doi:10.1006/jmbi.2000.4209 available online at http://www.idealibrary.com on J. Mol. Biol.

(2000) 304, 271±288

Yeast Chromosomes Have Been Significantly

Reshaped During Their Evolutionary History
R. B. Langkjñr, M. L. Nielsen, P. R. Daugaard, W. Liu and J. PisÏkur*

Department of Microbiology
Technical University of
Denmark, Building 301
DK-2800 Lyngby, Denmark
*Corresponding author
The structure of the ®rst eukaryotic genome, belonging to Saccharomyces
cerevisiae, has been deduced; however, very little is known about its ori-
gin. In order to trace events that led to the current state of the Saccharo-
myces nuclear genomes, random fragments of genomic DNA from three
yeasts were sequenced and compared to the S. cerevisiae database
sequence. Whereas, S. cerevisiae and Saccharomyces bayanus show perfect
synteny, a signi®cant portion of the analysed fragments from Saccharo-
myces servazzii and Saccharomyces kluyveri show a different arrangement
of genes when compared to S. cerevisiae. When the sequenced fragments
were probed to the corresponding karyotype, a group of genes present
on a single chromosome of S. servazzii and S. kluyveri had homologues
scattered on several S. cerevisiae chromosomes. Apparently, extensive
reorganisation of the chromosomes has taken place during evolution of
the Saccharomyces yeasts. In addition, while one gross duplication could
have taken place, at least a few genes have been duplicated indepen-
dently at different time-points in the evolution.
# 2000 Academic Press
Keywords: Saccharomyces; genome duplication; chromosome
rearrangements; synteny; molecular evolution

Introduction The S. cerevisiae genome is app. 13.2 megabases

A number of bacterial genomes have been
sequenced, providing a great deal of data on the
structure of the bacterial genome as well as infor-
mation to reconstruct the evolutionary history of
bacterial genomes, proteomes and metabolomes.
Recently eukaryote sequences at the genome scale
have accumulated. Apparently, there are large
differences, even among closely related organisms,
in genome size, number of genes, number of
chromosomes, and the arrangement of genes along
the chromosomes. Eukaryotic genomes are
dynamic structures, which have undergone dra-
matic changes during the evolution. However,
knowledge about the molecular mechanisms
responsible for evolving the eukaryotic genomes is
very limited. The complete sequencing of the yeast
Saccharomyces cerevisiae (Goffeau et al., 1996) has
supplied the ®rst eukaryotic genome sequence and
provides a tool to study the evolutionary history of
the yeast and eukaryotic genomes.
Abbreviations used: GSS, genome survey sequences;
ORF, open reading frame.
E-mail address of the corresponding author:
imjp@pop.dtu.dk
(Mb) in size, consists of 16 chromosomes, that con-
tain around 6000 open reading frames (ORFs), and
a separate 80 kb mitochondrial DNA molecule
(Goffeau et al., 1996; Foury et al., 1998). Some inter-
esting questions are: why are there 16 chromo-
somes in this yeast, and not more or less; and what
are the origin and the biological signi®cance of the
present gene order. The genome of S. cerevisiae
contains many large, paired chromosomal regions
consisting of duplicated gene pairs usually
arranged in the same order and with conserved
orientation on two chromosomes (Lalo et al., 1993;
Melnick & Sherman, 1993; Wolfe & Shields, 1997).
Wolfe & Shields (1997) proposed that these
regions, called blocks, are the result of a single
duplication of the entire genome in the progenitor
yeast. After the gross duplication, numerous del-
etions reduced the number of duplicated genes to
comprise 16 % of the S. cerevisiae proteome
(Seoighe & Wolfe, 1999a). Apparently, Candida
albicans and Kluyveromyces lactis do not contain
the same duplications as S. cerevisiae (Ozier-
Kalogeropoulos et al., 1998; Seoighe & Wolfe,
1999b), whereas Candida/Saccharomyces glabrata
does (Seoighe & Wolfe, 1999b). The alternative
hypothesis is that multiple independent dupli-

0022-2836/00/030271±18 $35.00/0 # 2000 Academic Press

272 Evolution of Yeast Genomes

cations of sections or whole chromosomes have
taken place.
Mechanisms that have operated during the evol-
ution can be deduced by comparisons of the struc-
ture of the modern genomes belonging to closely
related yeast species. The genus Saccharomyces
contains several species (Barnett, 1992; Kurtzman
& Robnett, 1998), which can be divided into a
group of petite-positive and a group of petite-
negative yeasts, the latter includes Saccharomyces
kluyveri (PisÏkur et al., 1998). Petite-positive Sacchar-
omyces yeasts are further divided into sensu stricto
yeasts, including S. cerevisiae and Saccharomyces
bayanus, and sensu lato yeasts, including Saccharo-
myces servazzii (Barnett, 1992). Some early reports
grouped S. kluyveri in the sensu lato group, but
recent reports have shown that this yeast is not clo-
sely related to other sensu lato yeasts (Kurtzman &
Robnett, 1998). Whereas the genome size of
Saccharomyces yeasts is relatively similar, the
number of chromosomes varies from seven to 16
(Petersen et al., 1999). So far, it has not been deter-
mined if the gene order is preserved among these
species; however, regarding the gene content, at
least some of these yeast species are known to con-
tain genes that are not found in S. cerevisiae
(Gojkovic et al., 2000).
In order to trace the events that led to the cur-
rent state of the yeast genomes, random fragments
of genomic DNA from close relatives of S. cerevi-
siae, namely S. bayanus, S. servazzii and S. kluyveri,
were sequenced, compared for homology to S. cere-
visiae, and mapped to the chromosomes. Analysis
of the data provided evidence for genomic
rearrangements and strongly suggests that the
yeast chromosomes have been signi®cantly
reshaped during the evolutionary history of the
Saccharomyces yeasts.
Results
Analysis of sequences obtained from
S. bayanus, S. servazzii and S. kluyveri
Several hundred plasmids containing genomic
DNA from each of the three species were isolated
from the corresponding libraries and the sizes of
the inserts were determined. Inserts, which were
over 1 kb, were used for sequence analysis. In the
case of S. bayanus, 69 inserts had the ends
sequenced, providing 127 genome survey
sequences (GSSs). In the case of S. servazzii and
S. kluyveri, 46 and 76 inserts, respectively, were
sequenced partially, providing 89 and 152 GSSs
(Table 1). The average size of the GSSs ranged
from 350 to 400 bp (Table 1). When the sequences
were compared to S. cerevisiae open reading frames
(ORFs), a majority showed signi®cant homology,
i.e. 76 % of S. bayanus GSSs, 63 % of S. servazzii
GSSs, and 61 % of S. kluyveri GSSs (Table 1). The
sequences that did not display any homology to
S. cerevisiae ORFs were intergenic sequences, pro-
moter and terminator regions, introns, transposons,
transfer RNAs (tRNAs), ribosomal DNA (rDNA)
loci, mitochondrial DNA (mtDNA), and ORFs not
found in S. cerevisiae (Table 2). The S. bayanus
GSSs did not contain any ORFs displaying hom-
ology to genes other than those found in S. cerevi-
siae. In addition, a few S. bayanus GSSs showed
homology to S. cerevisiae intergenic sequences
(data not shown). In the other two yeasts, a few
GSSs that did not give homology to S. cerevisiae
showed homology to genes that were previously

