [1 pages] ISBN: 9781464183393
34. /FERGUSON, L. R. "C hrom ium picolinate does not produce chom osom e dam age in
CHO cells" from MUTATION RESEARCH, (ELSEVIER BV, 1964) pp. 140-146 [7 pages]
IS B N :00275107
35. SKIPPER, MAGDALENA "Fig Epigenic Reprogram m ing in the zygote" from NATURE
REVIEWS GENETICS, (NATURE PUBLISHING GROUP, 2000) pp. 129 [1 pages] ISBN:
14710056
36. SKIPPER, MAGDALENA "Fig Functions of covalent histone m odification" from

NATURE REVIEWS GENETICS, (NATURE PUBLISHING GROUP, 2000) pp. 159 [1 pages]
IS B N :14710056
37. Kumar A, Abbas A, Aster J "Fig 7-25" from Robbins and Cotran Pathologic Basis o f
Disease 9th ed, 9 Ed.(Elsevier/Saunders, 2015) pp. 1 [1 pages] ISBN: 9781455726134
Disease 9th ed, 9 Ed.(Elsevier/Saunders, 2015) pp. 115 [1 pages] ISBN:
9781455726134
Disease 9th ed, 9 Ed.(Elsevier, 2015) pp. 1 [1 pages] ISBN: 9781455726134
Disease 9th ed, 9 Ed.(Elsevier, 2015) pp. 1 [1 pages] ISBN: 9781455726134
Permissions: We have made every effort to seek permission to use all borrowed material that appears in this course pack. If we have inadvertently used
anyone's material without permission, we will be happy to make the necessary arrangements at the first opportunity. Please contact the MSU Libraries
Course Materials Program (517.884.6468, coursematerials@lib.msu.edu).
P resen tation : Details of pack format and the names of rights holders who have granted permission for materials to be reproduced are in this list. The
bibliography is the result of a conversation between databases designed to automate the permissions process; neither format, nor punctuation, nor the
information in the citations conforms to scholarly standards. The bibliography should not be used either for reference or as a model.

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Post-translational Modifications

Ferrier: Chapter 14: Glycosaminoglycans, Proteoglycans, and Glycoproteins - from Oligosaccharide

Measurable Outcomes: What you should be able to do...

II. Post-translational Processing

Anything that crosses an intact

(S. Triezenberg and J. Wang, MSU)

1. Signal peptide (or signal sequence) consists of the

2. Signal recognition particle binds to the signal peptide as it is synthesized while it is

3. Co-translational transport: As the ribosome continues translation, it pushes the

C. Peripheral (tethered) Membrane Proteins

D. Targeting Signals for localization to different subcellular compartments.

Targeting Signal Chemical Nature Receptor for Signal Activitv

Nuclear Export leucine-rich Exportin-1 export from nucleus

Mitochondrial amphipathic helix involves receptors, im port into mitochondrial

Signal Sequence stretch of (~15) Signal Recognition im port into ER lumen

Lysosome Targeting mannose 6- Mannose 6-phosphate im port into lysosomes

Other ammo acids: P04-His; P04-Arg (s Triezenberg and j. Wang, Msu)

Example: kinase cascades (signal transduction in receptor as a tyrosine kinase pathway).

(S. Triezenberg and

Example: histone acetylation key mechanism in determining tightly packed

- lysine * 'Nl/' a-Ketoglutarate

a. It is catalyzed by specific hydroxylases: prolyl hydroxylase Succinate + C 0 2

Example: hydroxyproline and hydroxylysine collagen (Ferrier7_F4.6_p45)

Zymogens - is an inactive enzyme precursor which requires the removal of polypeptides to

o Proteosome degrades targeted protein and

B. ATP-independent degradative enzyme system of the

Ferrier: Chapter 33: Regulation of Gene Expression

Optional Suggested Videos:

Measurable Outcomes: What you should be able to do...

I. General Principles of Gene Regulation

II. Strategies of Gene Regulation in E. coli

IV. Specialized types of Recombination

A. Goal of gene regulation:

turn genes ON when theyre needed - activation

keep genes OFF when theyre not needed - repression or silencing

B. Gene regulatory mechanisms work in combinations

integration of all regulatory signals determines final output

C. Transcriptional initiation is a key step in gene For most genes

Gene expression is regulated at multiple levels:

Transcriptional regulation: transcription initiation

Post-translational regulation (at protein level):

A. Alternative sigma factors recognize distinct sets of gene promoters

E. c o li sigma factors recognize promoters with different consensus sequences

Factor Use -35 Sequence Separation -10 Sequence

a 70 general TTGACA 16-18 bp TATAAT

(S. Triezenberg and J. Wang, MSU)

(S. Triezenberg and J. Wang, MSU)

Nevertheless, close spatial localization in a subregion of the nucleus allows for

B. Levels of transcriptional regulation

There are two levels of transcriptional regulation:

(a) Eukaryotic promoter elements RNA pol II

-445 -114 -40 -30 +1

promoter-proximal regulatory sites (hundreds of bases from +1)

distant regulatory sites (thousands or tens of thousands of bases from +1)

(b) Transcriptional regulatory proteins

(R. Ritchie, MSU)

Regulatory proteins often have two distinct domains

Recognizes and b in d s to Site o f p ro te in -p ro te in in teractio n