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BB30055: Genes and genomes

BB30055: Genes and genomes. Genomes - Dr. MV Hejmadi (bssmvh). BB30055: Genomes - MVH. 3 broad areas Genomes, transcriptomes, proteomes Applications of the human genome project (C) Genome evolution. Why sequence the genome?. 3 main reasons

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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 3 broad areas • Genomes, transcriptomes, proteomes • Applications of the human genome project (C) Genome evolution

  3. Why sequence 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

  4. 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

  5. Human Genome Project (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

  6. 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 !!!)

  7. Strategies for sequencing the human genome

  8. sequencing larger genomes Mapping phase Sequencing phase

  9. Result…. ~30 - 40,000 protein-coding genes estimated based on known genes and predictions IHGSCCelera definite genes 24,500 26,383 possible genes 5000 12,000

  10. Nuclear genome(3.2 Gbp) 24 types of chromosomes Y- 51Mb and chr1 -279Mbp Mitochondrial genome Organisation of human genome

  11. General organisation of human genome

  12. Coding & regulatory - (mRNA)

  13. Non polypeptide–coding: RNA

  14. Gene organisation Rare bicistronic transcription units E.g. UBA52 transcription generates ubiquitin and a ribosomal protein S27a

  15. Pseudogenes () non functional copies of exonic sequences of an active gene. Thought to arise by genomic insertion of a cDNA as a result of retroposition Contributes to overall repetitive elements (<1%)

  16. processed pseudogenes -

  17. Pseudogenes in globin gene cluster

  18. 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

  19. Repetitive elements • Main classes based on origin • Tandem repeats • Interspersed repeats • Segmental duplications

  20. 1) Tandem repeats Blocks of tandem repeats at • subtelomeres • pericentromeres • Short arms of acrocentric chromosomes • Ribosomal gene clusters

  21. Tandem / clustered repeats Broadly divided into 4 types based on size HMG3 by Strachan and Read pp 265-268

  22. Satellites Large arrays of repeats Some examples Satellite 1,2 & 3 a (Alphoid DNA) - found in all chromosomes b satellite HMG3 by Strachan and Read pp 265-268

  23. Minisatellites Moderate sized arrays of repeats Some examples Hypervariable minisatellite DNA - core of GGGCAGGAXG - found in telomeric regions - used in original DNA fingerprinting technique by Alec Jeffreys HMG3 by Strachan and Read pp 265-268

  24. Microsatellites VNTRs - Variable Number of Tandem Repeats, SSR - Simple Sequence Repeats 1-13 bp repeats e.g. (A)n ; (AC)n • 2% of genome (dinucleotides - 0.5%) • Used as genetic markers (especially for disease mapping) Individual genotype HMG3 by Strachan and Read pp 265-268

  25. Microsatellite genotyping design PCR primers unique to one locus in the genome a single pair of PCR primers will produce different sized products for each of the different length microsatellites .

  26. 2) Interspersed repeats A.k.a. Transposon-derived repeats 45% of genome Arise mainly as a result of transposition either through a DNA or a RNA intermediate

  27. Interspersed repeats (transposon-derived) major types * Updated from HGP publications HMG3 by Strachan & Read pp268-272

  28. LINEs (long interspersed elements) Most ancient of eukaryotic genomes • Autonomous transposition (reverse trancriptase) • ~6-8kb long • Internal polymerase II promoter and 2 ORFs • 3 related LINE families in humans – LINE-1, LINE-2, LINE-3. • Believed to be responsible for retrotransposition of SINEs and creation of processed pseudogenes

  29. LINEs (long interspersed elements) Most ancient of eukaryotic genomes • Autonomous transposition (reverse trancriptase) • ~6-8kb long • Internal polymerase II promoter and 2 ORFs • 3 related LINE families in humans – LINE-1, LINE-2, LINE-3. • Believed to be responsible for retrotransposition of SINEs and creation of processed pseudogenes Nature (2001) pp879-880 HMG3 by Strachan & Read pp268-272

  30. SINEs (short interspersed elements) • Non-autonomous (successful freeloaders! ‘borrow’ RT from other sources such as LINEs) • ~100-300bp long • Internal polymerase III promoter • No proteins • Share 3’ ends with LINEs • 3 related SINE families in humans – active Alu, inactive MIR and Ther2/MIR3.

  31. LINES and SINEs have preferred insertion sites • In this example, yellow represents the distribution of mys (a type of LINE) over a mouse genome where chromosomes are orange. There are more mys inserted in the sex (X) chromosomes.