Table 1. General characteristics of the S. bayanus, S. servazzii and S. kluyveri inserts and GSSs
Yeast species S. bayanus S. servazzii S. kluyveri
Plasmid inserts sequenced in total 69 46 76
GSS sequences in total 127 89 152
Plasmid inserts with homology to S. cerevisiae mtDNA 6 2a 0
Plasmid inserts where only one end shows homology to S. cerevisiae 24 22 33
Plasmid inserts where both ends show homology to S. cerevisiae 34 17 30
Plasmid inserts where both ends show homology to the same ORF 10 1 2
Plasmid inserts that show synteny to S. cerevisiae 24 9 12
Plasmid inserts with no homology 3 4 10
GSSs with homology to S. cerevisiae rDNA 4 0 4
GSSs with homology to S. cerevisiae tRNA 1b 0 2c
GSSs with homology to S. cerevisiae transposon elements 1b 1 1d
GSSs showing homology to S. cerevisiae ORFs 96 56 93
GSSs not showing homology to S. cerevisiae 18 30 52
GSSs showing homology to other species than S. cerevisiae 0 2 4
Average length of sequence (n) 399 356 376
The actual number of inserts sequenced is higher than given here, but several sequences had overlaps and were pooled.
a
Only two GSSs show homology to S. cerevisiae mtDNA
b
Whereas one end (b063b092b181f) shows homology to a delta element and a tRNA gene, the other (b063b092b181r) shows
homology to GAL80 of S. cerevisiae.
c
Whereas k043f shows homology to a tRNA gene, k043r shows homology to ILV1 of S. cerevisiae. k120r also shows homology to
a tRNA gene.
d
Whereas k181r shows homology to the transposon LTR, k181f shows homology to RAD26 of S. cerevisiae.
Evolution of Yeast Genomes
described in other organisms. Two S. servazzii
GSSs out of 58 GSSs carrying ORFs, and four
S. kluyveri GSSs out of 97 GSSs carrying ORFs,
apparently contained a gene that has no close hom-
ologue in S. cerevisiae (Table 2A).
An interesting feature found in some of the
S. kluyveri sequence parts that did not show hom-
ology to S. cerevisiae ORFs, were long poly(A), or
poly(T), stretches. Out of 152 S. kluyveri GSSs, 17
(11 %) had at least one stretch that contained ten or
more consecutive A or T residues. Among these
sequences, 14 did not show any homology to the
S. cerevisiae coding sequences. Thus, about 25 % of
the GSSs not showing any homology contained a
poly(A) or poly(T) stretch. In the case of k042r, the
sequence contained 27 consecutive A residues
(Table 2B). Among S. bayanus GSSs, only four
poly(A) stretches of ten or 11 consecutive A resi-
dues, were found. These four stretches mapped to
four different ORFs and not to intergenic regions.
Only two S. servazzii GSSs contained a stretch hav-
ing more than ten consecutive A or T residues.
s178r, containing 26 consecutive A residues,
mapped to an Alu-like repeat (Table 2B).
Among the GSSs that gave matches to
S. cerevisiae ORFs, i.e. 96 from S. bayanus 56 from
S. servazzii and 96 from S. kluyveri, the average
amino acid identity for homology matches was
calculated. The average identity was 76 % for
S. bayanus, 60 % for S. servazzii and 61 % for
S. kluyveri. Taking into account the facts that sev-
eral GSSs did not have any homology, and that for
several GSSs only a part of the sequence had hom-
ology, the degree of the overall nuclear genome
identity with S. cerevisiae on the nucleic acid level
has been estimated to be 36 % for S. bayanus,
25 % for S. servazzii and 23 % for S. kluyveri.
A minor proportion of sequences showed hom-
ology to S. cerevisiae mtDNA, tRNA, rDNA loci,
and transposons (Table 2C and D). In all three
species transposon-like, Ty, sequences were found.
Twelve S. bayanus GSSs apparently originated
from mtDNA and had homology to six different
mitochondrial genes. Homology to S. cerevisiae
mtDNA was found for only two S. servazzii GSS,
whereas no S. kluyveri GSSs showed homology to
S. cerevisiae mtDNA (Table 2C). A single putative
intron sequence from S. servazzii, mapping to the
RPS23 homologue, and four putative S. kluyveri
introns, mapping to the SPT14, PUP3 and CIT1
homologues were found (Table 2E). The S. kluyveri
CIT gene contains two introns. Note that the
S. cerevisiae PUP3 and CIT1 genes do not contain
any intron. All ®ve putative introns had
S. cerevisiae-like intron consensus sequences.
Whereas some inserts had both ends displaying
homology to the S. cerevisiae ORFs, others had one
or none of the ends giving homology (Tables 1 and
3). From an evolutionary point of view, the most
interesting group of inserts is that showing hom-
ology to different S. cerevisiae ORFs at both ends,
i.e. 24 in the case of S. bayanus, 16 in the case of
S. servazzii and 28 in the case of S. kluyveri.
273
Synteny
If a pair of genes mapping to the same chromo-
some in one species also map to the same chromo-
some in another species, the pair of genes is
considered as syntenic. If the orientation of the
genes and the distance between the two genes are
preserved, then the observed situation is likely to
re¯ect the con®guration of the genes in the pro-
genitor of the two analysed species. The syntenic
pairs of genes from all three species are presented
in Table 3.
All gene pairs analysed in S. bayanus showed
synteny with S. cerevisiae (Tables 1 and 3A). The
S. bayanus insert size was compared to the map
distance in S. cerevisiae. The distance, 1.5 kb,
between the S. bayanus genes of the analysed pairs
®ts with the distance between the S. cerevisiae hom-
ologues (Table 3A). In addition, the transcriptional
orientation was preserved between the two species.
Thus, it is likely that the arrangement of genes
within the chromosome fragments presented in
Table 3A existed in the progenitor of both yeasts.
The degree of synteny was only 56 %
when S. servazzii gene pairs were compared to
S. cerevisiae (Figure 1(a) and Table 1). However,
whereas all syntenic gene pairs had the same
orientation in both species, the distance between
the two genes was not always preserved (Table 3B).
Two S. servazzii inserts s148 and s276 had a con-
®guration that was slightly different, ‡4.2 kb and
ÿ1.6 kb, respectively, from the situation in
S. cerevisiae (Table 3B). In some cases, even if the
S. cerevisiae homologues mapped to two different
chromosomes, they belonged to the same dupli-
cated block. These clones were likely to re¯ect the
progenitor con®guration of the genes prior to the
duplication of the block, and could therefore be
considered as syntenic-like. Among seven non-
syntenic clones, ®ve showed conservation of
synteny within the duplicated blocks (Figure 1(a)).
Thus, 88 % of S. servazzii clones show synteny with
S. cerevisiae. One of the non-syntenic clones,
namely s163, had opposite gene orientation when
compared to the corresponding S. cerevisiae
block. In addition, s146 had homology to a pair of
S. cerevisiae genes, which mapped to two different,
duplicated blocks, namely blocks 45 and 50.
Among S. kluyveri inserts that showed homology
to S. cerevisiae ORFs, 43 % were syntenic
(Figure 1(b) and Table 1). Almost half of these
showed a discrepancy, i.e. 1.5 kb, in the distance
between the syntenic genes of the two species
(Table 3C). In S. kluyveri the distance was larger in
the case of the inserts k054, k137 and k153, and
shorter in the case of k134 and k162. These frag-
ments either had an insertion of an additional
gene/intergenic region, or a deletion of a gene,
which is present in S. cerevisiae. Whereas syntenic
clones from S. bayanus and S. servazzii showed
preservation of gene orientation when compared to
S. cerevisiae, one out of 12 syntenic S. kluyveri
clones, namely k082, showed a different
274 Evolution of Yeast Genomes

Table 2. Characteristics of some special sequences from S. bayanus , S. servazzii, and S. kluyveri
Evolution of Yeast Genomes
Table 2. (continued)
aa, amino acid; n; nucleotides; S. cer., S. cerevisiae
orientation. Comparing the orientation of the TFB2
and DBP1 genes in the two species, S. kluyveri and
S. cerevisiae revealed that one of the TFB2 genes
had been inverted. Among non-syntenic clones,
50 % showed conservation of synteny within the
duplicated blocks (Figure 1(b)). Thus, 72 % of
clones were in a sense syntenic and re¯ected the
progenitor con®guration. Only 28 % of the clones
showed a completely new con®guration, and one
clone, k200, had homology to two different dupli-
cated S. cerevisiae blocks, namely block 9 (locus
adjacent to this block) and 43. The con®guration of
this fragment was con®rmed by PCR using the
S. kluyveri genomic DNA as the template (data not
shown). A discrepancy in the size was observed in
the case of k228, which covers only a part of the
GRE2 gene (Table 3C). The gene con®guration,
possibly because of an additional intron, is signi®-
cantly different than in S. cerevisiae.
A few non-syntenic inserts contained homol-
ogues at both ends that mapped closely to the
S. cerevisiae centromere regions of two different
chromosomes. Such inserts could help elucidating
the origin of the present centromers. One end of
275
the S. servazzii insert, s165, had homology to
YFR005C on chromosome VI, and the other end to
YPL012W on chromosome XVI (Table 3B). Simi-
larly, the S. kluyveri insert k183 showed homology
at one end to YFR002W from chromosome VI, and
at the other end homology to YPR002W from
chromosome XVI (Table 3C). However, even if
these two inserts connect the centromers of S. cere-
visiae chromosome VI and XVI, and thereby
suggest a common origin of these regions, so far
no duplicated block has been assigned to this
region. In addition, the transcriptional orientation
of these four genes is not the same among the
three species. The two sequences from S. servazzii
clone s163 showed homology to ORFs mapping in
the vicinity of the centromers of S. cerevisiae
chromosome XI and VIII, respectively (Table 3B).
Again, the orientation was not preserved between
the two species. In this case, the duplicated block
35 has already connected these centromers (Wolfe
& Shields, 1997). Finally, three S. kluyveri clones,
k086, k108, and k195, connect CEN10 and CEN12,
which are not part of a block. These also provide
evidence for rearrangements in the centromeric
276 Evolution of Yeast Genomes

