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BB30055: Genes and genomes

BB30055: Genes and genomes. Genomes - Dr. MV Hejmadi (bssmvh). BB30055: Genomes - MVH. Recommended texts: Genetics from genes to genomes 2e - Hartwell et al Human Molecular Genetics 3 – Strachan and Read 4) Genomes 2 - TA Brown 5) Genes VII – Benjamin Lewin Special issue Journals:

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BB30055: Genes and genomes

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

  2. BB30055: Genomes - MVH Recommended texts: • Genetics from genes to genomes 2e - Hartwell et al • Human Molecular Genetics 3 – Strachan and Read 4) Genomes 2 - TA Brown 5) Genes VII –Benjamin Lewin Special issue Journals: Nature (2001) 15th 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 • Genomes, transcriptomes, proteomes • 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 Entire DNA complement of any organism which include organelle DNA transcriptome All RNA transcribed from genome of a cell or tissue all proteins expressed by a genome, cell or tissue proteome

  6. 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

  7. 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

  8. HGP Goal: Obtain the entire DNA sequence of human genome Players: • 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

  9. Whose genome is it anyway? • 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 !!!)

  10. Genomicists looked at two basic features ofgenomes: sequence and polymorphism Major challenge - to determine sequence of each chromosome in genome and identify polymorphisms • 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%)

  11. 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

  12. Strategies for sequencing the human genome

  13. Strategies for sequencing the human genome

  14. Whole-genome shotgun sequencing Private company Celera used to sequence whole human genome • 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 • 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 Genetics by Hartwell Fig. 10.13

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

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

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

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

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

  20. 16S, 23S, 28S, 18S etc 22 types of mitochondrial & 49 cytoplasmic U1,U2.U4,U5,U6 etc > 100 types RNA genes - Nuclear genome organisation (human) Major classes of RNA involved in gene expression • rRNA • tRNA • snRNA • snoRNA • Other RNA classes • microRNA • XIST RNA • Imprinting associated RNA • Nervous system specific • Antisense RNA • Others

  21. Non-coding regions….. introns

  22. Non-coding regions….. 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

  23. Non-coding regions….. 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 b2

  24. Non-coding regions….. rabbit pseudogeneb2 Related to b1 Usual exon and intron organisation b1 b2

  25. Non-coding regions… Pseudogenes - processed

  26. Non-coding regions… Pseudogenes - processed 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

  27. Non-coding regions….. 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

  28. Nuclear genome organisation (human)

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

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

  31. 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

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