Introduction to Genomics and Proteomics - Historical Perspective and the Future Eleftherios P. Diamandis, M.D., Ph.D., F

1. Introduction to Genomics and Proteomics - Historical Perspective and the FutureEleftherios P. Diamandis, M.D., Ph.D., FRCPC (C) UNIVERSITY OF TORONTO (Course 1505S/Jan. 9, 2001 #1)

2. Organization of the LectureHistorical BackgroundThe Human Genome ProjectCritical Technologies: • Massive, automated sequencing•DNA and RNA analysis• Mass spectrometry• DNA and protein microarrays• Bioinformatics• Single nucleotide polymorphismsApplications:• Diagnostics• Therapeutics• PharmacogeneticsEthicsPatents(Course 1505S/Jan. 9, 2001 #2)

3. Historical MilestonesYearMilestone1866 Mendel’s discovery of genes1871 Discovery of nucleic acids1951 First protein sequence (insulin)1953 Double helix structure of DNA1960s Elucidation of the genetic code1977 Advent of DNA sequencing1975-79 First cloning of human genes1986 Fully automated DNA sequencing1995 First whole genome (Haemophilus Influenza)1999 First human chromosome(Chr #22)2000 Drosophila / Arabidopsis genomes2001 Human and mouse genomes(Course 1505S/Jan. 9, 2001 #3)

4. Terminology DNA Genomics mRNA Transcriptomics Protein Proteomics Metabolites Metabolomics Functional genomics, proteomics ----- etc.(Course 1505S/Jan. 9, 2001 #4)

5. HistoryOn June 26, 2000, at The White House, it was announced that the Human Genome Project was essentially completed by - Celera Genomics (private company)The National Human Genome Research Initiative and its International Partners (publicly funded)Work has yet to be published but Celera scientists submitted a paper to “Science” on December 6, 2000.(Course 1505S/Jan. 9, 2001 #5)

6. HistoryOn June 26, 2000, at The White House, it was announced that the Human Genome Project was essentially completed by - Celera Genomics (private company)The National Human Genome Research Initiative and its International Partners (publicly funded)Work has yet to be published but Celera scientists submitted a paper to “Science” on December 6, 2000.(Course 1505S/Jan. 9, 2001 #5)

7. Diagnostics / Prognostics• Does my DNA predispose me to a specific disease?• Do I want to know? (Ethics)• Genetic mutations ® disease ®cancer® diabetes®Alzheimer’s® heart disease• Whole genome scans for identification of mutations/polymorphisms?AACC2000-#2 - 1

8. Pharmacogenetics and PharmacogenomicsGoal is to associate human sequence polymorphisms with:• Drug metabolism• Adverse effects• therapeutic efficacyß* Decrease drug development cost* Optimize selection of clinical trial participants* Increase patient benefitAACC2000-#2 - 3

9. Critical Protein TechnologiesProtein• Make pure form (recombinant)• Activity• Reagents (antibodies)• Identification (sequencing)• Identify post-translational modification (glycation, phosphorylation, etc.)• Protein-protein interactions (physiological function)• Gene ® protein knockout / transgene AACC2000 -#2 - 13

10. Models of Human Disease• Identify natural human knockouts• Develop mice with every gene (or gene combination) being knocked out (this project is now underway!) AACC2000 -#2 -14

11. Expressed Sequence Tags (ESTs)• Cloned cDNAs from various tissues (cDNA libraries)• Can search through by BLAST analysis• Can purchase them, fully sequence and characterize themGreat help for new gene identification.AACC2000 -#2 -16

12. Gene Patents• Gene fragments• Whole genes without function• Whole genes with function• Whole genes with function and utility (enablement) AACC2000 -#2 - 18

13. Where Do We Stand Today? (July 2000)Public Consortium: 85% of Genome is done* 24% finished form* 22% near finished* 38% draft* rest is being doneCelera: Claims to have more than 99% of genome now!Incyte: They may have all the genes! AACC2000 -#2 -25

14. Where Does the Individual Researcher Stand?• At the end of the day, each gene must be looked at in great detail: - structure - function - physiology/pathways - pathophysiology - connection to disease - tools• Individual researchers can make the big discoveries on a very specific gene or a very specific gene family• Great time for individual researchers AACC2000 -#2 - 20

15. The Future of Genome Projects Human¯ Mouse (just started)¯ Rat¯ Zebra Fish¯ Dog¯ Other Primates* The Era of Comparative Genomics(you can learn a lot about humans by studying the yeast, drosophila, mouse, etc.) AACC2000 -#2 - 21

16. The Impact of the Human Genome Project in Medicine• You can’t make a car if you are missing parts• Once all genes are known, we will start understanding their function ® PATHWAYS• We will then be able to correlate disease states to certain genes (Pathobiology) DISEASE ® GENE (S) GENE (S) ® DISEASE• We will then find ways for rational treatments (designer drugs), prevention, diagnosis……AACC2000 -#2 - 22