  32. Try the link below to do an online experiment which shows how an Alu insertion polymorphism has been used as a tool to reconstruct the human lineage http://www.geneticorigins.org/geneticorigins/pv92/intro.html

  33. Long Terminal Repeats (LTR) Repeats on the same orientation on both sides of element e.g. ATATATNNNNNNNATATAT • contain sequences that serve as transcription promoters • as well as terminators. • These sequences allow the element to code for an mRNA molecule that is processed and polyadenylated. • At least two genes coded within the element to supply essential • activities for the retrotransposition mechanism. • The RNA contains a specific primer binding site (PBS) for initiating reverse transcription. • A hallmark of almost all mobile elements is that they form small direct repeats formed at the site of integration.

  34. Long Terminal Repeats (LTR) • Autonomous or non-autonomous • Autonomous retroposons encode gag, pol genes which encode the protease, reverse transcriptase, RNAseH and integrase Nature (2001) pp879-880 HMG3 by Strachan & Read pp268-272

  35. DNA transposons (lateral transfer?) DNA transposons Inverted repeats on both sides of element e.g. ATGCNNNNNNNNNNNCGTA Nature (2001) pp879-880 From GenesVII by Levin

  36. 3) Segmental duplications • Closely related sequence blocks at different genomic loci • Transfer of 1-200kb blocks of genomic sequence • Segmental duplications can occur on homologous chromosomes (intrachromosomal) or non homologous chromosomes (interchromosomal) • Not always tandemly arranged • Relatively recent

  37. Segmental duplications Intrachromosomal duplications occur within a chromosome / arm Interchromosomal segments duplicated among non-homologous chromosomes Nature Reviews Genetics2, 791-800 (2001);

  38. Segmental duplications Segmental duplications in chromosome22

  39. Segmental duplications - chromosome 7.

  40. Nature Reviews Genetics 2, 791-800 (2001)

  41. Major insights from the HGP • Gene size, content and distribution • Proteome content • SNP identification • Distribution of GC content • CpG islands • Recombination rates • Repeat content Nature (2001) 15th Feb Vol 409 special issue; pgs 814 & 875-914.

  42. 1) Gene size

  43. Gene content…. More genes: Twice as many as drosophila / C.elegans Uneven gene distribution: Gene-rich and gene-poor regions More paralogs: some gene families have extended the number of paralogs e.g. olfactory gene family has 1000 genes More alternative transcripts: Increased RNA splice variants produced thereby expanding the primary proteins by 5 fold (e.g. neurexin genes)

  44. Gene distribution Genes generally dispersed (~1 gene per 100kb) Class III complex at HLA 6p21.3 Overlapping genes (transcribed from 2 DNA strands) - Rare Genes- within genes E.g. NF1 gene HMG3 Fig 9.8

  45. Uneven gene distribution Gene-rich E.g. MHC on chromosome 6 has 60 genes with a GC content of 54% Gene-poor regions 82 gene deserts identified ? Large or unidentified genes What is the functional significance of these variations?

  46. 2) Proteome content proteome more complex than invertebrates Protein Domains (sections with identifiable shape/function) Domain arrangements in humans largest total number of domains is 130 largest number of domain types per protein is 9 Mostly identical arrangement of domains A A B B B C C C C C Protein X

  47. Proteome more complex than invertebrates…… • no huge difference in domain number in humans • BUT, frequency of domain sharing very high in human proteins (structural proteins and proteins involved in signal transduction and immune function) • However, only 3 cases where a combination of 3 domain types shared by human & yeast proteins. • e.g carbomyl-phosphate synthase (involved in the first 3 steps of de novo pyrimidine biosynthesis) has 7 domain types, which occurs once in human and yeast but twice in drosophila

  48. 3) SNPs (single nucleotide polymorphisms) • Sites that result from point mutations in individual base pairs • biallelic • ~60,000 SNPs lie within exons and untranslated regions (85% of exons lie within 5kb of a SNP) • May or may not affect the ORF • Most SNPs may be regulatory More than 1.4million SNPs identified One every 1.9kb length on average Densities vary over regions and chromosomes e.g. HLA region has a high SNP density, reflecting maintenance of diverse haplotypes over many MYears Nature (2001) 15th Feb Vol 409 special issue; pgs 821-823 & 928

  49. How does one distinguish sequence errors from polymorphisms? sequence errors Each piece of genome sequenced at least 10 times to reduce error rate (0.01%) Polymorphisms Sequence variation between individuals is 0.1% To be defined as a polymorphism, the altered sequence must be present in a significant population Rate of polymorphisms in diploid human genome is about 1 in 500 bp Nature (2001) 15th Feb Vol 409 special issue; pgs 821-823 & 928

  50. SNPs and disease

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