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YEAST CHROMOSOME DYNAMICS: FROM EXPERIMENTAL ANALYSES TO COMPARATIVE GENOMICS AND BACK

YEAST CHROMOSOME DYNAMICS: FROM EXPERIMENTAL ANALYSES TO COMPARATIVE GENOMICS AND BACK. DEA of Bioinformatics - Bruxelles 17 th December 2003. 1 - Sequence data available:. Saccharomyces cerevisiae : complete genome sequence Goffeau et al., Science, 1996

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YEAST CHROMOSOME DYNAMICS: FROM EXPERIMENTAL ANALYSES TO COMPARATIVE GENOMICS AND BACK

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  1. YEAST CHROMOSOME DYNAMICS:FROM EXPERIMENTAL ANALYSESTO COMPARATIVE GENOMICSAND BACK DEA of Bioinformatics - Bruxelles 17th December 2003

  2. 1 - Sequence data available: • Saccharomyces cerevisiae: complete genome sequence • Goffeau et al., Science, 1996 • Yeast Genome Directory, Nature 1997 (633 scientists) • Génolevures 1: partial genome sequence of 13 yeast species • FEBS Letters, 2000, special issue (43 scientists) • Génolevures 2: complete genome sequence of 4 yeast species • to be release in 2004

  3. 200 400 600 800 1000 1200 1400 1600 I II III IV V VI VII VIII IX X XI XII - 16 chromosomes XIII XIV mt XV XVI Le génome de S. cerevisiae - 13 Megabases - 5803 protein-coding genes 140 rRNA genes 275 tRNA genes - only 4% of intron-containing genes - 30 to 40% of the genes belong to families - ORFs occupy 75% of the genome!

  4. Hemiascomycete Yeasts S. cerevisiae sensu stricto S. bayanus S. exiguus S. servazzii sensu lato Z. rouxii S. kluyveri K. thermotolerans K. lactis kluyveromyces K. marxianus P. angusta D. hansenii P. sorbitophila C. tropicalis Y. lipolytica

  5. The « Génolevures » Program, phase 1 (1999-2000) Strategy Fragmentation of genomic DNA Cloning fragments of 3 to 5 kb Left sequence Internal part not sequenced Right sequence Insert 2 à 4 kb

  6. Sequences and genes: • About 50,000 very long Random Sequence Tags (av. 910 bases) • Blast comparisons with S. cerevisiae and with other sequenced organisms • Approximately 20, 000 genes identified • Re-annotation of the S. cerevisiae genome • Detailed study of yeast genome evolution (redundancy, synteny, introns, transposable elements, evolutionary rate…)

  7. Nb of genes identified Saccharomycescerevisiae Saccharomyces sensu-stricto 3240 Saccharomyces uvarum 0.4X 1501 Saccharomyces exiguus 0.2X Saccharomyces sensu-lato 1548 Saccharomyces servazzii 0.2X 2540 Hemiascomycetes Zygosaccharomyces rouxii 0.4X 1564 Saccharomyces kluyveri 0.2X 1603 Kluyveromyces thermotolerans 0.2X Candida glabrata 2871 Kluyveromyces lactis 0.4X 1479 Kluyveromyces marxianus 0.2X 2787 Pichia angusta 0.4X 1241 Debaryomyces hansenii 0.2X 1548 Pichia sorbitophila 0.4X 1038 Candida tropicalis 0.2X Candida albicans 1311 Yarrowia lipolytica 0.4X Euascomycetes Schizosaccharomycespombe 24 271 The « Génolevures » Program, phase 1 (1999-2000) http://cbi.labri.fr/Genolevures/

  8. 0 20 = 0 Saccharomycescerevisiae Saccharomyces sensu-stricto 42 Saccharomyces uvarum 42 Saccharomyces exiguus Saccharomyces sensu-lato 40 Homo sapiens Saccharomyces servazzii 40 Hemiascomycetes Zygosaccharomyces rouxii 43 Saccharomyces kluyveri 16 Mus musculus Kluyveromyces thermotolerans 42 Candida glabrata 43 Kluyveromyces lactis 50 Kluyveromyces marxianus 52 38 Pichia angusta 48 Debaryomyces hansenii 52 Takifugu rubripes Pichia sorbitophila Candida tropicalis 52 Candida albicans Yarrowia lipolytica Euascomycetes Schizosaccharomycespombe Average amino-acid replacement (in %) between orthologous proteins Despite their morphological similarities, hemiascomycetes are more diversified than vertebrates