Table 3. Clones and GSSs from S. bayanus, S. servazzii and S. kluyveri having S. cerevisiae homologues.
A. Analysis of S. bayanus clones and GSSs
S. bayanus GSSs S. cerevisiae S. bayanus /S. cerevisiae comparison S. bayanus insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.
b057b775 s(s) 3.2 3.0 9
b057b775f AZ124528 402 YOR198C BFR1 15 7.0E-53 131 79
b057b775r AZ124529 381 YOR202W HIS3 15 5.0E-64 126 91
b059r AZ124530 227 YCL040W GLK1 3 9.0E-25 66 77 1 3.0 2
b063b092b181r AZ124532 399 YML051W GAL80 13 5.0E-48 125 72 1 1.6 n.d.
b064r AZ124533 418 YLR427W 12 5.0E-45 84 80 1 3.8 n.d.
b068b179 AZ124534 278 YIL130W 9 4.0E-48 92 99 1 0.3 3
b069 s(s) 12.9 11.5 5
b069f AZ124535 224 YJL085W EXO70 10 3.0E-13 35 89
b069r AZ124536 243 YJL080C SCP160 10 4.0E-25 68 76
b070 s(s) 3.9 3.3 15
b070f AZ124537 386 YLR310C CDC25 12 2.0E-56 77 90
b070r AZ124538 368 YLR312C 12 1.0E-16 57 60
b071r AZ124539 327 YDR247W 4 2.0E-49 97 86 1 6.3 14
b082 AZ124540 227 YBR244W 2 4.0E-23 56 84 1 0.3 n.d.
b088r AZ124541 426 YCL052C PBN1 3 8.0E-21 53 85 1 8.4 n.d.
b089b091 s(s) 8.9 8.5 7
b089b091f AZ124542 409 YKR006C MRPL13 11 8.0E-50 111 81
b089b091r AZ124543 415 YKR010C TOF2 11 2.0E-13 91 37
b103 s(s) 5.2 4.5 10
b103f AZ124544 511 YPR097W 16 6.0E-59 136 82
b103r AZ124545 437 YPR095C 16 8.0E-58 143 73
b105 s(s) 7.2 5.9 8
b105f AZ124546 390 YHR086W NAM8 8 1.0E-35 53 91
b105r AZ124547 376 YHR091C MSR1 8 1.0E-56 124 85
b108 s(s) 10.8 10.0 12
b108f AZ124550 409 YDL122W UBP1 4 4.0E-24 59 81
b108r AZ124551 404 YDL116W NUP84 4 2.0E-58 126 84
b109 s(s) 6.8 6.5 13
b109f AZ124552 434 YGL099W 7 4.0E-33 68 65
b109r AZ124553 412 YGL097W SRM1 7 3.0E-36 79 84
b110 s(s) 4.2 4.2 9
b110f AZ124554 408 YOR244W TAS1 15 8.0E-08 19 79
b110r AZ124555 404 YOR246C 15 2.0E-63 73 90
b113 s(s) 2.5 2.5 8
b113f AZ124556 289 YNR057C BIO4 14 3.0E-18 47 79
b113r AZ124557 265 YNR058W BIO3 14 7.0E-21 40 85
b120 s(s) 8.2 8.9 11
b120f AZ124558 372 YML103C NUP188 13 2.0E-22 93 55
b120r AZ124559 346 YML099C ARGR2 13 1.0E-37 114 64
b121 s(s) 6.2 5.9 8
b121f AZ124560 339 YNL064C YDJ1 14 3.0E-05 19 95
b121r AZ124561 358 YNL061W NOP2 14 2.0E-23 104 57
b139 Same 1.2 1.3 13
b139f AZ124562 409 YGL195W GCN1 7 1.0E-50 135 76
b139r AZ124563 385 YGL195W GCN1 7 1.0E-36 71 65
b141b249b1031 Same 2.2 2.0 11
b141b249b1031f AZ124564 304 YMR259C 13 4.0E-29 98 64
b141b249b1031r AZ124565 306 YMR259C 13 4.0E-34 98 62
b143 AZ124566 180 YNL277W MET2 14 1.0E-27 59 86 1 n.d.
b160b526 Same 1.0 1.0 n.d.
b160b526f AZ124567 401 YDR314C 4 7.0E-51 124 71
b160b526r AZ124568 384 YDR314C 4 2.0E-43 66 73
b165f AZ124569 417 YAL068C 1 2.0E-30 49 94 1 2.2 n.d.
b201 AZ124570 732 s(s) 0.7 0.7 n.d.
b201f YDR199W 4 3.0E-14 71 57
b201r YDR200C 4 2.0E-35 122 57
b210b874r AZ124571 387 YDR011W SNQ2 4 1.0E-59 128 84 1 3.1 n.d.
b218 s(s) 4.4 4.5 10
b218f AZ124572 327 YPL242C IQG1 16 3.0E-47 100 83
b218r AZ124573 340 YPL243W SRP68 16 5.0E-05 19 100
b219 AZ124574 453 YOL006C TOP1 15 8.0E-86 151 97 1 0.5 n.d.
b344r AZ124575 454 YGR295C COS6 7 1.0E-32 144 51 1 6.5 n.d.
b416 AZ124576 208 YPR114W 16 4.0E-30 69 87 1 n.d. n.d.
b466 s(s) 4.7 4.7 6
b466f AZ124577 372 YER140W 5 2.0E-42 111 71
b466r AZ124578 376 YER143W DDI1 5 4.0E-19 107 54
b468 s(s) 3.8 3.4 n.d.
Evolution of Yeast Genomes 277

Table 3. (continued)
S. bayanus GSSs S. cerevisiae S. bayanus /S. cerevisiae comparison S. bayanus insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.
b468f AZ124579 357 YNR052C POP2 14 4.0E-20 117 51
b468r AZ124580 465 - SNR49 14 2.0E-34a 307a 78a
b489 Same 0.9 1.0 4
b489f AZ124581 481 YJR109C CPA2 10 9.0E-64 104 96
b489r AZ124582 454 YJR109C CPA2 10 2.0E-73 132 96
b503 s(s) 1.6 1.1 n.d.
b503f AZ124583 384 YJR097W 10 3.0E-23 57 72
b503r AZ124584 372 YJR096W 10 1.0E-17 45 76
b511 s(s) 2.2 2.3 n.d.
b511f AZ124585 389 YLL053C 12 6.0E-27 72 79
b511r AZ124586 438 YLL051C FRE6 12 2.0E-32 77 66
b527 Same 1.0 1.0 2
b527f AZ124587 412 YCR017C 3 7.0E-69 131 89
b527r AZ124588 412 YCR017C 3 3.0E-49 131 70
b533b687f AZ124591 426 YHR203C RPS4B 8 3.0E-67 141 87 1 6.7 n.d.
b617b680b1045 AZ124592 767 YBR216C 2 1.0E-104 255 71 1 1.6 12
b628 Same 1.4 1.1 n.d.
b628f AZ124593 343 YDR379W RGA2 4 1.0E-26 56 71
b628r AZ124594 398 YDR379W RGA2 4 8.0E-50 132 70
b635f AZ124595 387 YER069W ARG5,6 5 1.0E-26 79 71 1 3.8 6
b665b949 s(s) 1.6 1.4 n.d.
b665b949f AZ124597 393 YGL073W HSF1 7 7.0E-40 130 63
b665b949r AZ124598 396 YGL071W AFT1 7 5.0E-48 132 67
b669 s(s) 3.7 3.8 12
b669f AZ124599 580 YBR136W MEC1 2 1.0E-60 127 80
b669r AZ124600 577 YBR137W 2 5.0E-87 148 91
b702f AZ124603 418 YOR153W PDR5 15 6.0E-63 135 84 1 1.1
b771 s(s) 3.4 3.5 3
b771f AZ124604 544 YIL153W RRD1 9 6.0E-52 162 64
b771r AZ124605 575 YIL151C 9 8.0E-72 103 84
b780 Same 1.0 1.1 15
b780f AZ124606 388 YLR290C 12 9.0E-53 108 87
b780r AZ124607 342 YLR290C 12 2.0E-07 39 56
b786 AZ124608 334 YLR467W 12 1.0E-52 99 98 1 0.4 n.d.
b805r AZ124609 468 YDR103W STE5 4 3.0E-48 151 64 1 6.3 n.d.
b844 AZ124610 520 YIL144W TID3 9 7.0E-44 109 78 1 0.5 n.d.
b852b1101 AZ124613 736 s(s) 0.7 0.8 n.d.
b852b1101f YGR255C COQ6 7 1.0E-62 118 93
b852b1101r YGR254W ENO1 7 8.0E-12 33 94
b892 Same 1.8 1.5 n.d.
b892f AZ124614 426 YER129W PAK1 5 6.0E-74 141 89
b892r AZ124615 443 YER129W PAK1 5 7.0E-08 43 56
b896f AZ124616 446 YHL034C SBP1 8 8.0E-37 128 59 1 1.6 9
b927r AZ124619 391 YGR030C 7 2.0E-29 60 65 1 1.3 n.d.
b946 s(s) 1.8 1.8 n.d.
b946f AZ124620 322 YER091C MET6 5 2.0E-40 84 95
b946r AZ124621 221 YER091C-A 5 3.0E-07 64b 67b
b962 Same 1.2 1.2 n.d.
b962f AZ124622 420 YDR443C SRB9 4 3.0E-28 54 100
b962r AZ124623 361 YDR443C SRB9 4 1.0E-05 58 53
b1061 s(s) 3.1 2.6 7
b1061f AZ124624 341 YKL064W MNR2 11 5.0E-10 94 31
b1061r AZ124625 240 YKL063C 11 2.0E-20 65 63
b1071 Same 0.9 1.1 n.d.
b1071f AZ124628 431 YBR141C 2 4.0E-18 56 70
b1071r AZ124629 383 YBR141C 2 2.0E-38 50 84
b1117 AZ124630 490 YIL145C 9 2.0E-27 62 85 1 0.6 n.d.
b1138 AZ124631 487 YFR030W MET10 6 3.0E-26 63 84 1 0.8 n.d.
Sum 35877 8369
278 Evolution of Yeast Genomes

