Biochemistry 412 Overview of Genomics & Proteomics 17 January 2006

1. Biochemistry 412 Overview of Genomics & Proteomics 17 January 2006

3. 2006 Biochemistry 412 Syllabus con’d:

4. DNA Sequencing & the Human Genome Project

5. Timeline: The Foundations of Genomics 1953 • Model for 3D structure of DNA - J. Watson & F. Crick • First protein sequence (insulin) - F. Sanger 1965 • First RNA sequences - R. W. Holley & colleagues; F. Sanger & colleagues 1970 • Restriction endonucleases discovered - D. Nathans & H. O. Smith 1972 • First recombinant DNA molecule - P. Berg & colleagues 1975 • “Plus-minus” method of DNA sequencing - F. Sanger & A. R. Coulson 1977 • Chemical method of DNA sequencing - A. Maxam & W. Gilbert • Dideoxy method of DNA sequencing - F. Sanger & A. R. Coulson • First bioinformatics software for DNA sequences - R. Staden 1978 • Single-stranded phage vectors developed - J. Messing & colleagues 1980 • “Shotgun cloning” strategy for DNA sequencing - J. Messing & colleagues; F. Sanger & colleagues 1981 • Random shotgun cloning method developed - S. Anderson 1985 • Polymerase chain reaction (PCR) method developed - K. Mullis 1986 • Development of first automated DNA sequencer - L. Hood & colleagues >>> For the past 25+ years, the size of the largest genome sequenced (from X174 to human) has doubled approximately every 18 months!

6. Lander et al (2001) Nature409, 860.

9. How the human genome was sequenced The Random “Shotgun” DNA Sequencing Strategy Allows sequence information about a target genome to be accumulated rapidly and in a non-biased and semi-automatable fashion.

10. Random shotgun DNA Sequencing Fragmentation by DNAase I digestion of target DNA in the presence of Mn++ Anderson (1981) Nucleic Acids Res.9, 3015.

11. Random fragmentation yields clones covering the target DNA region (in this case, a portion of the bovine mitochondrial genome) • Coverage is reasonably complete and uniform • Most regions are sequenced more than once, improving overall accuracy Anderson (1981) Nucleic Acids Res.9, 3015.

12. >>> The recursive, identical steps involved in random shotgun DNA sequencing allowed automation of the sequencing process (even for very large genomes). Anderson (1981) Nucleic Acids Res.9, 3015.

13. Lander et al (2001) Nature409, 860.

14. The “Public” Consortium Approach: Lander et al (2001) Nature409, 860.

15. The Celera (“whole genome shotgun”) Approach: Venter et al (2001) Science291, 1304.

16. Venter et al (2001) Science291, 1304. [Note: individual “B” is Craig Venter!!]

17. Differences between the Public (Lander et al) and Celera (Venter et al) Human Genome Sequencing Efforts Public Project: • Mapped BACs and YACs from the genome first (to sort out where the repeats were), then shotgun sequenced these • Started earlier (~1990) • Initial (2001) draft not as accurate as Celera’s (see below)* • Finished later (~2003) • Final draft more accurate than Celera’s • Cost: ca. $3 billion Celera Project: • Shotgun sequenced entire human genome in one go • Used sequenced end pairs from linking clones to address the repeat problem (also used public project data) • Started in the late ‘90s • Initial (2001) draft more accurate than public’s (see below)* • Quit working before final draft was finished • Cost: ca. $300 million**

18. Differences between the Public (Lander et al) and Celera (Venter et al) Human Genome Sequencing Efforts (footnotes from previous slide) *Celera’s sequence, which was proprietary, incorporated all of the public data, which was available on the internet, so initially Celera’s genome sequence was more complete and accurate than the public consortium’s sequence (Duh!!). At the time, this fact escaped most journalists who reported on the competition with the public consortium’s effort, and the consortium scientists did not help their cause by dumping on Celera’s data! **Applied Biosystems (ABI, Celera’s parent company) more than recouped all of its expenses by selling DNA sequencing machines to -- among others -- the panicked public sequencing consortium members (and also by selling Celera stock to Wall Street). Some observers have even suggested that the entire Celera human genome sequencing effort was nothing more than a Machiavellian marketing ploy by ABI! >>> Nevertheless, the race between the public and private sectors delivered a high quality finished human genome sequence to the scientific community yearsearlier than would have been the case without such a competition!

19. The Results….

20. Lander et al (2001) Nature409, 860.

21. 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*: Cow Chicken Polyp hydra Dog Clawed frog Starlet sea anemone Coyote Mosquito (several) Acorn worm Wolf Honey bee Blood fluke Guinea pig Silkworm Flat worm Nine-banded armadillo Multiple fruit fly species Surf clam Cat Tobacco budworm Rat Lemur Red flour beetle Orangutan Elephant Zebrafish Baboon Rhesus monkey Pufferfish Chimpanzee Wallaby Freshwater snail Duck-billed platypus Opposum Sea squirt Rabbit …. plus literally hundreds of microbial and other lower organism genomes! *As of January, 2005.

22. 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) • Proteomics

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

24. “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!!

25. 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!