  9. Hemiascomycetes Saccharomyces cerevisiae Saccharomyces bayanus Yarrowia lipolytica Diptera Vertebrates 0 Drosophila melanogaster 0 0 0 Homo sapiens 0 10 10 10 60 Mus musculus 20 20 20 30 30 30 Fugu rubripes 40 40 40 100 250 Anopheles gambiae 50 50 50 % amino acid divergence time Mya

  10. Saccharomyces sensu-stricto Saccharomyces uvarum Saccharomyces exiguus Saccharomyces sensu-lato Saccharomyces servazzii Hemiascomycetes Zygosaccharomyces rouxii Saccharomyces kluyveri Kluyveromyces thermotolerans Candida glabrata Kluyveromyces lactis Kluyveromyces marxianus Pichia angusta Debaryomyces hansenii Pichia sorbitophila Candida tropicalis Complete sequences finishing, annotation phase Yarrowia lipolytica Euascomycetes The « Génolevures » Program, phase 2 (2001-2003) Saccharomycescerevisiae 1996 Candida albicans 10 X shotgun Stanford Schizosaccharomycespombe 2002

  11. Total DNA nebulized and size fractionated (3.5 - 6 kb) Paired end sequencing Ligation into SmaI digested plasmid pBAM3 Total DNA, short fragment random libraries BAC-end sequencing (scaffolds assembly) EcoRI and/or HindIII BAC fingerprints (colinearity check) High coverage BAC libraries Partial HindIII-digests of total DNA cloned into pBeloBAC11 Assignment of scaffolds to chromosomes Pulsed-field gel electrophoresis Separation of intact chromosomes and size measurement Sequencing and mapping methods Phrap assembly, contig verification, colinearity of sequence with BACs, final quality check

  12. total total assembly Nb of Nb of N50 N50 Nb of reads coverage size (w/o rDNA) contigs scaffolds contigs scaffolds gaps (rDNA reads) + (rDNA size) Candida glabrata 172 644 9.3X 12 300 kb 64 13 352 kb 953 kb 51 (strain CBS 138) (7 876) + (700 kb) 13 chromosomes Kluyveromyces lactis 152 071 11.4X 10 625 kb 6 6 1 670 kb 1 670 kb 0 (strain CLIB210) (7 534) + (649 kb) 6 chromosomes Debaryomyces hansenii 141 037 9.1X 12 240 kb 214 16 102 kb 995 kb 207 (strainCBS767) (2 711) + (293 kb) 7 chromosomes Yarrowia lipolytica 231 161 9.0X 20 390 kb 82 6 545 kb 3 600 kb 76 (strain CLIB89) (15 517) + (1 379 kb) 6 chromosomes BAC end average genome sequences insert coverage size Candida glabrata 5 144 75 14 X Kluyveromyces lactis 8539 75 27X Debaryomyces hansenii in progress Yarrowia lipolytica 5154 107 12X 3635 75 6X Sequencing status

  13. S. kudriavzavii 2-3X WU S. paradoxus 7.7X MIT S. mikatae 5.9X MIT - »- 2-3 X WU S. bayanus 6.4X MIT S. castellii 2-3X WU S. kluyveri 2-3X WU MIT (Kellis et al., 2003, Nature 423: 241-254) WU. (Cliften et al., 2003 Science 301: 71-76) Saccharomyces sensu-stricto Saccharomyces uvarum Saccharomyces exiguus Saccharomyces sensu-lato Saccharomyces servazzii Hemiascomycetes Zygosaccharomyces rouxii Saccharomyces kluyveri Kluyveromyces thermotolerans Candida glabrata Kluyveromyces lactis Kluyveromyces marxianus Pichia angusta Debaryomyces hansenii Pichia sorbitophila Candida tropicalis Complete sequences finishing, annotation phase Yarrowia lipolytica Euascomycetes Saccharomycescerevisiae 1996 Ashbya gossypii Basel Candida albicans 10 X shotgun Stanford Schizosaccharomycespombe 2002

  14. 2 - Genome Evolution: • 2.1 - Chromosomal translocations in Saccharomyces sensu stricto : • 2.2 - Gene order evolution between related species • 2.3 - Gene relics in intergenic regions • 2.4 - Duplication of chromosomal segments • 2.5 - Genome redundancy and synteny conservation

