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Text authored by Dr. Peter J. Russell
Slides authored by Dr. James R. Jabbur
CHAPTER 9
Functional and
Comparative Genomics
Functional Genomics
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Functional genomics is interested in the function of
genes, their genetic control and their expression
This field is dependent on experimental molecular
biology laboratory research and sophisticated computer
analyses by statisticians
This fusion of biology with math and computer science is
used to study many things, including…
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Finding genes within a genomic sequence
Aligning sequences in databases to determine matching
Predicting structure and function of gene products
Describing interactions between genes and gene products in the
cell, between cells, and between organisms
Considering phylogenetic relationships
Computational Assignment of Gene Function
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Genome sequencing can identify genes but does not reveal their
functions
Computationally generated, tentative identification is based on
homology with genes of known function. The best way to identify
gene function is to look at their proteins (i.e. BLASTp search)
Sometimes only part of a protein is a homologous match
There are many “genes” that are classified as orphans, but in reality,
they do not exist
Experimental Assignment of Gene Function
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An approach to determine gene function is to delete
the gene and observe the phenotype when that
gene’s function is knocked out
 RNA
interference (RNAi, also called RNA silencing)
uses small regulatory RNAs to silence gene
expression. It does not create a permanent
chromosomal change
 PCR can be used to produce a gene knockout that
results in a permament DNA change
Polymerase Chain Reaction
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The polymerase chain reaction (PCR) can produce
many copies of a specific target segment of DNA
A three-step cycle of heating, cooling, and replication
brings about a chain reaction that produces an
exponentially growing population of identical DNA
molecules
Kary Mullis was awarded the Nobel Prize for this
discovery (he is a super freak – really!)
Which is Kary?
Animation: Polymerase Chain Reaction
5
PCR METHOD
3
Target
sequence
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Limitations of PCR:
Sequence specific primers
require the template DNA
sequence be known
The DNA polymerizing
enzyme does not
proofread; thus, mutations
are incurred in the nascent
DNA (Taq polymerase)
The remarkable sensitivity
of the technique can result
in the amplification of
contaminating sequences,
a specific hazard in
forensic applications
3
Genomic DNA
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Gene Knockouts in Yeast
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Sequence specific primers are
designed to construct a
replication deficient, artificial
linear DNA deletion module
(see figure a)
The amplified linear DNA is
tranformed into yeast
Since the deletion module can
not reproduce itself, only
recombinants will survive
G418 selection (G418 is an
antibiotic that the kanR gene
destroys) (see figure b)
The recombinants have also
lost target gene function, due
to insertion within the
sequence
Gene Knockouts in Mice
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Knockout mice are used to study genes with human
analogs. This procedure is similar to that in yeast
The deletion module is the disrupted target gene with two
selectable markers encoded on the vector:
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neoR gene (neomycin resistance; inserts into the target gene seq)
tk gene (thymidine kinase; inserts outside the target gene seq)
The module is transformed into mouse embryonic stem
(ES) cells and grown in tissue culture with neomycin
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Two types of transformants containing neoR are selected:
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Homologous recombinants: Those with homologous recombination
between target vector and target gene result in the desired knockout,
which is validated by PCR assay
Random integrants: Those with random integration, generated by
nonhomologous recombination, are more common but are selected
against with a subsequent round of ganciclovir exposure
But how does ganciclovir work?
…what does thymidine kinase do to gancyclovir?
deoxyT + ATP → TMP + ADP
Thymidine kinase catalyzes this:
where deoxyT is deoxythymidine
ATP is adenosine 5’-triphosphate
TMP is deoxythymidine 5’-phosphate
ADP is adenosine 5’-diphosphate
Ganciclovir (nucleoside analog) makes use of the specificity
for thymidine kinase. These drugs act as prodrugs, which in
themselves are not toxic, but are converted to toxic drugs by
phosphorylation by thymidine kinase. Thus, random integranttransformed cells produce highly-toxic triphosphates that lead
to cell death (the analogs are incorporated into nascent DNA
causing the disruption of DNA synthesis).
…the next step in generating a transgenic mouse
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Knockout ES cells are injected into the blastocysts of a
mouse strain having a different coat color (e.g., agouti
ES cells are injected into a black-coated recipient)
The gestating offspring will become genetic chimera,
readily identified by patches of agouti and black hair
(agouti is dominant over black)
The offspring mice are cross-bred and in-bred to
generate a complete knockout genotype. PCR is also
used at this step for verification of genotype
The ko/ko genotype may be lethal during development
Analysis of embryos can give an indication of the gene’s
role in normal development
What is the probability of
generating a +/ko mouse:
Mate +/+ with +/ko; 50%
May? Why may?
