1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 25
Genomics II: Functional Genomics, Proteomics, and Bioinformatics
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 25.1 Functional Genomics: Gene Expression on a Genomic Scale
Functional genomics refers to those areas that deal with the function or expression of genomes
All transcripts an organism makes at any given time is an organism’s transcriptome
Use of genomic information to block expression systematically is called genomic functional profiling
Study of structures and functions of the protein products of genomes is proteomics

3. Transcriptomics
This area is the study of all transcripts an organism makes at any given time
Create DNA microarrays and microchips that hold 1000s of cDNAs or oligos
Hybridize labeled RNAs from cells to these arrays or chips
Intensity of hybridization at each spot reveals the extent of expression of the corresponding gene
Microarray permits canvassing expression patterns of many genes at once
Clustering of expression of genes in time and space suggest products of these genes collaborate in some process

4. Oligonucleotides on a Glass Substrate

5. Serial Analysis of Gene Expression
Serial Analysis of Gene Expression (SAGE) allows us to determine:
Which genes are expressed in a given tissue
The extent of that expression
Short tags, characteristic of particular genes, are generated from cDNAs and ligated together between linkers
These ligated tags are then sequenced to determine which genes are expressed and how abundantly

6. SAGE

7. Cap Analysis of Gene Expression (CAGE)
CAGE gives the same information as SAGE about which genes are expressed and how abundantly, in a given tissue
It focuses on the 5’-ends of mRNAs, which allows for the identification of transcription start sites and may help in locating pormoters

8. Whole Chromosome Transcription Mapping
High density whole chromosome transcriptional mapping studies have shown a majority of sequences in cytoplasmic poly(A)RNAs derive from non-exon regions of human chromosomes
Almost half of the transcription from these same chromosomes is nonpolyadenylated
Results indicate that great majority of stable nuclear and cytoplasmic transcripts in these chromosomes come from regions outside exons
Helps to explain the great differences between species whose exons are almost identical

9. Transcription maps of 10 Human Chromosomes

10. Genomic Functional Profiling
Genomic functional profiling can be performed in several ways
A type of mutation analysis, deletion analysis - mutants created by replacing genes one at a time with antibiotic resistance gene flanked by oligomers serving as barcode for that mutant
A functional profile can be obtained by growing the whole group of mutants together under various conditions to see which mutants disappear most rapidly

11. RNAi Analysis
Another means of genomic functional analysis on complex organisms can be done by inactivating genes via RNAi
An application of this approach targeting the genes involved in early embryogenesis in C. elegans has identified:
661 important genes
326 are involved in embryogenesis

12. Tissue-Specific Functional Profiling
Tissue-specific expression profiling can be done by examining a spectrum of mRNAs whose levels are decreased by an exogenous miRNA
Then compare to the spectrum of expression of genes at the mRNA level in various tissues
If that miRNA causes a decrease in the levels of mRNAs naturally low in cells expressing the miRNA
Suggests that the miRNA is at least a partial cause of those natural low levels
This type of analysis has implicated
miR-124 in destabilizing mRNAs in brain tissue
miR-1 in destabilizing mRNAs in muscle tissue

13. Locating Target Sites for Transcription Factors
ChIP-chip analysis can be used to identify DNA-binding sites for activators and other proteins
Small genome organisms - all of the intergenic regions can be included in the microarray
If genome is large, that is not practical
To narrow areas of interest can use CpG islands
These are associated with gene control regions
If timing/conditions of activator’s activity are known, control regions of genes known to be activated at those times, or under those conditions, can be used

14. Locating Target Sites for Transcription Factors
Tag sequencing, or ChIPSeq, in which chromatin pieces precipitated by ChIP are repeatedly sequenced, can also be used to identify transcription factor-binding sites
Knowledge of the sequence of multiple mammalian genomes allows one to narrow the search for human transcription factor binding sites by beginning with conserved regions of the genome
In addition, it is easier to search for cis-regulatory modules (CRMs), which contain several transcription factor binding sites

