Biochemistry 412 Overview of Genomics & Proteomics (con’d) 21 January 2005

After the human genome and the genomes of the model organisms S. cerevisiae, C. elegans, D. melanogaster, and M. musculus, what is left to sequence?? Other animal genome sequences also in progress: CowChicken Polyp hydra DogClawed frog Starlet sea anemone CoyoteMosquito (several) Acorn worm WolfHoney bee Blood fluke Guinea pigSilkworm Flat worm Nine-banded armadilloMultiple fruit fly species Surf clam CatTobacco budworm Rat LemurRed flour beetle Orangutan ElephantZebrafish Baboon Rhesus monkeyPufferfish Chimpanzee WallabyFreshwater snail Duck-billed platypus OpposumSea squirt Rabbit Note: plus literally hundreds of microbial and other lower organism genomes!

The human genome sequence is finished…. >>> But what other genome-based studies have been enabled by this achievement? Some examples: Human variation and evolution (e. g., “SNPs”) Somatic mutations (e. g., loss-of-heterozygosity in cancer) RNA expression profiling (cf. “DNA chips”) Methylation patterns (e. g., epigenetics and gene silencing)

Single Nucleotide Polymorphisms (“SNPs”)

Roses (2000) Nature 405, 857.

Microarrays (DNA Chips)

Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, Note: 4N masks required to make an array of oligonucleotides each of length N.

Pease et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, Note: this is the photolabile blocking group, “X”, indicated schematically in Figure 1.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20. Key feature: known oligo sequence at each “address” on the chip.

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20.

RNA Profiling

Lockhart & Winzeler (2000) Nature 405, 827. a) In situ synthesis of oligonucleotides on slide b) Post-synthesis spotting of cDNAs on slide

Lipshutz et al (1999) Nature Genet. (suppl.) 21, 20. mRNA Expression Profiling with Oligonucleotide Microarrays (Affymetrix chip) …some tricks of the technique

Lockhart & Winzeler (2000) Nature 405, 827. Note that the technique is sensitive enough to detect as little as one mRNA molecule per cell!

Highly parallel genomics-based analysis methods are ideal for identifying cross-correlations (e.g., co-expression, co-regulation, sharing of binding partners, etc.) and other associations among disparate genes or gene products. Clustering algorithms can be used to highlight these relationships. The desire to understand the biological relevance of such data has been one of the strong driving forces behind the development of the field of systems biology.

Bassett et al (1999) Nature Genet. (suppl.) 21, 51. An example from yeast

Ref: Lee et al (1999) Science 285, Note: caloric restriction gene chip experiment w/ rats.

Lee et al (1999) Science 285, 1390.

Pharmaceutical companies also use mRNA profiling methods to search for new drug targets. For example, one might compare mRNA extracted from normal vs. cancerous liver tissue and look for genes that are up-regulated in the tumor. If such genes code for proteins that are necessary for maintenance of the tumor but are less important to normal, slowly-dividing cells, then these proteins may be candidates for novel drug discovery and development programs. Examples of such genes are those involved in vascularization of the tumor, maintenance of telomeres, etc. [Note: some genes identified in this way may have biochemical functions that are as yet unknown, which has motivated the field of functional genomics.] DNA Chips and Drug Discovery

Kapranov et al (2002) Science 296, 916. Surprise: not all (or even most) transcription comes from protein coding regions of the genome!

“Proteomics” The study of the complete complement of proteins found in an organism

“Degrees of Freedom” for Protein Variability Covalent Modifications in Proteins Post-translational modifications (e.g., phosphorylation, glycosylation, etc.) - more than 200 such modifications are known, and they can occur at multiple sites in a single protein Alternative splicing of a primary transcript - in extreme cases, a single gene can produce tens of thousands of different mRNAs! Proteolytic processing Protein aging Thus, there are probably many millions of different proteins in our bodies!!

More Reality Therapy re Proteins They have “personalities”: each behaves differently They exist in different concentrations, ranging over a million-fold It will be extremely difficult to even identify them all (see previous slide) Take-home message: Proteomics presents challenges that are orders-of-magnitude more difficult than those presented by genomics!

“Classic” Proteomics: 2-Dimensional Gel Electrophoresis

Pandey & Mann (2000) Nature 405, 837. Mass spectrometry is a major tool in proteomics.

Pandey & Mann (2000) Nature 405, 837. Comparative Protein Profiling Using 2D Gels and Mass Spectrometry Note: sometimes looking at the proteins gives a different impression than one gets from looking at the mRNAs!

Pandey & Mann (2000) Nature 405, 837. Protein chips

Pandey & Mann (2000) Nature 405, 837. Yeast Two-Hybrid System (Song and Fields)