Table 3. (continued)

B. Analysis of S. servazzii clones and GSSs

S. servazzii GSSs S. cerevisiae S. servazzii /S. cerevisiae comparison S. servazzii insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.

s034r AZ124409 320 YER024W 5 2.0E-11 100 38 1 5.1 n.d.

s046 Same 1.1 1.0 n.d.
s046f AZ124410 370 YGR094W VAS1 7 1.0E-46 101 83
s046r AZ124411 410 YGR094W VAS1 7 3.0E-63 134 81
s050r AZ124413 390 YGL049C TIF4632 7 7.0E-28 82 71 1 5.5 7
s054f AZ124414 380 YDL082W RPL13A 4 2.0E-53 126 78 1 8.4 3
s059 ns s(s)b 11.5 10
s059f AZ124415 342 YOR232W MGE1 15 3.0E-35 76 86
s059r AZ124416 360 YPL147W PXA1 16 3.0E-55 87 83
s061r AZ124417 259 YEL004W YEA4 5 4.0E-08 60 42 1 7.3 n.d.
s067r AZ124418 360 YHR118C ORC6 8 4.0E-22 111 43 1 10.6 1
s071f AZ124419 390 YGL084C 7 1.0E-34 96 68 1 6.8 9
s073r AZ124420 340 YDL215C GDH2 4 5.0E-39 112 67 1 9.1 6
s077 ns s(s)b 7.9 10
s077f AZ124421 308 YDR463W STP1 4 2.0E-05 62 42
s077r AZ124422 367 YML035C AMD1 13 9.0E-42 121 64
s094 AZ124423 172 YLR072W 12 3.0E-14 55 49 1 0.2 n.d.
s098 s(s) 1.8 2.9 10
s098f AZ124424 310 YGR082W TOM20 7 8.0E-07 83 36
s098r AZ124425 363 YGR080W TWF1 7 5.0E-25 107 47
s110 ns s(s)b 3.5 9
s110f AZ124426 386 YMR001C CDC5 13 2.0E-62 128 83
s110r AZ124427 384 YKL104C GFA1 11 8.0E-65 127 93
s118 s(s) 9.0 9.0 4
s118f AZ124428 451 YPL231W FAS2 16 3.0E-74 149 83
s118m AZ124429 343 YPL231W FAS2 16 9.0E-20 114 49
s118r AZ124430 422 YPL235W 16 4.0E-58 137 82
s120 s(s) 8.4 9.0 11 ‡ 12
s120f AZ124431 405 YNL262W POL2 14 7.0E-70 135 87
s120m AZ124432 310 YNL265C 14 2.0E-15 101 41
s120r AZ124433 358 YNL267W PIK1 14 3.0E-30 116 58
s125r AZ124434 182 YGR111W 7 1.0E-15 60 60 1 14.0 n.d.
s129 s(s) 7.4 8.1 9
s129f AZ124435 386 YBR176W ECM31 2 6.0E-46 128 66
s129r AZ124436 400 YBR170C NPL4 2 6.0E-50 73 79
s144 ns s(s)b 13.0 4
s144f AZ124437 369 YKL215C 11 6.0E-26 101 55
s144r AZ124438 360 YJR076C CDC11 10 8.0E-55 119 82
s146 ns cb 9.2 n.d.
s146f AZ124439 300 YOR171C LCB4 15 6.0E-13 43 67
s146r AZ124440 400 YNL101W 14 7.0E-17 107 45
s148 s(s) 13.4 9.2 10
s148f AZ124442 340 YGR118W RPS23A 7 1.0E-60 112 99
s148r AZ124443 340 YGR110W 7 3.0E-40 112 56
s150f AZ124444 330 YDR268W MSW1 4 6.0E-27 109 53 1 15.0 9
s154 s(s) 11.1 12.4 n.d.
s154f AZ124446 250 YNL292W PUS4 14 3.0E-29 51 78
s154r AZ124447 230 YNL297C 14 9.0E-07 75 33
s161f AZ124448 400 YDR061W 4 4.0E-12 36 75 1 11.5 3
s163 ns s(o)b 7.5 8
s163f AZ124450 351 YKR002W PAP1 11 1.0E-29 81 75
s163r AZ124451 370 YHL007C STE20 8 9.0E-29 102 62
s165 ns ob 3.5 4
s165f AZ124452 410 YFR005C 6 2.0E-16 127 37
s165r AZ124453 450 YPL012W 16 6.0E-41 146 59
s178f AZ124454 419 YKR023W 11 6.0E-15 84 46 1 8.0 5
s197r AZ124456 349 YDR046C BAP3 4 3.0E-13 114 38 1 4.5 7
s198f AZ124457 383 YMR235C RNA1 13 2.0E-34 125 57 1 4.8
s215 s(s) 6.9 6.0 n.d.
s215f AZ124458 390 YLR071C RGR1 12 9.0E-36 116 60
s215r AZ124459 400 YLR067C PET309 12 2.0E-04 119 24
s242f AZ124461 370 YGL047W 7 8.0E-24 116 51 1 7.5 3‡9
s247r AZ124462 270 YIL156W UBP7 9 3.0E-18 79 54 1 2.5 6
s262 AZ124463 146 YCR048W ARE1 3 2.0E-16 47 74 1 0.2 9
s265f AZ124464 463 YLR007W 12 4.0E-09 45 44 1 6.7 n.d.
s276 s(s) 7.5 9.1 n.d.
s276f AZ124465 439 YGL116W CDC20 7 3.0E-09 47 47
s276r AZ124466 451 YGL113W 7 6.0E-18 95 43
s281r AZ124467 330 YFL036W RPO41 6 1.0E-34 106 61 1 5.5 n.d.
Evolution of Yeast Genomes 279

Table 3. (continued)
S. servazzii GSSs S. cerevisiae S. servazzii /S. cerevisiae comparison S. servazzii insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.
s293r AZ124468 370 YPL122C TFB2 16 2.0E-42 122 68 1 4.3 n.d.
s308r AZ124470 370 YBR055C PRP6 2 1.0E-21 121 41 1 6.3 7
s319r AZ124471 390 YPL032C SVL3 16 4.0E-34 126 55 1 8.7 n.d.
s320 s(s) 10.4 10.0 n.d.
s320f AZ124472 384 YIL115C NUP159 9 2.0E-04 45 42
s320r AZ124473 370 YIL112W 9 1.0E-10 83 41
Sum 20662 5692