  15. Saccharomycescerevisiae Saccharomyces sensu-stricto S. cerevisiae Saccharomyces uvarum Saccharomyces exiguus S. cariocanus Saccharomyces sensu-lato Saccharomyces servazzii Hemiascomycetes Zygosaccharomyces rouxii S. paradoxus Saccharomyces kluyveri Kluyveromyces thermotolerans S. mikatae Candida glabrata Kluyveromyces lactis S. kudriavzevii Kluyveromyces marxianus Pichia angusta S. uvarum S. bayanus Debaryomyces hansenii Pichia sorbitophila Candida tropicalis Candida albicans Yarrowia lipolytica Euascomycetes Schizosaccharomycespombe 2.1 - Chromosomal evolution in Saccharomyces sensu stricto Saccharomyces uvarum

  16. ELECTROPHORETIC KARYOTYPES => size polymorphism, chromosomal rearrangements and speciation? • monophyletic group of species • closely related • 16 chromosomes:

  17. chromosomal translocations => reduced viability of hybrids v=1/2n => post-zygotic isolation=> speciation

  18. Fischer et al., Nature, 2000

  19. Moderate genome reorganization: • 10 chromosomal translocation • unique to each species • 13 out 16 chromosomes involved • Crucial role for repeated sequences: • duplicate genes • transposon sequences • No chromosomal speciation: • different species with colinear genomes • no correlation between phylogenetic distance and number of rearrangments • Genome instability not constant over time

  20. 1- RECIPROCAL TRANSLOCATION chr. A gene B2 gene A1 gene A2 gene A1 chr. B gene B1 gene B2 gene B1 gene A2 Translocated chromosome segment 2- SINGLE GENE TRANSPOSITION D chr. A gene A1 gene A2 gene A2 chr. B gene B1 gene B2 gene B1 gene A1 gene B2 transposed gene 3- SEGMENTAL DUPLICATION AND GENE LOSS chr. A gene A1 gene A2 D chr. A gene A1-1 gene A2-1 gene A2-1 D chr. B gene A1-2 gene A2-2 gene A1-2 2.2 - Evolution of gene order between related species Mechanisms resulting in loss of synteny :

  21. Left sequence tag Right sequence tag Yeast species X gene 1 gene 2 Average distance between neighbour genes in S. cerevisiae neighbours Question: where are the homologs of gene 1 and gene 2, respectively, on the S. cerevisiae map ? Total number of gene couples Yeast species S. bayanus 2167 S. exiguus 500 Answer: S. servazzi 639 Conservation of synteny Z. rouxii 819 either gene 1 gene 2 S. kluyveri 651 K. thermotolerans 719 neighbours K. lactis 1188 K. marxianus 481 P. angusta 1112 gene 1 or D. hansenii 329 loss of synteny gene 2 P. sorbitophila 112 C. tropicalis 193 Y. lipolytica 158 or gene 1 gene 2 Total 9068 distant and separated by many other genes Genolevures 1:

  22. S. cerevisiae S. bayanus 97.9 (97.2-98.6) 70.7 ±4.1 S. exiguus S. servazzii 70.2 ±3.8 71.3 ±3.3 Z. rouxii 53.5 ±4.1 S. kluyveri 56.5 ±3.7 K. thermotolerans K. lactis 47.3 ±3.1 K. marxianus 49.8 ±4.6 18.9 ±2.4 P. angusta D. hansenii 16.2 ±4.1 59.2 ±9.7 P. sorbitophila C. tropicalis 18.1 ±5.5 Y. lipolytica 10.1 ±4.8 CONSERVATION OF SYNTENY % synteny

  23. S. cerevisiae S. bayanus var. uvarum Evolution of gene order between S. cerevisiae and S. bayanus: • Saccharomyces sensu stricto • Genome coverage 0.4X • About 2,000 gene couples • 20% aa divergence (man-mouse) • 98% conservation of synteny

  24. 4 chromosomal translocations = 8 synteny breakpoints • Génolevures : 35 synteny breakpoints (80 breakpoints in total predicted) • 3 couples corresponded to translocation breakpoints • 32 breakpoints left??

