Professional Documents
Culture Documents
Gilles Fischer
INTRODUCTION:
Comparative genomics
Yeasts as model organisms
GENOME EVOLUTION:
DNA duplications
Chromosome dynamics
Nucleotide composition
"Tout ce qui est vrai pour le colibacille est vrai pour l'lphant"
identical
divergent
different
time
or
quantity of evolutionary
changes
Looking for differences
identical
divergent
different
time
or
quantity of evolutionary
changes
Looking for differences
Genome sequences
Rules governing
genome evolution
Experimental Biology
Molecular
mechanisms
Genetic screens
functional genomics
Mechanistic
hypotheses
Genome sequences
Rules governing
genome evolution
SMALL GENOMES
AND
EXPERIMENTALLY TRACTABLE
Experimental Biology
Molecular
mechanisms
Genetic screens
functional genomics
Mechanistic
hypotheses
Saccharomycotina
A brief introduction to the field of Yeast Genomics
The first eukaryotic genome sequence:
The genome of S. cerevisiae
Andr Goffeau
8 years, 120 labs,
641 people
Saccharomyces paradoxus
Saccharomyces mikatae
Saccharomyces cerevisiae
Saccharomyces kudriavzevii
Saccharomyces bayanus
Saccharomyces pastorianus
Saccharomyces exiguus
Saccharomyces servazzii
Saccharomyces castellii
Candida glabrata
Vanderwaltozyma polyspora
Zygosaccharomyces rouxii
Lachancea thermotolerans
Lachancea waltii
Lachancea kluyveri
Kluyveromyces lactis
Kluyveromyces marxianus
Eremothecium gossypii
Saccharomycodes ludwigii
Brettanomyces bruxellensis
Pichia angusta
Candida lusitaniae
Debaryomyces hansenii
Pichia stipitis
Pichia sorbitophila
Candida guilliermondii
Candida tropicalis
Candida parapsilosis
Lodderomyces elongisporus
Candida albicans
Candida dubliniensis
Arxula adeninivorans
Yarrowia lipolytica
Schizosaccharomyces pombe
Saccharomycotina
A brief introduction to the field of Yeast Genomics
Gain of Megasatellites
Gain of HO gene
Saccharomyces paradoxus
Saccharomyces mikatae
Saccharomyces cerevisiae
Saccharomyces kudriavzevii
Saccharomyces bayanus
Saccharomyces pastorianus
Saccharomyces exiguus
Saccharomyces servazzii
Saccharomyces castellii
Candida glabrata
Vanderwaltozyma polyspora
Zygosaccharomyces rouxii
Lachancea thermotolerans
Lachancea waltii
Lachancea kluyveri
Kluyveromyces lactis
Kluyveromyces marxianus
Eremothecium gossypii
Saccharomycodes ludwigii
Brettanomyces bruxellensis
Pichia angusta
Candida lusitaniae
Debaryomyces hansenii
Pichia stipitis
Pichia sorbitophila
Candida guilliermondii
Candida tropicalis
Candida parapsilosis
Lodderomyces elongisporus
Candida albicans
Candida dubliniensis
Arxula adeninivorans
Yarrowia lipolytica
Schizosaccharomyces pombe
size (Mb)
# genes
# tRNA
# introns
Saccharomyces cerevisiae
16
12,1
5769
274
287
Candida glabrata
13
12,3
5204
207
131
Zygosaccharomyces rouxii
9,8
4998
272
167
Lachancea kluyveri
11,3
5308
258
322
Lachancea thermotolerans
10,4
5104
231
286
Kluyveromyces lactis
10,7
5084
162
175
Debaryomyces hansenii
12,1
6273
200
475
Yarrowia lipolytica
20,5
6434
510
1070
Evolutionary scale
Saccharomyces cerevisiae
100 *
Candida glabrata
65
100 *
100 MYr
100 MYr
amino acid
identity %
Homo sapiens
100 - 300 MYr
90
Lachancea kluyveri
Lachancea thermotolerans
Mus musculus
550 MYr
450 MYr
Zygosaccharomyces rouxii
70
Kluyveromyces lactis
60
Debaryomyces hansenii
51
Yarrowia lipolytica
48
Takifugu rubripes
Tetraodon negroviridis
50
Ciona intestinalis
Genome redundancy
WGD
Saccharomyces cerevisiae
1.40
1.35
1.30
Candida glabrata
1.25
1.20
Zygosaccharomyces rouxii
1.15
Lachancea kluyveri
YA
LI
DE
HA
A
KL
L
LA
TH
LA
KL
ZY
RO
CA
GL
SA
CE
1.10
Lachancea thermotolerans
Kluyveromyces lactis
Debaryomyces hansenii
Yeast Genomes
evolutionary
novelties
in
Pseudogenization
Loss of function
(most frequent
fate)
C. e.
65%
49%
Neofunctionalization
Gain of a
new
function
D. m.
40%
duplication
Conservation
Degeneration
Complementation
A duplication assay:
XV
3days - YPD - 30
RPL20A
???