C. Analysis of S. kluyveri clones and GSSs

S. kluyveri GSSs S. cerevisiae S. kluyveri /S. cerevisiae comparison S. kluyveri insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.
k007r AZ124474 268 YDL205C HEM3 4 2.0E-30 87 74 1 4.1 n.d.
k017r AZ124475 309 YKR024C DBP7 11 3.0E-35 99 68 1 3.8 4
k024f AZ124478 327 YDR087C RRP1 4 3.0E-18 104 44 1 3.3 n.d.
k037r AZ124481 344 YPR031W 16 1.0E-45 113 66 1 3.2 n.d.
k039f AZ124482 354 YOR036W PEP12 15 1.0E-31 105 61 1 3.4 6
k042f AZ124483 267 YOR059C 15 1.0E-25 87 63 1 6.0 n.d.
k043r AZ124486 419 YER086W ILV1 5 2.0E-20 57 77 1 2.1 n.d.
k051r AZ124487 383 YGR167W CLC1 7 1.0E-12 45 62 1 4.3 6
k054 s(s) 1.4 5.2 4
k054f AZ124488 304 YKL046C 11 5.0E-15 62 63
k054r AZ124489 409 YKL045W PRI2 11 7.0E-37 104 69
k059 ns s(s)b 4.7 1
k059f AZ124490 407 YPL207W 16 2.0E-08 72 44
k059r AZ124491 330 YGL064C 7 1.0E-20 54 76
k062 ns s(s)b 3.1 2
k062f AZ124492 350 YPR082C 16 4.0E-27 55 93
k062r AZ124493 328 YBR123C TFC1 2 1.0E-36 103 60
k063 s(s) 3.2 3.0 7
k063f AZ124494 372 YPL176C 16 4.0E-08 51 49
k063r AZ124495 449 YPL175W SPT14 16 1.0E-52 112 79
k064 ns ob 5.2 3‡7
k064f AZ124496 306 YAL063C FLO9 1 4.0E-11 99 38
k064r AZ124497 351 YPL171C OYE3 16 4.0E-44 116 69
k067f AZ124498 275 YNL016W PUB1 14 3.0E-11 41 66 1 4.3 4
k068r AZ124500 333 YHL039W 8 3.0E-14 107 42 1 5.3 7
k074f AZ124501 358 YGR194C 7 1.0E-35 113 59 1 3.8 6
k078 ns s(s)b 3.1 7
k078f AZ124502 362 YDL077C VAM6 4 2.0E-27 111 50
k078r AZ124503 379 YLR407W 12 9.0E-06 74 38
k079r AZ124504 409 YCR093W CDC39 3 2.0E-31 136 42 1 3.1 1
k081f AZ124505 364 YBR251W MRPS5 2 5.0E-04 23 78 1 3.9 n.d.
k082 s(o) 4.4 5.0 all
k082f AZ124506 371 YPL122C TFB2 16 2.0E-42 117 69
k082r AZ124507 376 YPL119C DBP1 16 1.0E-47 120 76
k086 ns ob 5.5 n.d.
k086f AZ124509 389 YJL005W CYR1 10 3.0E-06 52 36
k086r AZ124510 415 YLL007C 12 2.0E-10 99 33
k087 s(s) 3.3 4.3 7
k087f AZ124511 434 YDL057W 4 7.0E-16 131 34
k087r AZ124512 423 YDL056W MBP1 4 6.0E-34 141 50
k088r AZ124513 338 YNL137C NAM9 14 3.0E-51 112 83 1 4.2 4
k098f AZ124514 367 YGL058W RAD6 7 2.0E-15 35 100 1 3.7 n.d.
k101 ns ob 3.5 7
k101f AZ124516 384 YAL002W VPS8 1 3.0E-19 122 33
k101r AZ124517 358 YLR316C 12 2.0E-18 101 46
k102f AZ124518 368 YNL297C 14 8.0E-22 122 39 1 4.0 3
k108 ns ob 3.1 6
k108f AZ124520 349 YJL034W KAR2 10 1.0E-47 115 80
k108r AZ124521 395 YLR016C 12 2.0E-27 122 52
k121 s(s) 2.8 2.7 6
k121f AZ124524 384 YDR295C 4 2.0E-22 122 48
k121r AZ124525 409 YDR294C DPL1 4 4.0E-09 72 33
k125f AZ124632 414 YBR208C DUR1,2 2 1.0E-52 136 66 1 5.3 7
k126 ns s(s)b 4.7 4
280 Evolution of Yeast Genomes

Table 3. (continued)
S. kluyveri GSSs S. cerevisiae S. kluyveri /S. cerevisiae comparison S. kluyveri insert
GSS Alignment % S.cer. Insert
Accession length Systematic Gene S.cer. BLASTX length identity Synteny size size S.k.
Clones/GSSs number (bp) name name chr. P-value (aa) (aa) features (kb) (kb) chr.
k126f AZ124633 403 YAL042W 1 7.0E-15 49 71
k126r AZ124634 369 YOR356W 15 7.0E-24 32 78
k130f AZ124635 374 YDR283C GCN2 4 8.0E-14 110 40 1 5.6 n.d.
k131f AZ124636 256 YIL031W SMT4 9 2.0E-31 84 67 1 2.7 5
k134 s(s) 12.4 2.2 2
k134f AZ124638 373 YMR049C 13 9.0E-38 61 72
k134r AZ124639 397 YMR053C STB2 13 1.0E-06 73 41
k137 s(s) 4.2 7.1 4
k137f AZ124640 357 YBR281C 2 5.0E-46 118 69
k137r AZ124641 353 YBR283C SSH1 2 6.0E-34 116 58
k150 Same 5.0 3.7 3
k150f AZ124642 402 YBL004W 2 1.0E-18 128 35
k150r AZ124643 365 YBL004W 2 3.0E-30 121 51
k151f 348 YNL292W PUS4 14 3.0E-20 75 63 1 4.4 3
k153 s(s) 2.4 4.1 5
k153f AZ124645 356 YLR193C 12 5.0E-15 48 65
k153r AZ124646 364 YLR195C NMT1 12 2.0E-26 72 71
k160 ns ob 5.9 4
k160f AZ124647 397 YLR039C RIC1 12 4.0E-18 131 36
k160r AZ124648 397 YMR130W 13 2.0E-42 130 61
k162 s(s) 8.2 4.2 2
k162f AZ124649 400 YOR326W MYO2 15 2.0E-45 133 65
k162r AZ124650 402 YOR329C SCD5 15 9.0E-15 70 54
k166r AZ124651 392 YOR349W CIN1 15 2.0E-09 69 41 1 3.7 n.d.
k167r AZ124653 446 YNL011C 14 2.0E-53 133 74 1 5.6 n.d.
k168r AZ124655 378 YAL054C ACS1 1 2.0E-55 125 80 1 4.7 n.d.
k170r AZ124657 417 YPR006C ICL2 16 1.0E-60 138 77 1 3.7 n.d.
k174r AZ124658 403 YNR017W TIM23 14 6.0E-36 77 86 1 3.1 n.d.
k175f AZ124659 407 YMR304W 13 6.0E-48 134 62 1 6.5 n.d.
k176f AZ124661 414 YPR147C 16 4.0E-35 135 50 1 5.0 n.d.
k177 s(s) 4.8 5.6 7
k177f AZ124662 408 YDL075W RPL31A 4 4.0E-05 20 95
k177r AZ124663 381 YDL077C VAM6 4 5.0E-26 124 48
k179 ns s(s)b 3.5 2
k179f AZ124664 377 YBR136W MEC1 2 2.0E-24 125 38
k179r AZ124665 399 YPR091C 16 7.0E-26 78 63
k181f AZ124666 375 YJR035W RAD26 10 6.0E-12 107 41 1 4.3 all
k183 ns ob 3.4 2
k183f AZ124669 400 YFR002W NIC96 6 1.0E-53 132 75
k183r AZ124670 402 YPR002W 16 7.0E-19 74 54
k186 s(s) 3.9 3.8 4
k186f AZ124671 422 YHL002W 8 2.0E-18 140 37
k186r AZ124672 442 YHL004W MRP4 8 8.0E-36 144 51
k188f AZ124673 393 YER073W 5 9.0E-17 45 82 1 2.7 n.d.
k190r AZ124676 416 YMR309C NIP1 13 3.0E-49 91 69 1 3.8 n.d.
k193 ns s(o)b 3.4 5
k193f AZ124677 423 YER094C PUP3 5 1.0E-13 35 97
k193r AZ124678 403 YBL052C SAS3 2 2.0E-22 128 42
k195 ns ob 4.7 6
k195f AZ124679 377 YLR018C 12 3.0E-08 42 52
k195r AZ124680 380 YJL005W CYR1 10 8.0E-47 66 80
k197 ns s(s)b 5.3 1
k197f AZ124681 410 YPL236C 16 1.0E-31 136 49
k197r AZ124682 396 YMR185W 13 4.0E-09 49 47
k200 ns cb 3.6 7
k200f AZ124683 375 YKL078W JA2 11 6.0E-43 105 68
k200r AZ124684 382 YDR536W STL1 4 1.0E-47 125 67
k211 ns s(s)b 3.6 7
k211f AZ124685 407 YDR096W GIS1 4 1.0E-18 92 54
k211r AZ124686 385 YER168C CCA1 5 1.0E-55 126 81
k212r AZ124687 399 YJL214W HXT8 10 8.0E-57 132 78 1 3.9 n.d.
k213 s(s) 4.8 5.0 7
k213f AZ124688 375 YLR394W 12 2.0E-06 35 63
k213r AZ124689 382 YLR397C AFG2 12 1.0E-08 36 67
k228 Same 0.5 3.5 n.d.
k228f AZ124693 325 YOL151W GRE2 15 1.0E-18 72 56
k228r AZ124694 350 YOL151W GRE2 15 4.0E-12 54 59
k232r AZ124695 375 YLR240W VPS34 12 4.0E-62 124 87 1 3.0 n.d.
k240f AZ124696 336 YOL030W 15 1.0E-13 36 89 1 5.9 n.d.

