1. Functional and Comparative Genomics
Text authored by Dr. Peter J. Russell
Slides authored by Dr. James R. Jabbur
CHAPTER 9
Functional and Comparative Genomics

2. Functional Genomics
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…
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

3. Computational Assignment of Gene Function
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

4. Experimental Assignment of Gene Function
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

5. Polymerase Chain Reaction
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!)

6. Animation: Polymerase Chain Reaction
Which is Kary?
Animation: Polymerase Chain Reaction

7. molecules; 2 molecules (in white boxes) match target sequence
PCR METHOD 5 3
Target sequence
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
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

8. Gene Knockouts in Yeast
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
Figure 8.21 The polymerase chain reaction (PCR) for selective amplification of DNA sequences.

9. Gene Knockouts in Mice
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:
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
Two types of transformants containing neoR are selected:
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

10. But how does ganciclovir work?

11. …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 integrant- transformed cells produce highly-toxic triphosphates that lead to cell death (the analogs are incorporated into nascent DNA causing the disruption of DNA synthesis).

12. …the next step in generating a transgenic mouse
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

13. 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%

14. 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 knock-down gene expression while leaving the gene itself unchanged
Introduction of the siRNA includes transformation, microinjection, absorption or injestion (C. elegans is an example)

15. Organization of the Genome
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?)

16. Describing Patterns of Gene Expression
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

17. DNA microarrays are used to study global gene expression
Samples of mRNA were derived from a sporulating yeast at different stages of meiosis, converted to cDNA and analyzed on microarrays of PCR-amplified 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

19. Pharmacogenomics
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…)

20. Proteomics
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)

21. After 2DE, take purification to the next level!

22. Animation: HPLC

23. http://www.youtube.com/watch?v=dsiGxfrJnU0&feature=related WHA? 
Animation: QTOF MS
WHA? 

25. Comparative Genomics Studies
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?
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:
FOXP2 protein: important in speech production
ASPM protein: a regulator of brain size

26. Recent Changes in the Human Genome
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)
Mutations are associated with haplotypes
Recombination within the block is rare
The haplotype is heritable for generations
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
Lactose tolerance in dairying societies
Skin and eye color is geographically relevant

27. e
DNA Microarray Characterization of Gene Amplification and Deletion in Cancer
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

28. Identifying a Virus in a Viral Infection Using DNA Microarray Analysis
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
Virochip analysis was used in 2003 to identify the cause of the emerging SARS (sudden acute respiratory syndrome) outbreak; a coronavirus.