1. BB30055: Genes and genomes Genomes - Dr. MV Hejmadi (bssmvh)

2. BB30055: Genomes - MVH Recommended texts: 1)Genetics from genes to genomes 2e - Hartwell et al 2)Human Molecular Genetics 3 – Strachan and Read 4) Genomes 2 - TA Brown 5) Genes VII – Benjamin Lewin Special issue Journals: Nature (2001) 15 th Feb Vol 409 Science (2001) Vol 291 No 5507 Full text of both above journals available at http://www.bath.ac.uk/library/subjects/bs/links.html#hgp

3. BB30055: Genomes - MVH 3 broad areas (A)Genomes, transcriptomes, proteomes (B)Applications of the human genome project (C) Genome evolution

4. A) Genomes, transcriptomes, proteomes Genome projects - Human Genome Project (HGP): a history - Other genome projects: why do it - Genome organisation -insights from HGP -Repeat elements -Transposable elements -Mitochondrial genomes -Y chromosome Post-genomics -transcriptomes - proteomes

5. (A) Genomes, transcriptomes and proteomes genome transcriptome proteome Entire DNA complement of any organism which include organelle DNA All RNA transcribed from genome of a cell or tissue all proteins expressed by a genome, cell or tissue

7. Why study the genome? 3 main reasons description of sequence of every gene valuable. Includes regulatory regions which help in understanding not only the molecular activities of the cell but also ways in which they are controlled. identify & characterise important inheritable disease genes or bacterial genes (for industrial use) Role of intergenic sequences e.g. satellites, intronic regions etc

8. History of Human Genome Project (HGP) 1953 – DNA structure (Watson & Crick) 1972 – Recombinant DNA (Paul Berg) 1977 – DNA sequencing (Maxam, Gilbert and Sanger) 1985 – PCR technology (Kary Mullis) 1986 – automated sequencing (Leroy Hood & Lloyd Smith 1988 – IHGSC established (NIH, DOE) Watson leads 1990 – IHGSC scaled up, BLAST published (Lipman+Myers) 1992 – Watson quits, Venter sets up TIGR 1993 – F Collins heads IHGSC, Sanger Centre (Sulston) 1995 – cDNA microarray 1998 – Celera genomics (J Craig Venter) 2001 – Working draft of human genome sequence published 2003 – Finished sequence announced

9. HGP Goal: Obtain the entire DNA sequence of human genome Players: (A)International Human Genome Sequence Consortium (IHGSC) - public funding, free access to all, started earlier - used mapping overlapping clones method (B) Celera Genomics – private funding, pay to view - started in 1998 - used whole genome shotgun strategy

10. Whose genome is it anyway? (A)International Human Genome Sequence Consortium (IHGSC) - composite from several different people generated from 10-20 primary samples taken from numerous anonymous donors across racial and ethnic groups (B) Celera Genomics – 5 different donors (one of whom was J Craig Venter himself !!!)

11. Genomicists looked at two basic features of genomes: sequence and polymorphism –How does one sequence a 500 Mb chromosome 600 bp at a time? –How accurate should a genome sequence be? DNA sequencing error rate is about 1% per 600 bp –How does one distinguish sequence errors from polymorphisms? Rate of polymorphism in diploid human genome is about 1 in 500 bp –Repeat sequences may be hard to place –Unclonable DNA cannot be sequenced (30%) Major challenge - to determine sequence of each chromosome in genome and identify polymorphisms

12. Divide and conquer strategy meets most challenges Chromosomes are broken into small overlapping pieces and cloned Ends of clones sequenced and reassembled into original chromosome strings Each piece is sequenced multiple times to reduce error rate –10-fold sequence coverage achieves a rate of error less than 1/10,000

13. Strategies for sequencing the human genome

15. Whole-genome shotgun sequencing Whole genome randomly sheared three times –Plasmid library constructed with ~ 2kb inserts –Plasmid library with ~10 kb inserts –BAC library with ~ 200 kb inserts Computer program assembles sequences into chromosomes No physical map construction Only one BAC library Overcomes problems of repeat sequences Fig. 10.13 Whole genome randomly sheared three times –Plasmid library constructed with ~ 2kb inserts –Plasmid library with ~10 kb inserts –BAC library with ~ 200 kb inserts Computer program assembles sequences into chromosomes No physical map construction Only one BAC library Overcomes problems of repeat sequences Private company Celera used to sequence whole human genome Fig. 10.13 Genetics by Hartwell

16. sequencing larger genomes Mapping phase Sequencing phase http://www.DNAi.org

17. Other genomes sequenced 2002 36,000 genes Sept 2003 18,473 human orthologs 1997 4,200 genes 1998 19,099 genes 2002 38,000 genes Science (26 Sep 2003)Vol301(5641)pp1854-1855

18. Human genome – size and structure Nuclear genome (3.2 Gbp) 24 types of chromosomes Y- 51Mb and chr1 -279Mbp Base composition – 41% GC Mitochondrial genome

19. Nuclear genome organisation (human) Genomes 2 by TA Brown pg 23

20. Nuclear genome organisation (human) 1) Gene and gene related sequences Coding regions – Exons (5%) Non-coding regions RNA genes Introns Pseudogenes Gene fragments

21. Nuclear genome organisation (human) 16S, 23S, 28S, 18S etc 22 types of mitochondrial & 49 cytoplasmic U1,U2.U4,U5,U6 etc > 100 types RNA genes - rRNA tRNA snRNA snoRNA Major classes of RNA involved in gene expression Other RNA classes microRNA XIST RNA Imprinting associated RNA Nervous system specific Antisense RNA Others

22. introns Non-coding regions…..

23. Pseudogenes ( ) A non functional copy of most or all of a gene Inactivated by mutations that may cause either inhibition of signal for initiation or transcription prevent splicing at exon-intron boundary premature termination of translation Human Mol Gen 3 by Strachan & Read pgs 262-264 Non-coding regions…..

24. Pseudogenes ( ) Different classes include Non-processed: contain non functional copies of genomic DNA sequence incl exons and introns arise from gene duplication events E.g. rabbit pseudogene 2 Non-coding regions…..

25. rabbit pseudogene 2 Related to 1 Usual exon and intron organisation Non-coding regions….. 1 2

26. Pseudogenes - processed Non-coding regions…

27. Pseudogenes - processed Non-coding regions… non functional copies of exonic sequences of an active gene. Thought to arise by genomic insertion of a cDNA as a result of retroposition Expressed processed: processed pseudogene integrated adjacent to a promoter site Contribute to overall repetitive elements

28. Gene fragments or truncated genes Gene fragments: small segments of a gene (e.g. single exon from a multiexon gene) Truncated genes: Short components of functional genes (e.g. 5 or 3 end) Thought to arise due to unequal crossover or exchange Non-coding regions…..

29. Nuclear genome organisation (human)

30. 2) Extragenic (intergenic) DNA (~62% of genome) A) Unique or low copy number sequences B) Repetitive sequences (~ 53%)

31. A) Unique or low copy number sequences Non –coding, non repetitive and single copy sequences of no known function or significance

32. B) Repetitive sequences Significance Evolutionary signposts Passive markers for mutation assays Actively reorganise gene organisation by creating, shuffling or modifying existing genes Chromosome structure and dynamics Provide tools for medical, forensic, genetic analysis