Sum 34996 8684

Evolution of Yeast Genomes 281

region (Table 3C). On the contrary, two inserts,
s110 and k101, contained at one end a S. cerevisiae
homologue mapping close to the centromeres of
chromosome XIII and I, respectively, whereas the
other end showed homology to an ORF that
mapped far away from the centromeric region on
another chromosome (Table 3B and C). Thus, in
general, the regions mapping in the vicinity of cen-
tromers have apparently undergone several
rearrangements during their evolution.
Organisation of chromosomes
The above data show that many regions found
in the modern Saccharomyces species are likely to
re¯ect the original organisation of the genome at
the local level. In the following section, we exam-
ine whether whole chromosomes or large pieces
of chromosomes have been preserved during
evolution.
Karyotypes of S. bayanus, S. servazzii and
S. kluyveri were produced. The S. bayanus chromo-
somes were separated into 15 distinctive bands,
where band number 8 possibly contained two
chromosomes. The S. servazzii chromosomes were
separated into 12 distinctive bands, and S. kluyveri
chromosomes into seven bands (Petersen et al.,
1999). However, the S. kluyveri band 4 seemed to
consist of two chromosomes, which could not be
separated (data not shown). By hybridising
the genomic inserts to the corresponding karyo-
type, the native chromosomes of 29 S. bayanus,
24 S. servazzii, and 41 S. kluyveri inserts were deter-
mined. In general, the probes carrying ORFs hybri-
dised strongly to only one chromosomal band
determining the position of the corresponding
genes (Figure 2 and Table 3). Two S. servazzii
inserts, s120 and s242, and one S. kluyveri insert,
k064, hybridised to two different chromosomal
bands (Figure 2 and Table 3). Insert k181, carrying
a transposon-like element (Table 2), hybridised to
all S. kluyveri chromosomes (Table 3C). So did
S. kluyveri insert k082, containing the inverted
TFB2, indicating the presence of some kind of
repeats that likely could have been involved in the
inversion. Two inserts, b107 and k018, which con-
tain a part of the rDNA locus, revealed that the
rDNA is located on the largest chromosome, band
15 and band 7 of S. bayanus and S. kluyveri,
respectively.
The hybridisation results were plotted showing
the position of the homologous genes in the tested
yeast and S. cerevisiae (Figure 2). In the case of syn-
teny, the corresponding plasmid fragment was
regarded as a single unit, and contributed only a
single dot in Figure 2. In the case of non-synteny,
each insert end was treated as a separate unit, and
thus the corresponding insert contributed two dots
to the graph (Figure 2). According to these results,
the three yeasts fall into two different groups. A
majority of S. bayanus chromosomes correspond to
only one of the S. cerevisiae chromosomes. In
addition, the sizes of these matching chromosomes
are similar in most cases. In other words, a small
chromosome from S. bayanus matches a small
chromosome from S. cerevisiae, medium chromo-
somes also match together, and so do large
chromosomes. In the case of the S. bayanus band 8,
which presumably contains two chromosomes, the
genes match homologues on S. cerevisiae chromo-
some VIII and XIV. In the case of the S. bayanus
band 9, the three tested loci map to S. cerevisiae
chromosome VIII and XV, and in the case of the
S. bayanus band 12, the three genes match S. cere-
visiae chromosome II and IV.
In general, the position of homologous genes
originating from the same S. cerevisiae chromosome
on the S. servazzii and S. kluyveri karyotypes did
not show any signi®cant order. For example, the
16 genes mapping to the S. kluyveri band 7, which
is the largest chromosome, show homology to
eight different S. cerevisiae chromosomes. Even in
the case of the smallest S. kluyveri chromosome,
band 1, the ®ve tested inserts showed that their
homologues belonged to four different S. cerevisiae
chromosomes. In other words, genes belonging to
one chromosome in one species did not show sig-
ni®cant clustering to a limited number of chromo-
somes in other species (Figure 2(b) and (c)).
Duplicated regions
A radical explanation for the presence of
duplicated regions in S. cerevisiae is that a yeast
progenitor, presumably just after separation of the
S. cerevisiae/bayanus/servazzii lineage from the
S. kluyveri lineage, underwent complete genome

a
The BLASTN P-value, the alignment in nucleotides, and % identity in nucleotides
b
The sequence contains several frameshifts compared to S. cerevisiae, thus % aa identity has been calculated from all alignments.
Each clone is ®rst introduced as an insert with its synteny characteristics, the experimentally obtained size, the size of the corre-
sponding orthologous S. cerevisiae fragment and ®nally the chromosome origin. Afterwards, both ends of the insert, f and r, are
introduced as GSSs with the corresponding sequence accession number, the length of the sequenced part, and then a homologous
S. cerevisiae gene with the highest level of amino acid identity. The BLASTX P-value, alignment length and the degree of identity
characterise the homologous part. The synteny parameter is characterized as: s(s), syntenic with conserved gene orientation; s(o)
syntenic without conserved gene orientation; ns s(s)b, non-syntenic with conservation of synteny within blocks and conserved gene
orientation; ns s(o)b, non-syntenic with conservation of synteny within blocks but without conserved gene orientation; ns cb, non-
syntenic that contradicts blocks; ns ob, non-syntenic that neither con®rms blocks nor contradicts blocks; Same, both insert ends have
homology to the same S. cerevisiae gene; 1, only one insert end has homology to a S. cerevisiae gene.
S. cer., S. cerevisiae; chr., chromosome; aa, amino acid; n.d., not determined; S.b., S. bayanus ; S.s., S. servazzii; S.k., S. kluyveri.
282 Evolution of Yeast Genomes

Figure 1. The degree of synteny among analysed gene

pairs in (a) S. servazzii and (b) S. kluyveri. The clones
that show non-synteny can be divided into three
groups. For each insert, the two ends were compared
regarding their location within the duplicated regions in
S. cerevisiae (Wolfe & Shields, 1997; Seoighe & Wolfe,
1999a). When two ends that do not show synteny were
located in the same block number, the clone is said to
show conservation of synteny within blocks (ns sb).
When one end of the insert was located in one block
number and the other end was located in another, the
clone is said to show contradiction of blocks (ns cb).
Finally, there is a group of non-syntenic clones that does
not show conservation of synteny within blocks or con-
tradiction of blocks (ns ob).