15. Locating enhancers that bind unknown proteins
There are still many enhancers whose protein partners are unknown
Pennachio and colleagues started the search for vertebrate enhancers by looking for highly conserved non-coding DNA regions in 2006
The strategy had a remarkably high success rate but has a drawback in that it only detects highly conserved sequences and not all important control regions are conserved

16. Locating promoters that bind unknown proteins
Ren and colleagues performed a genome-wide search for human promoters and were surprised to find that many genes have alternative promoters located hundreds of bases away from their primary promoters
Class II promoters can be identified using ChIP-chip analysis with an anti-TAF1 antibody
In one study using human fibroblasts, over 9,000 promoters were identified and over 1600 genes had multiple promoters

17. In Situ Expression Analysis
The mouse can be used as a human surrogate in large-scale expression studies that would be ethically impossible to perform on humans
Scientists have studied the expression of almost all the mouse orthologs of the genes on human chromosome 21
Expression followed through various stages of embryonic development
Catalogued the embryonic tissues in which these genes are expressed

18. Single-Nucleotide Polymorphisms (SNPs)
Single-nucleotide polymorphisms can probably account for many genetic conditions caused by single genes and even some by multiple genes
Might be able to predict response to a drug
Haplotype map with over 1 million SNPs makes it easier to sort out important SNPs from those with no effect

19. Structural Variation
Structural variation is a prominent source of variation in human genomes
Insertions
Deletions
Inversions
Rearrangements of DNA chunks
Some structural variation can, in principle, predispose certain people to contract diseases
Some variation is presumably benign
Some also is demonstrably beneficial

20. 25.2 Proteomics
The sum of all proteins produced by an organism is its proteome
Study of these proteins, even smaller subsets, is called proteomics
Such studies give a more accurate picture of gene expression than transcriptomics studies do

21. Protein Separations and Analysis
Current research in proteomics requires first that proteins be resolved, sometimes on a massive scale
Best tool for separation of many proteins at once is 2-D gel electrophoresis
After separation, proteins must be identified
Best method of identification involves digestion of proteins one by one with proteases
Then identify the peptides by mass spectrometry
In the future, microchips with antibodies attached may allow analysis of proteins in complex mixtures without separation

22. Quantitative Proteomics
To determine the changes in protein levels upon perturbation of a cell culture, one can label the cells under the first condition with a light isotopic tag, and under the second condition with a heavy isotopic tag
If the proteins are labeled in vivo, the cell cultures can be mixed, the proteins can be extracted and fragmented by proteolysis and upon further separation can be subjected to mass spectronomy
The ratio of heavy to light peak areas will reflect the change in protein concentration as the growth conditions change

23. Comparative Proteomics
What makes a worm a worm and a fly a fly?
Mass spectrometry data can be used to compare protein concentrations in two different organisms
This type of analysis was applied to C.elegans and Drosophila to reveal that the concentrations of orthologous proteins are correlated much better than the orthologous mRNAs in the two organisms

24. Protein Interactions
Most proteins work with other proteins to perform their functions
Several techniques are available to probe these interactions
Yeast two-hybrid analysis has been used for some time, now other methods are available
Protein microarrays
Immunoaffinity chromatography with mass spectrometry
Other combinations

25. Detecting Protein-Protein Interactions

26. 25.3 Bioinformatics
Bioinformatics involves the building and use of biological databases
Some of these databases contain the DNA sequences of genomes
Essential for mining the massive amounts of biological data for meaningful knowledge about gene structure and expression

27. Finding Regulatory Motifs in Mammalian Genomes
Using computational biology techniques, Lander and Kellis have discovered highly conserved sequence motifs in 4 mammalian species, including humans:
In the promoter regions, these motifs probably represent binding sites for transcription factors
3’-UTRs motifs probably represent binding sites for miRNAs

28. Using the Databases
The National Center for Biological Information (NCBI) website contains a vast store of biological information, including genomic and proteomic data
Start with a sequence and discover the gene to which it belongs, then compare that sequence with that of similar genes
Query the database with a topic for information
View structures of protein in 3D by rotating the structure on your computer screen