XV
XIII
RPL20B RPL20B
rpl20A
dltion
==>slow growth
and so on
RPL20B
XIII
Hybridization
RPL20B
IV - XII
XV
RPL20B
143 kb
VII, XV
V, XIII
II
XIV
X
XI
V - VIII
IX
Molecular combing
direct tandem
III
VI
I
A A C C T A G A G C T T ( G T T ) 14 G T G G A T T G T T T
Despite the selection of a single gene duplication event, only large segmental duplications were recovered
strain
REPLICATION
WT
pol32
time
(min)
rate of SDs
(/cell/division)
10-7
type of SDs
Intra-chromosomal
(1)
Raghuraman
2001
0 et al. Science,
(<0.07)
Inter-chromosomal
42
-T
T T -
LTRs
(300bp)
microhomologies
(2 to 11 bp)
microsatellites
(poly A/T or
rpt trinucleotides)
48
52
62
38
clb5
7x 10-5
(730)
66
CPT
3 x 10-5
(320)
22
rad52
3 x 10-7
(3)
70
10
15
DSB REPAIR
20
25
30
35
40
rad52
rad1
dnl4
8 x 10-8
(0.8)
15
a connection with
replication?
0
100
Replication-based mechanisms
strain
WT
clb5
rate of SDs
(/cell/division)
type of SDs
Intra-chromosomal
Inter-chromosomal
LTRs
microhomologies
microsatellites
10-7
(1)
42
48
52
7x 10-5
(730)
66
62
38
Clb5
Pol32
(<0.07)
Replication-based mechanisms
strain
rate of SDs
(/cell/division)
type of SDs
Intra-chromosomal
Inter-chromosomal
LTRs
microhomologies
microsatellites
WT
10-7
(1)
42
48
52
CPT
3 x 10-5
(320)
22
54
56
Top1
Top1
CPT
Dnl4
NHEJ
Resection
HR
Rad52
Rad51
pas dhomologies,
religature simple
Rad1
Pol32
SSA
MMEJ
BIR
Microhomologies (5-12pb
>30pb dhomologies
SDSA
DSBR
rate of SDs
(/cell/division)
type of SDs
Intra-chromosomal
Inter-chromosomal
LTRs
microhomologies
microsatellites
WT
10-7
(1)
42
48
52
rad52
3 x 10-7
(3)
70
100
====>
=>
HR-dependent
HR-independent
Dnl4
X
Resection
Rad52
X
Rad1
X
rate of SDs
(/cell/division)
type of SDs
Intra-chromosomal
Inter-chromosomal
LTRs
microhomologies
microsatellites
WT
10-7
(1)
42
48
52
rad52
3 x 10-7
(3)
70
100
rad52
rad1
dnl4
8 x 10-8
(0.8)
15
100
HR requires Rad52
MMEJ requires Rad1
NHEJ requires Dnl4
SD are still being formed in the absence of all known DSB repair pathways
existence of a new DSB repair pathway?
Dnl4
X
Resection
Rad52
X
Rad1
X
Dnl4
X
Resection
Rad52
X
Rad1
X
A new pathway?
MMIR
- independent from all known DSB repair pathways (HR, NHEJ, MMEJ)
- dependent from Pol32
- Replication template switching between microhomologies and microsatellites
SDs are spontaneously generated at high frequency: 10-7 SD/cell/division for the RPL20B locus
SDs arise from two alternative replication-based mechanisms: BIR and MMIR
MMIR represents a new mechanism different from known DSB repair pathways (HR, NHEJ):
between microhomologie (between 2 to 11 nt) and microsatellites (poly A/T, trinucleotide
repeats)
independent from Rad52
requires Pol32
MMIR induces the formation of chimerical genes at the rearrangement junctions
Species 1
translocations
Inversions
duplications
Species 2
deletions
#
#
rates of rearrangements
Sensu stricto
S. serevisiae
S. bayanus
Candida glabrata
Zygosaccharomyces rouxii
S. bayanus
Saccharomyces sensu stricto complex:
- monophyletic group
- very closely related species
- hybrids viable but sterile
- 16 chromosomes
Lachancea kluyveri
Lachancea thermotolerans
Kluyveromyces lactis
Debaryomyces hansenii
Yarrowia lipolytica
(0)
S. mikatae
(2)
S. kudriavzevii (0)
S. bayanus
S. cerevisiae S. paradoxus
(4)
S. kudriavzevii
S. mikatae
S. cariocanus
S. bayanus
S.cerevisiae
S.bayanus
8 15
C.glabrata
1
7 9 11 13