duplication (Keogh et al., 1998). To test this hypoth-

esis, two S. kluyveri ORFs that showed homology
to two duplicated S. cerevisiae genes, CIT1 and
PDC1, were cloned, sequenced and analysed for
their phylogenetic relationship with homologous
genes.
The duplicated gene pair CIT1/CIT2 belongs to
the duplicated block 11, found on S. cerevisiae
chromosome XIV and III. When S. kluyveri geno-
mic DNA was hybridised with the S. cerevisiae
CIT1 probe, only a single band gave a positive sig-
nal. Afterwards, the genomic library was screened,
and the S. kluyveri plasmid P248 that produced a
Figure 2. Hybridisation of different (a) S. bayanus,
(b) S. servazzii and (c) S. kluyveri clones to the karyotype
of the corresponding species to determine the chromo-
some from which the fragment had originated. The
three parts of the ®gure show the combined results
from the homology search and the Southern blot hybrid-
isation. The smallest chromosome of the investigated
species is numbered as 1. Band number 8 of S. bayanus
consists of two chromosomes. Two S. servazzii and one
S. kluyveri clones hybridised to two different chromo-
somes and are marked with ~ (see also Table 3).
Evolution of Yeast Genomes
hybridisation signal with CIT1 was sequenced
(AF193854). The putative S. kluyveri orthologue
was aligned with the S. cerevisiae CIT1 gene, giving
nucleotide identity of 77 % and amino acid identity
of 86 %. The S. kluyveri CIT gene contained
two putative introns that were not present in the
S. cerevisiae gene (Table 2E). The splice sites and
branch-point sequences were similar to the consen-
sus sequences of S. cerevisiae introns. The phyloge-
netic tree shown in Figure 3(a) was constructed on
the basis of amino acid alignment of CIT ortholo-
gues from ten species, including CIT1, CIT2 and
CIT3 from S. cerevisiae. Although CIT1 and CIT2 of
S. cerevisiae are duplicates, the S. kluyveri ortholo-
283
Figure 3. Phylogenetic trees
showing the relationship between
(a) various citrate synthase genes
from ten different organisms and
(b) various pyruvate decarboxylase
genes from ten different fungi.
Bootstrap values out of 1000 are
indicated at each node. The
measuring bar indicates a 10 %
deviation in the protein sequence.
The accession number is stated
beside each gene.
gue groups with CIT1, indicative of a common ori-
gin after the duplication of CIT1/CIT2. The third
gene, CIT3, appears to be very distantly related to
the other two and it has been suggested that this is
the result of an independent duplication event (Jia
et al., 1997).
Whereas K. lactis possesses only one structural
PDC gene (Bianchi et al., 1996), the structural
PDC genes in S. cerevisiae appear in triplicate, i.e.
PDC1, PDC5 and PDC6 (Hohmann, 1991). When
S. kluyveri genomic DNA, digested with differ-
ent restriction enzymes, was screened with the
S. cerevisiae PDC1 probe, usually two restriction
fragments gave a signal, suggesting that this gene
284
is at least duplicated in S. kluyveri (data not
shown). Upon screening of the genomic library
with the same probe, the P249 plasmid was iso-
lated and the sequence of a S. kluyveri PDC gene
determined (AF193853). The orthologous gene
showed 83 % and 80 % identity to S. cerevisiae
PDC1, in nucleotide and putative amino acid com-
parisons, respectively. To make an evolutionary
assessment of the PDC gene, 13 orthologous pro-
tein sequences from ten different fungi were
aligned in order to construct the phylogenetic tree
shown in Figure 3(b). The tree indicates that genes
of S. kluyveri and Kluyveromyces progenitors
branched off from the S. cerevisiae progenitor gene
before the latter was duplicated.
Discussion
Recently, a number of Saccharomyces yeasts
have been characterised for the structure and ori-
gin of their mitochondrial genomes (Groth et al.,
2000). In order to understand the origin and
molecular mechanisms that have been involved in
the generation of the modern S. cerevisiae chromo-
somes, we analysed the organisation of the genome
of three other Saccharomyces species. One,
S. bayanus, is a very close relative, which appar-
ently has the same number of chromosomes as
S. cerevisiae (Nguyen et al., 2000; Fischer et al.,
2000), and also belongs to the sensu stricto group.
This yeast can easily mate with S. cerevisiae, and
gives viable hybrids (Marinoni et al., 1999). The
next closest phylogenetic relative that we analysed
was S. servazzii, which has fewer chromosomes
(Petersen et al., 1999). This yeast belongs to the
sensu lato group, which sexually is almost comple-
tely isolated from the sensu stricto group
(Marinoni et al., 1999). The third species, S. kluyveri,
is the least closely related to S. cerevisiae
(Kurtzman & Robnett, 1991). It displays the lowest
number of chromosomes, and has phenotypic
traits, like the petite phenotype, catabolism of
purines and pyrimidines, glucose repression, etc.
that are signi®cantly different from those of other
Saccharomyces yeasts (Barnett, 1992; PisÏkur et al.,
1998; Gojkovic et al., 2000).
A large proportion of analysed GSSs showed
homology to S. cerevisiae ORFs (Table 1). The per-
centage of non-homologous GSSs is the lowest,
14 %, for S. bayanus, and much higher, 34 %, for
S. servazzii and S. kluyveri. Similarly, the amino
acid identity of ORFs, 76 %, as well as the overall
nuclear genome identity, 36 %, is greatest between
S. cerevisiae and S. bayanus. The low percentage of
the overall genome identity among the different
Saccharomyces species was expected from pre-
vious results based on DNA reassociation exper-
iments (Martini & Kurtzman, 1985). With respect
to the identity of ORFs, 60 % and 61 %, and the
overall nuclear genome identity, 25 % and 23 %,
the relationships between S. cerevisiae and either
S. servazzii or S. kluyveri do not show a pro-
Evolution of Yeast Genomes
nounced difference. This suggests that, upon separ-
ation from the S. cerevisiae lineage, both species
accumulated many point mutations in ORFs as
well as intergenic sequences. However, one should
keep in mind that in our analysis only very small
portions, 2-4-, of each genome were compared
and that the results have some degree of statistical
uncertainty. The S. kluyveri genome, and especially
the intergenic sequences, exhibit a unique feature
regarding the presence of poly(A)/poly(T)
stretches. Apparently, a stretch of ten or more
consecutive A or T residues is present at least once
per kilobase of intergenic sequences (Table 2B).
Similar stretches appear at much lower frequency
in S. servazzii, S. bayanus and S. cerevisiae. So far,
the function of these elements is not understood.
These sequences found in S. kluyveri may either
have a regulatory role in gene expression, a gen-
ome structural role, or they could simply be
``junk'' DNA. Another feature of the S. servazzii
and S. kluyveri genomes is the presence of ORFs,
which do not show homology to S. cerevisiae ORFs,
but instead to ORFs from other organisms
(Table 2A). These non-cerevisiae ORFs are likely to
be responsible for the presence of some character-
istic phenotypes found in S. servazzii and S. kluy-
veri. It seems that upon speciation from the
common Saccharomyces progenitor, several genes
have been lost in some lineages or, alternatively,
some lineages may have adopted novel genes
through horizontal transfer or evolution of already
existing genes. A well-characterised family of
genes that apparently has been lost upon separ-
ation of the S. kluyveri and S. bayanus/cerevisiae/
servazzii lineages, includes pyrimidine catabolic
genes. These genes are found in S. kluyveri and in
some closely related genera, like Kluyveromyces,
but not in other Saccharomyces species (GojkovicÂ
et al., 2000). On the other hand, S. cerevisiae con-
tains three PDC genes, whereas S. kluyveri and
K. lactis may have only two and one, respectively.
The PDC1 and PDC5 genes appear to have evolved
from a common progenitor upon separation of the
S. kluyveri lineage from the rest of the Saccharo-
myces yeasts (Figure 3(b)).
While the introns found in this study have
S. cerevisiae-like consensus sequences (Table 2E), a
previously described S. kluyveri intron was found
to have non-cerevisiae consensus sequences
(Gojkovic et al., 2000). This intron could not be
ef®ciently spliced out of the PYD2 transcript when
introduced into S. cerevisiae (GojkovicÂ, 1999). Thus,
the speci®city of the splicing machinery appears to
have diverged upon separation of the two yeast
lineages. Another interesting feature of all three
genomes is the presence of Ty-like elements
(Table 2D). However, a detailed sequence analysis
will be necessary to ®nd out if these elements are
transmitted horizontally, or if they were present
already in the common progenitor.
The yeast K. lactis, which branched off the
S. cerevisiae lineage just prior to speciation of the
Saccharomyces yeasts (Kurtzman & Robnett, 1998),
Evolution of Yeast Genomes
has been studied by sequencing of short genomic
tags (Ozier-Kalogeropoulos et al., 1998). While the
amino acid identity of this yeast, 64 %, is very simi-
lar to those obtained for S. servazzii and S. kluyveri,
almost half, 46 %, of the sequence tags did not
show homology to S. cerevisiae. In yet another
yeast, Candida albicans, which phylogenetically is
even more distantly related to Saccharomyces
yeasts, only 30 % of sequences gave a positive
match with the S. cerevisiae sequences (Ozier-
Kalogeropoulos et al., 1998). Thus, the portion of
homologous sequences and the degree of synteny
show a correlation with the degree of the phyloge-
netic relationship.
The data on gene order and the position of
single genes on the karyotypes (Figure 2 and
Table 3) provided useful information on diver-
gence at the chromosome level. A great proportion
of gene pairs that were studied in the three species
have a similar organisation in S. cerevisiae.
However, the degree of synteny decreases as the
phylogenetic distance increases. In our study, all
S. bayanus gene pairs showed perfect synteny,
even the distance between genes and their
orientation was preserved when compared to
S. cerevisiae (Table 3A). This suggests, in
addition to the hybridisation results (Figure 2(a)), a
high degree of co-linearity regarding the organis-
ation of the chromosomes in S. bayanus and
S. cerevisiae, and con®rms the previous obser-
vations that the S. bayanus and S. cerevisiae gen-
omes differ by only a limited number of
translocations between a few chromosome pairs
(Ryu et al., 1996; Fischer et al., 2000). Even if the
nucleotide sequence within intergenic regions had
diverged signi®cantly between the two species, the
organisation of the coding parts is well preserved
at the local as well as the chromosomal level. For
S. servazzii and S. kluyveri, the degree of synteny is
lower when compared to S. cerevisiae. For S. servaz-
zii, 88 % of gene pairs showed synteny (Figure 1(a))
and therefore probably the original con®guration
that was present in the common progenitor just