What is the probability of
generating a ko/ko mouse:
Mate +/ko with +/ko; 25%
Knocking Down the
Expression of a Gene by
RNA Interference
RNA
interference (RNAi) is a
normal regulatory process in
eukaryotic cells (we already
covered this stuff previously)
Small RNA molecules specifically
silence gene expression and is
dependent on sequence homology
between the regulatory RNA
(siRNA) and the target mRNA
This system is used to knockdown gene expression while
leaving the gene itself unchanged
Introduction of the siRNA includes
transformation, microinjection,
absorption or injestion (C. elegans
is an example)
Organization of the Genome
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Highly transcribed genes occur in clusters where gene
density is high and introns are small. SINEs are more
common in these areas (SINEs are short interspersed
elements; Alu is an example)
Less frequently transcribed genes also cluster, but
gene density is lower and introns are larger. LINEs (long
interspersed elements) occur more often in these areas
In the interphase nucleus (G1, S, G2), high-density
chromosomal regions are centrally located, while
chromosome regions with low gene density are found
near the nuclear membrane (think about it; it makes
sense, right?)
Describing Patterns of Gene Expression
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Sequencing makes it possible to determine all the
genes that are expressed in a cell by analyzing the
total RNA transcripts produced (transcriptome)
In addition, the complete set of proteins in a cell,
termed it’s proteome, indicates the disposition of the
cell
The transcriptone is an indicator of cell phenotype,
and is analyzed with a DNA microarray
DNA microarrays are
used to study global
gene expression
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Samples of mRNA were
derived from a sporulating
yeast at different stages of
meiosis, converted to
cDNA and analyzed on
microarrays of PCRamplified ORF sequences
Control (green) and
experimental (red) samples
were labeled
Red spot = induced gene
Green spot = not induced
Yellow spot = intermediate
Animation: DNA Microarray Analysis
Pharmacogenomics
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Pharmacogenomics investigates how the individual
genome affects the body’s response to medication
The goal of pharmacogenomics is to tailor treatment
according to individual genetic factors
 Develops drugs associated with RNA molecules
and proteins associated with genes and diseases.
 Particularly useful for cancer patients in their
response to chemotherapy (2 recent
developments at AACR in June…)
Proteomics
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Proteomics is the cataloging and analysis of the
complete set of expressed proteins in a cell at a
given time
 Questions
asked include which proteins are made, in
what quantities, how are they modified and what are
their interactions with other proteins (interactomes)
 Conventional analysis employs 2-D acrylamide gel
electrophoresis, liquid chromatography and mass
spectrometry (quadrupole TOF MS)
 Protein arrays, which are similar to DNA microarrays,
are used to detect, quantify, and characterize proteins
on a large scale (capture array, Celera; immobilized
antibody, labeled target protein)
After 2DE, take purification to the next level!
Animation: HPLC
http://www.youtube.com/watch?v=kz_egMtdnL4
Animation: QTOF MS
http://www.youtube.com/watch?v=dsiGxfrJnU0&feature=related
WHA? 
http://www.youtube.com/watch?v=dufXvl-EM6c&feature=related
Comparative Genomics Studies
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Comparative genomics provides a way to study the
functions of human genes by working with nonhuman
homologues. examples of studies & uses follow…
Finding the genes which make us human?
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What are the differences between human and chimp gene
expression. human accelerated region-1 gene is an example
 Humans differ from chimps with only 100 out of 118 bases
matching (compared to chickens with 116/118 matching)
 This gene encodes a small, noncoding RNA and is expressed
in a region of the brain that develops uniquely in humans
 Other proteins have been identified this way:
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FOXP2 protein: important in speech production
ASPM protein: a regulator of brain size
Recent Changes in the Human Genome
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Changes after human populations split can be
studied by linkage disequilibreum, in which specific
alleles occur together more often than chance
predicts (think about flipping a coin)
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Mutations are associated with haplotypes
 Recombination within the block is rare
 The haplotype is heritable for generations
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Natural selection acts on individuals with a
haplotype that confers an advantage. A large
haplotype block in a population indicates recent
positive selection for a gene that it contains
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Lactose tolerance in dairying societies
 Skin and eye color is geographically relevant
DNA Microarray
Characterization of Gene
Amplification and
Deletion in Cancer
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Cancer cell genomes are
unstable and incur copy
number changes
Representational
Oligonucleotide Microarray
Analysis (ROMA) compares
whole genomes between
normal and tumor cells
DNA is isolated, digested,
amplified and labeled
Label tumor DNA with Cy5
(red), normal with Cy3 (green)
Laser scan and analyze data
Identifying a Virus in a Viral Infection Using DNA
Microarray Analysis
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The Virochip is a DNA microarray with
oligonucleotide probes for about 20,000 sequences
from an assortment of viruses.
 It
is used to screen mRNA from a patient specimen
likely to contain infected cells
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Virochip analysis was used in 2003 to identify the
cause of the emerging SARS (sudden acute
respiratory syndrome) outbreak; a coronavirus.