6 8 10 12
K.lactis
D.hansenii
Sensu stricto
3 5
2 4 6
S. serevisiae
S. bayanus
G IJ
Y.lipolytica
2 45 6
Candida glabrata
chrVIII
Zygosaccharomyces rouxii
Lachancea kluyveri
Lachancea thermotolerans
Kluyveromyces lactis
Debaryomyces hansenii
Yarrowia lipolytica
98%
88%
77%
11%
5%
S.cerevisiae
S.bayanus
8 15
C.glabrata
1
7 9 11 13
6 8 10 12
K.lactis
3 5
2 4 6
D.hansenii
A
G IJ
Y.lipolytica
2 45 6
chrVIII
98%
88%
77%
11%
5%
S.cerevisiae
S.bayanus
8 15
C.glabrata
1
7 9 11 13
6 8 10 12
K.lactis
3 5
2 4 6
D.hansenii
A
G IJ
Y.lipolytica
2 45 6
chrVIII
Fischer
F. Brunet
98%
88%
77%
Fischer et al. , PLoS Genet 2006
S.cerevisiae
S.bayanus
8 15
C.glabrata
1
7 9 11 13
6 8 10 12
K.lactis
3 5
2 4 6
D.hansenii
A
G IJ
Y.lipolytica
2 45 6
chrVIII
98%
88%
77%
11%
5%
S.cerevisiae
S.bayanus
8 15
C.glabrata
1
7 9 11 13
6 8 10 12
K.lactis
3 5
2 4 6
D.hansenii
A
G IJ
Y.lipolytica
2 45 6
chrVIII
98%
88%
77%
11%
5%
Candida glabrata
Zygosaccharomyces rouxii
C. glabrata
- comprehensive reshuffling
- 509 translocations, 104 inversions
- no homologous chromosomes
"UNSTABLE" GENOMES
Lachancea kluyveri
S.cerevisiae
Lachancea thermotolerans
L. thermotolerans
"STABLE" GENOMES
L. kluyveri
GOC (gene
=5
# neighboring orthologues
If yes: +1
If no: 0
GOC =
Total # orthologues
species 2
=5
GOL
- Rate of rearrangements =
Dist phylogntique
mean rate
1.5
0.7
2.7
1.3
0.4
Candida glabrata
D. hansenii
0.6
0.6
Zygosaccharomyces rouxii
1.7
0.3
S. cerevisiae
Lachancea kluyveri
(WashU seq center M. Jonhston)
0.4
Lachancea thermotolerans
0.0
0.9
1.7
1.7
Kluyveromyces lactis
C. glabrata
0.5
Z. rouxii
K. lactis
L. kluyveri
0.4
L. thermot
0.3
Debaryomyces hansenii
Yarrowia lipolytica
Fischer et al. , PLoS Genet 2006
moderate
massive
low
Sensu stricto
S. serevisiae
S. bayanus
Candida glabrata
Unstable genome
Zygosaccharomyces rouxii
Lachancea kluyveri
(WashU seq center M. Jonhston)
Lachancea thermotolerans
Stable genomes
Kluyveromyces lactis
TGA expansion
Debaryomyces hansenii
No synteny
Y. lipolytica
38.3
Candida glabrata
38.8
Zygosaccharomyces rouxii
39.1
QuickTime et un
dcompresseur
sont requis pour visionner cette image.
Lachancea kluyveri
41.5
T transversions : 8-oxo-guanine
Shibutani et al., Nature, 1991
Lachancea thermotolerans
47.3
Kluyveromyces lactis
38.8
Global AT-enrichment
Eremothecium gossypii
52.0
>
Debaryomyces hansenii
36.3
<
Yarrowia lipolytica
49.0
not in yeast?
GC mutations
Global GC-enrichment
Marsolier-Kergoat and Yeramian, Genetics, 2009
Lachancea thermotolerans
GC%
80
60
47.3
40
39.1
QuickTime et un
dcomp resseur
so nt req uis pour vision ner cett e image.
20
1
10
Mb
Zygosaccharomyces rouxii
Lachancea kluyveri
GC%
80
1 Mb
C-left
60
52.9
40
41.5
20
10
11
Mb
DNA
GC% in C-left:46.1
GC% out of C-left:37.4
RNA
1st
54.2
42.0
2nd
3rd
46.8
36.5
1st
2nd
global GC increase
3rd
1st
Protein
2nd
3rd
AAAAAA
84
84
84
72
11
16
16
16
1.3
1.2
1.1
1.2
0.7
0.8
0.9
100
C. glabrata
Z. rouxii
L. kluyveri
100
100
0.05
96
100
100
100
100
L. waltii
L. thermotolerans
K. lactis
98
E. gossypii
C-left has the same phylogentic origin than the rest of the g
LAWA_S33
670 kb
LATH_F
LAWA_S27
LAWA_S56
LAWA_S55
LAKL_C
LATH_G
LATH_C
LATH_E
LATH_A
200bp fragments
G1
DNACy3
DNACy5
G2
ChrA
ChrB
ChrC
ChrD
Merci
- Unit de Gntique Molculaire des Levures, Institut Pasteur
Celia Payen
Romain Koszul
- Gnolevures consortium:
Jean-Luc Souciet