  25. (i) Gene transposition: S. cerevisiae S. uvarum

  26. YML051w YJR054w YJR052w YJR057w YJR053w YJR055w X YJR056c YJR058c

  27. YML051w YML051w YML049w YML046w YML051w YJR052w YJR055w Kt : YML046w YJR052w YJR053w Kl : YML048w YJR054w YJR052w YJR055w YJR057w YJR053w YML049w YJR056c YJR058c YML046w YML048w YJR054w YJR052w YJR057w YJR053w YJR055w X YJR056c YJR058c

  28. YML051w YML051w DUPLICATION YJR054w YJR054w YJR052w YJR052w YJR055w YJR055w YJR057w YJR057w YJR053w YJR053w YML049w YML049w YJR056c YJR056c YJR058c YJR058c YML046w YML046w YML048w YML048w

  29. YML051w YML051w YJR054w YJR052w YJR055w YJR057w YJR053w YML049w YJR056c YJR058c YML046w YML048w YJR054w YJR052w YJR055w YJR057w YJR053w YML049w YJR056c YJR058c YML046w YML048w ALTERNATIVE GENE LOSS

  30. YML051w X XIII YJR054w YJR052w YJR055w YJR057w YJR053w YJR056c YJR058c ALTERNATIVE GENE LOSS YML047c YML049w YML046w YML048w

  31. Chr IV Chr XV Chr VI Chr VII K. l K. m K. t K. l S. s S. e S. k YDL0020c YFR044c YOL056w YGL234w YDL021w YFR045w YGL238w YDL0022w YOL059w YFR047c YGL240w YDL0033c YGL244w YOL064w YFR048w YDL035c YGL245w YDL036c YOL066c YGL246c YDL042c YOL068c YFR049w YGL248w YDL043c YFR050c YOL069w YGL249w YDL044c YFR051c YOL071w YGL252c duplicated blocks unrelated regions Duplicated chromosome segments : Chr X Chr XIII S.u K. l K. t YJR052W YML046w YJR053w YML047c YML048w YJR054w YML049w YJR055w YML051w depicted as: duplicated gene pair

  32. A B C D E A C D A C D A B C E B’ C’ E’ B’ E’ A’ B’ C’ D’ E’ A’ C’ D’ E’ Block of duplication Duplicated gene pair Unrelated regions Variable rate of loss of duplicated gene pairs

  33. Gene transposition: = Duplication + speciation + alternative loss Common ancestor S. c S. u

  34. (ii) Species specific gene duplication: S. cerevisiae S. uvarum

  35. S. bayanus YDR037w YDR038c YDR037w YBR060c YBR061c YDR037w S. cerevisiae IV YDR036c YDR038c II YBR060c YBR061c 1000 2000 3000 4000 5000 1000 2000 3000 4000 5000 YDR037w ) 1000 2000 3000 4000 5000 Relic of YDR037w between YBR060c and YBR061c

  36. i.e. One copy of the two ancestral genes has undergone several hundred events of: nucleotide substitutions single nucleotide deletions or insertions, microdeletions. Average sequence identity between relic and gene = 62 % (1127 / 1818)

  37. (ii) Species specific gene duplication: = Duplication + speciation + loss relic Common ancestor S. c S. u

  38. DUPLICATION LOSS • 90% of the synteny breakpoints between S. cerevisiae and S. bayanus are due to gene duplications and losses (gene relics, anciently duplicated blocks) • only 10% are due to chromosomal translocations

  39. 2.3 - Gene relics in the S. cerevisiae genome pseudogene relic relic

  40. Method Comparison ORF - intergenic regions BLASTN no relic Elimination of the alignments at the median left Conservation of the alignments at the median right DOTPLOT analysis alignments > 23 bp Selection of alignments containing a relic number of matches length (bp) median Dotplot analysis of the 10 best alignments at the median level relic

  41. Genomic localization of the relics - genes IV XII VII XV XIII XVI II XIV X XI V VIII IX III VI I genes relics

  42. Distribution of the sequence identity between relics and cognate ORFs in function of their relative length

  43. Discovery of a triplication

  44. Intermingled segmental blocks

  45. 124 gene relics => a total of 278 pseudogenes identified • High plasticity of subtelomeric regions • Detection of successive duplication events mechanisms? <- DUPLICATION LOSS -> sequence degeneration deletion

  46. 2.4 - Duplication of chromosomal segments: How was redundancy generatedin the yeast genome ? • Whole genome duplication • Segmental duplications • both, other?

  47. Wolfe and Schields, Nature, 1997

  48. The genome of S. cerevisiae as of 1996

  49. Deleted strain RPL20B alone : Slow growth XV XV RPL20B RPL20B (D) XIII x 2 (RPL20B duplication) (D) XIII Recovery of a normal growth rate Genetic screen based on gene dosage recovery XV RPL20B RPL20A + RPL20B : Normal growth WT (haploid) XIII RPL20A

  50. Growth rate assay for selection of duplication events 100 independent slow growth subclones (deleted for RPL20A) rich medium for 80 generations 83 cultures were overtaken by normal growth revertants Reversion rate estimation : 10-10 reversion per cell per division

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