prior to the duplication event(s). Only in a few
cases were the distances between the two syntenic
genes not the same in both species. In general,
whereas the organisation at the local level is
relatively highly preserved, the organisation
of chromosomes has completely diverged
(Figure 2(b)). The degree of synteny between
S. kluyveri and S. cerevisiae was determined to be
72 % (Figure 1(b)). For comparison, the degree of
synteny was estimated to be 78 % for K. lactis
when adjacent gene pairs, as well as gene pairs
conserved between duplicated blocks, were taken
into account (Ozier-Kalogeropoulos et al., 1998;
Seoighe & Wolfe, 1999a). However, the distance
between syntenic genes in S. kluyveri and S. cerevi-
siae is often signi®cantly different,  1.5 kb. In
addition, the orientation of genes in the syntenic
pairs is not always preserved (Table 3C).
Thus, whereas the local organisation of approxi-
mately half of the loci is almost identical between
285
S. cerevisiae and S. kluyveri, many other loci have
undergone small changes, such as insertions
and deletions. In addition, several original gene
pairs have been translocated from each other
during evolution (Figure 1). As for S. servazzii, the
S. kluyveri chromosomes do not show co-linearity
with the S. cerevisiae chromosomes (Figure 2(c))
and, as deduced from the syntenic pairs, similarity
is limited to small fragments that presumably
rarely extend more than 10 - 20 kb. However, con-
sidering the relatively small number of clones used
in our study, further sequence data on the three
genomes is necessary to obtain statistically more
signi®cant ®gures.
The variation in the number of chromosomes
found in the modern yeast species (Petersen et al.,
1999) and our mapping results (Figure 2) suggest
that chromosomes have been very dynamic during
their evolutionary history. Whereas the local con-
®guration of some loci still re¯ects the progenitor
situation, one cannot talk about homologous
chromosomes within the modern Saccharomyces
yeast. Apparently, fragments of various sizes have
been shuf¯ed around. Even centromeric regions
have been drastically reshaped.
In addition to the shuf¯ing of fragments, as well
as insertions and deletions of single genes, gene
duplications have played a signi®cant role in the
evolution of yeast chromosomes. At present, the
central question is whether there was one large
genome duplication and, if so, when did it happen.
Keogh et al. (1998) proposed that the gross genome
duplication happened once and just after the separ-
ation of S. cerevisiae and the S. kluyveri lineages.
Alternatively, a series of duplications, for example
duplications of single chromosomes or duplications
of even shorter fragments, could have occurred. To
get an idea of which of the two hypotheses is cor-
rect, two different S. kluyveri genes for which the
orthologues in S. cerevisiae are duplicated were
completely sequenced and analysed. Judging from
the large size of the duplicated regions containing
CIT1 and CIT2 (Lalo et al., 1993), it is likely that
this duplication was part of the proposed gross
duplication event (Wolfe & Shields, 1997). The
phylogenetic analysis reveals that the duplication
of the CIT gene that gave rise to the current S. cere-
visiae CIT1 and CIT2 genes happened before separ-
ation of the S. cerevisiae and S. kluyveri lineages.
The duplication/triplication of the PDC genes,
resulting in the modern S. cerevisiae PDC1, PDC5
and PDC6 genes, occurred after the two species
had diverged. The fact that all three PDC genes are
present in different duplicated regions where these
themselves are not duplicated, indicates that these
genes were not involved in a larger duplication
event that most likely produced the regions; thus,
suggesting that the PDC genes were duplicated/
triplicated independently during a second smaller,
or perhaps even local, duplication event occurring
later in evolution.
The data presented in Figure 3(a) and (b) suggest
that at least some chromosomal duplications have
286
occurred independently several times in the evol-
utionary history of Saccharomyces yeasts. It has
been reported that different regions of chromo-
some III are duplicated in different S. cerevisiae
strains (Wicksteed et al., 1994). Thus, short dupli-
cations may occur regularly in the yeast genomes.
However, a more systematic analysis of different
yeast species and of several regions that are hom-
ologous to duplicated regions in S. cerevisiae will
be necessary to understand the origin of the dupli-
cated genes.
Materials and Methods
Genomic libraries
Genomic DNA from S. bayanus NRRL Y-12624 and
S. servazzii NRRL Y-12661 strains was subcloned in to
the shuttle vector YEp352 (Green-Willms et al., 1998).
The two libraries were kindly provided by M. Costanzo
(Cornell University, USA). A genomic library of
S. kluyveri NRRL Y-12651 strain subcloned in to the
plasmid pBR322 (Egel-Mitani & Hansen, 1987) was
kindly provided by M. Egel-Mitani (Novo Nordisk,
Denmark).
Sequencing of selected plasmids
Several hundred colonies containing genomic DNA
from each of the three species were selected at random.
The corresponding plasmids were isolated and the size
of the inserts was determined on agarose gel using stan-
dard molecular biology techniques (Sambrook et al.,
1989). Inserts larger than 1 kb were sequenced manually
from both ends using forward and reverse primers map-
ping to the vector in the immediate vicinity of the clon-
ing site. Each insert gave two sequences that were
named after the plasmid insert and the primers, forward
(f) or reverse (r), which were used in the sequencing
reaction.
Sequence analysis
The genomic DNA sequences, GSSs from all three
yeast species were compared for homology to database
sequences of S. cerevisiae using variations of BLAST
(basic local alignment search tool) (Altschul et al., 1990),
BLASTN (nucleotide homology-based search) and
BLASTX (amino acid homology-based search) available
on the World Wide Web (Cherry et al., 1997). Positive
hits are presented as the corresponding S. cerevisiae
ORFs with the lowest P-value. In some cases, several
positive hits with low P-values were obtained. These
sequences were then further analysed with special
respect for homology to the duplicated regions (blocks)
of the S. cerevisiae genome (Wolfe & Shields, 1997;
Seoighe & Wolfe, 1999a).
Synteny
The term synteny describes genes arranged in the
same order on the chromosomes of different species, and
the degree of synteny re¯ects the degree of chromosome
fragmentation during the evolutionary history upon sep-
aration of two lineages. When a pair of genes, deduced
from the two sequences, f and r, originating from the
Evolution of Yeast Genomes
ends of the same plasmid insert, showed homology to a
pair of S. cerevisiae genes that were located on the same
chromosome, the couple was designated as syntenic. In
further analysis, special attention was given to the pres-
ervation of the gene orientation and to the correlation
between gene distances in S. cerevisiae and the studied
species. When the two ends of a plasmid insert gave
homology to two ORFs originating from two different
S. cerevisiae chromosomes, this pair was designated as
non-syntenic. However, this group could be further
divided into three subgroups. (1) A group of non-
syntenic clones, of which the insert ends showed hom-
ology to the same duplicated block. This combination
actually exhibited conservation of synteny within blocks.
(2) A group of non-syntenic inserts, contradicting the
existence of blocks, showed homology to ORFs located
in two different duplicated blocks. (3) The group of
non-syntenic clones including all other clones, which
neither show conservation of synteny within blocks nor
contradict the duplicated blocks.
Chromosome mapping
Several of the analysed inserts were mapped to the
karyotype of the corresponding yeast. Chromosomes iso-
lated from S. bayanus Y166 (MCYC 623-6C) (Marinoni
et al., 1999) and the type strains of S. servazzii and
S. kluyveri were separated by pulse ®eld electrophoresis
as described (Petersen et al., 1999). Note that the type
strain of S. bayanus is a partial hybrid containing, apart
from the complete set of S. bayanus chromosomes, a few
S. cerevisiae-like chromosomes (Nguyen et al., 2000).
Thus, a genuine isolate of S. bayanus, Y166, was used for
chromosome mapping. Separated chromosomes were
blotted onto nylon membranes and hybridised with
probes that were made by random primed labeling of
some of the sequenced plasmids. Hybridisation was per-
formed overnight at >55  C using the standard hybridis-
ation mix containing 2  SSC and 0.5 % (w/v) SDS
(Sambrook et al., 1989). After hybridisation, the
membrane was washed for 15 minutes with 0.1  SSC,
0.1 % SDS at 55  C. The chromosome nomenclature for
non- cerevisiae yeasts can be found elsewhere (Ryu et al.,
1996; Petersen et al., 1999) and is used to assign the
chromosome localisation of the hybridising fragments.
Cloning of two genes homologous to S. cerevisiae
duplicated genes
The pBR322 library containing S. kluyveri genomic
DNA was screened for genes homologous to CIT1 and
PDC1 using the standard colony lifting procedure
(Sambrook et al., 1989). The probes were made from tem-
plate DNA from S. cerevisiae CIT1 and PDC1, which was
obtained by PCR on S. cerevisiae total DNA using gene-
speci®c PCR primers. Hybridisation was performed
overnight at 55  C using standard hybridisation mix con-
taining 6  SSC and 0.5 % SDS (Sambrook et al., 1989).
After hybridisation, the membrane was washed for 15
minutes with 2  SSC, 0.5 % SDS at 55  C. Plasmid DNA
was isolated from two clones, P248 and P249, that gave
positive hybridisation signals with CIT1 and PDC1,
respectively, and the corresponding inserts were
sequenced.
Evolution of Yeast Genomes
Phylogenetic relationship
Sequencing originating from P248 and P249 were ®rst
searched against genomic S. cerevisiae DNA using vari-
ations of BLAST (Altschul et al., 1990). Once potential
ORFs were located, the reduced sequences were com-
pared to the corresponding S. cerevisiae ORFs using both
BLASTN and BLASTX with default settings, and puta-
tive introns were spliced out using in silico tools. The cor-
responding amino acid sequences were then aligned to
known sequences in GenBank, using the multiple align-
ment software Clustal W version 1.74 (Thompson et al.,
1994). Phylogenetic trees were calculated from these
alignments, excluding positions with gaps and drawn
using Tree View (Page, 1996).
Acknowledgements
The authors thank K. H. Wolfe, T. Nilsson-Tillgren, R.
F. Petersen, Z. GojkovicÂ, G. Marinoni and J. Hvidtfeldt
for their interest, suggestions, and help with this project.
This work has been funded by the Danish Research
Council, the Carlsberg Foundation, the Plasmid
Foundation, the Daloon Foundation and the Novo
Nordisk Foundation.
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Edited by J Karn