17. Gene Manipulation (Ethics??)Gene modulation (­¯ regulation)Gene repairGene excisionGene replacement/transplantationGene improvement AACC -#2 -23

18. Celera’s Whole Genome Shotgun Strategy• Doe not use BAC clones; cuts whole DNA into millions of pieces which are sequenced• Computer assembles pieces together• Achieve high accuracy with X6 coverage• Lots of relatively short gaps AACC -#2 - 26

19. Strategy to Sequence Human GenomeConstruct a human genomic library in an appropriate vector (BAC)Assemble overlapping BAC clones in order to obtain full coverage of the distance (restriction map) DNABACClonesStart sequencing each BAC until you finish the jobAACC -#2 - 27

20. How are these BACs Sequenced?Shotgun SequencingBAC clone is broken down to small pieces which have overlapping endsSmall pieces are sequenced and a computer assembles the pieces based on the overlapping sequence informationConstruct contigs (contiguous areas of sequence)Larger contigs ------------------------ AACC -#2 -28

21. Other Important Genomic Technologies• Recombinant DNA (cloning)• PCR• Pulsed Field Gel Electrophoresis (PFGE)• Chromosome microdissection• Somatic hybrid cell lines (mapping) [rodent x human]• Radiation hybrid cell lines [rodent x human]• DNA sequencingAACC2000- #2- 32

22. AnnotationWhat is annotation?Make sense out of a linear sequence ® identify genes, intron/exon boundaries, regulatory sequences, predict protein structure, identify motifs, predict function, etc.Annotation will likely go on for a few years.Major annotation tool Þ BIOINFORMATICS (hardware & software)

23. Celera Genomics• The publicly funded project started around 1990 with a goal to produce a highly accurate sequence by 2005• Celera started in 1998 and within 2 years sequenced more DNA than the publicly funded consortium!Why?• No bureaucracy• Facility (300 sequencers x 24h/day)• Powerful supercomputer• Lots of money• More efficient sequencing approach (no BACs necessary)• Use of data from the publicly funded project AACC2000- #2 -30

24. Cloning Vectors• Replicable units of DNA which can carry exogenously inserted DNA; size of insert varies with vector type:• plasmid 5-10 kb•l phage 20 kb• cosmid 45 kbPAC/BAC (P1- or bacterial artificial chromosome) 100 - 200 kbYAC (yeast artificial chromosome) 1,000 kb AACC2000- #2- 31

25. Human Genome• 3 x 109 base pairs• Approximately 100,000 genes• < 10% of DNA encodes for genes; the rest represents introns/repetitive elements• Importance of non-coding sequences currently not understoodAACC2000 -#2 -33

26. Quality of Sequencing• Clones are sequenced more than once to verify the sequence many times: x 4 ® rough draft ® 1 error per 100 bases x 8-11 ® finished draft ® 1 error per 10,000 basesAACC2000 -#2 -34

27. The Next Race• It will not be who has the sequence• It will be how you can use the sequence to arrive at products* DIAGNOSTICS* THERAPEUTICSAACC2000 -#2- 35

28. Genomics and Drug DiscoveryGenomic technologies are involved in all aspects of the drug discovery process from target validation though to the marketed drug, which include:• Molecular target identification• Drug target characterization and validation• Lead discovery• Lead optimization• Clinical candidate to marketed drugAACC2000- #2- 37

29. Key Corporate Players in ProteomicsCompay Location ApproachCelera Rockville, MD DatabasesIncyte Pharmaceuticals Palo Alto, CA DatabasesGeneBio Geneva, Switzerland DatabasesProteome Inc. Beverly, MA DatabasesPE Biosystems Framingham, MA InstrumentationCiphergen Biosystems Palo Alto, CA Protein arraysOxford GlycoSciences Oxford, UK 2D gel/MS*Protana Odense, Denmark 2D gel/MSGenomic Solutions Ann Arbor, MI 2D gel/MSLarge Scale Proteomics Corp. Rockville, MD 2D gel/MS ______________________________________________________* 2D gel electrophoresis and mass spectrometry AACC2000- #2-381

30. Pharmacogenetics and Pharmacogenomics in Drug Discovery_______________________________________________________Aspect of Drug Development Approach _______________________________________________________Drug-drug interactions Examine polymorphism in metabolic enzymesEfficacy Differentiate responders from nonrespondersSide Effects Examine variation in gene or genes involved in mediating the effects (may be mechanism related or unrelated)Toxicity Gene expression profiling in cells treated with compound. Look for toxicity signatures. AACC2000- #2- 39

31. The Biography of the Year 2000(Francis Collins and J.Craig Venter)

32. Creating an Array of Contigous BAC Clones

33. The ….omics

35. Introduction to Genomics and Proteomics - Historical Perspective and the FutureEleftherios P. Diamandis, M.D., Ph.D., FRCPC (C) UNIVERSITY OF TORONTO (Course 1505S/Jan. 9, 2001 #1)

36. Organization of the LectureHistorical BackgroundThe Human Genome ProjectCritical Technologies: • Massive, automated sequencing•DNA and RNA analysis• Mass spectrometry• DNA and protein microarrays• Bioinformatics• Single nucleotide polymorphismsApplications:• Diagnostics• Therapeutics• PharmacogeneticsEthicsPatents(Course 1505S/Jan. 9, 2001 #2)

37. Historical MilestoneYearMilestone1866 Mendel’s discovery of genes1871 Discovery of nucleic acids1951 First protein sequence (insulin)1953 Double helix structure of DNA1960s Elucidation of the genetic code1977 Advent of DNA sequencing1975-79 First cloning of human genes1986 Fully automated DNA sequencing1995 First whole genome (Haemophilus Influenza)1999 First human chromosome2000 Drosophila / Arabidopsis genomes2001 Human and mouse genomes(Course 1505S/Jan. 9, 2001 #3)

38. Technologies DNA Genomics mRNA Transcriptomics Protein Proteomics Metabolites Metabolomics Functional genomics, proteomics ----- etc.(Course 1505S/Jan. 9, 2001 #4)

39. HistoryOn June 26, 2000, at The White House, it was announced that the Human Genome Project was essentially completed by - Celera Genomics (private company)The National Human Genome Research Initiative and its International Partners (publicly funded)Work has yet to be published but Celera scientists submitted a paper to “Science” on December 6, 2000.(Course 1505S/Jan. 9, 2001 #5)

40. Predicting the FutureWhat is going to happen now that the human and other genomes are completed?How quickly the next steps will happen?What are the potential difficulties?Are we expecting too much?(Course 1505S - Jan. 15/01 - #6)

41. Grand PlanFind all the genesTranslate genes to proteins“Compute” function by similarity search and comparison to known proteins“Compute” structure(Course 1505S - Jan. 15/01 - #7)

42. Difficulties• Gene prediction programs are unreliable• Function inference by just similarity search may be fallacious• Computation of structure is still unreliable Our databases may get contaminated with “wrong” information.(Course 1505S - Jan. 15/01 - #8)

43. Gene Prediction• Programs were designed based on knowledge of already cloned genes (ORFs; splice sites; start/stop codons, etc.)• These programs provide excellent clues for gene presence but they never or rarely predict the complete gene structure• The computer prediction must be taken as a “starting point” to experimentally clone a gene How many genes in the genome? Estimate: 27,462 to 312, 278! (Course 1505S - Jan. 15/01 - #9)

44. What is a Gene?• Heritable unit corresponding to a phenotype?• DNA that encodes for a protein?• DNA that encodes RNA?• What if RNA is not translated?• What if a “gene” is not expressed?(Course 1505S - Jan. 15/01 - #10)

45. Prediction of FunctionWhat is function? This is not a simple termFunction may be: • a biological process (e.g. serine protease activity)• a molecular event (e.g. proteolysis of a specific substrate)• a cellular structure (e.g. membrane; chromatin; mitochondrion; etc.)• relevance to a whole process (e.g. cell cycle)• relevance to the whole organism (e.g. ovulation)* Some scientists have now initiated projects to “compute” function of whole organisms. (Course 1505S - Jan. 15/01 - #11)

46. Pattern Recognition• Looks for motifs that may have functional relevance (family signatures):* Membrane anchoring* Catalytic site* Nucleotide binding* Nuclear localization signal* Hormone response element* Calcium binding, etc.• Protein family resources (being created now)(Course 1505S - Jan. 15/01 - # 12)

47. Homology• What is “homology”?Definition: Two proteins are homologous if they are related by divergence from a common ancestor. B Divergent A C Evolution Ancestor D Homologous(Course 1505S - Jan. 15/01 - # 13)

48. Analogy• What is “analogy”?Definition: Two proteins are “analogous” if they acquired common structural and functional features via convergent evolution from unrelated ancestors. Convergent A B Evolution C D Unrelated Analogous (similar structure and/or function)(Course 1505S - Jan. 15/01 - # 14)

49. Serine Proteases (Convergent Evolution)Trypsin-like Subtilisin-like Analogous proteinsMany homologous Many homologousmembers membersTrypsin and subtilisin share groups of catalytic residues with almost identical spatial geometries but they have no other sequence or structural similarities.(Course 1505S - Jan. 15/01 - # 15)

50. Human Kallikrein Gene Family (Divergent Evolution)15 homologous genes on human chromosome 19q13.4 Divergence in tissue expression and substrate specificity(Course 1505S - Jan. 15/01 - # 16)