Saccharomyces sensu lato, Biotechnologia, Genomika porównawcza
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International Journal
of
Systematic Bacteriology
(1
999),
49,
1925-1 93 1
Printed in Great Britain
Karyotypes of
Saccharomyces sensu
lato
species
Randi Fans Petersen,’n2 Torsten Nilsson-Tillgren2and Jure Pijkurl
Author for correspondence:
Jure
PiSkur. Tel: +45
45 25
25 18.
Fax:
+45 45
93
28
09.
e-mail: imjp@pop.dtu.dk
Department
of
Microbiology, Technica
I
University of Denmark,
DTU-301, DK-2800 Lyngby,
Denmark
*
Department of Genetics,
Institute of Molecular
Biology, University of
Copenhagen, Denmark
An improved pulsed-field electrophoresis program was developed to study
differently sized chromosomes within the genus
Saccharomyces.
The number
of chromosomes in the type strains was shown to be nine in
Saccharomyces
castellii
and
Saccharomyces dairenensis,
12 in
Saccharomyces servazzii
and
Saccharomyces unisporus,
16 in
Saccharomyces exiguus
and seven in
Saccharomyces kluyveri.
The sizes of individual chromosomes were resolved
and the approximate genome sizes were determined by the addition of
individualchromosomes of the karyotypes. Apparently, the genome of
5.
exiguus,
which is the only
Saccharomyces sensu lato
yeast to contain small
chromosomes, is larger than that of
Saccharomyces cerevisiae.
On the other
hand, other species exhibited genome sizes that were 10-25% smaller than
that of
5.
cerevisiae.
Well-defined karyotypes represent the basis for future
genome mapping and sequencing projects, as well as studies of the origin of
the modern genomes.
Keywords
:
Saccharomyces, karyotypes, genome structure, pulsed-field
electrophoresis, genome duplication
S.
cerevisiae parent (Nilsson-Tillgren et al.,
1983; Pedersen, 1986a, b; Vaughan Martini
&
Kurtzman, 1985
;
Vaughan-Martini
&
Martini, 1987).
INTRODUCTION
Pulsed-field electrophoresis has made possible the
separation of large yeast DNA molecules, thus
allowing determination of the number and size of
nuclear chromosomes, denoting the nuclear karyotype
(Carle
&
Olson, 1985;Johnston
&
Mortimer, 1986).In
addition, pulsed-field electrophoresis studies can serve
as a rapid and relatively easy approach to the charac-
terization and identification of yeast species. Yeasts
belonging to the genus Saccharomyces can be divided
into the sensu strict0 and sensu lato groups. The sibling
species of the Saccharomyces cerevisiae complex have
been shown to exhibit uniform karyotypes (Cardinali
&
Martini, 1994; Carle
&
Olson, 1985; Naumov et al.,
1992, 1995; Vaughan-Martini et al., 1993). Their
chromosomes have been divided into three different
classes on the basis of their size: light
(<
500 kb),
medium (500-1000 kb) and heavy
(>
1000 kb)
(Vaughan-Martini et al., 1993). Karyotype patterns of
the type strains of S. cerevisiae and Saccharomyces
paradoxus are very similar, while Saccharomyces
bayanus has a species-specific karyotype (Naumov et
al., 1992). Saccharomyces pastorianus (syn. Saccharo-
myces carlsbergensis) has been reported to be a hybrid
and has been shown to possess one chromosome set
originating from
S.
cerevisiae and another from a non-
The karyotypes of the Saccharomyces sensu lato yeasts
exhibit much heterogeneity. When these species were
analysed in different laboratories, the number of bands
ranged from five to 14.
Saccharomyces kluyveri
chro-
mosomes may be resolved into five bands, according to
Jager
&
Philippsen (1989); however, in another study,
it was found to contain seven chromosomes of the
heavy class that were all well above 1000 kb (Vaughan-
Martini et al., 1993). In two different studies, the
Saccharomyces exiguus chromosomes were separated
into 11bands (Jager
&
Philippsen, 1989) and 14 bands,
of which some were double (Naumova et al., 1996).
The remaining four
Saccharomyces sensu lato
species,
Saccharomyces castellii, Saccharomyces dairenensis,
Saccharomyces servazzii and Saccharomyces unisporus,
were shown to contain numbers
of
chromosomes
within these two extremes. S. unisporus and
S.
servazzii
strains yielded chromosome banding patterns very
similar to each other, exhibiting between nine and 13
bands from
580 to 2200 kb (Jager
&
Philippsen,
1989; Naumov et al., 1995).
S.
dairenensis and
S.
castellii chromosomes were resolved into eight to 11
bands with sizes from 460 to 2200 kb (Jager
&
1925
01 104
0
1999
IUMS
R.
F.
Petersen,
T.
Nilsson-Tillgren
and
J.
Pigkur
chromosomes. The first run (Run 1) lasted for 18 h. Run 1
was divided into block Al, pulse-time 240 s for 8 h, and
block B1, pulse-time 160 s for 10 h. The second run (Run 2)
lasted for 22 h. Run 2 was divided into block A2, pulse-time
90 s for 14 h, and block B2, pulse-time 60 s for 8 h. Run 2
followed Run 1 immediately and was performed in the same
buffer. It was essential to restart the CHEF-DRII between
Run 1 and Run 2. The power supply was set to 150
V.
Separation program for
Saccharomyces sensu lato
chro-
mosomes on Bio-Rad Chef Mapper-multistate.
The Chef
Mapper-multistate program (Bio-Rad) allows for program-
ming of several blocks with different parameters. The
program used for separation of the Saccharomyces sensu lato
chromosomes resembled the program designed for the
CHEF-DRII. Again it was found that separation of the
large chromosomes resulted in better total separation and
the program was designed starting out with long pulse-times.
The Chef Mapper program was divided into four blocks
(A
+
B
+
C
+
D) and the total run time was 52
h.
Block A was
pulse-time 240 s for 12 h, block B was pulse-time 160 s for
16 h, block C was pulse-time 90 s for 16 h and block D was
pulse-time 60 s for 8 h.
Determination of chromosome and genome sizes.
The sizes
of single chromosomes were calibrated against the sizes of
the marker strain,
S.
cerevisiae YPH755. It was assumed that
logarithm of the molecular mass is a linear function of the
gel mobility. The total genome size was calculated by adding
the sizes of single chromosomes.
Philippsen, 1989; Naumov et al., 1995). The results
obtained in the two main studies (Vaughan-Martini
et al., 1993; Naumov et al., 1995) were similar in most
instances. However, until this point, chromosome
identification within the Saccharomyces sensu
lato
yeasts was not adequate because several bands in the
previous studies apparently contained overlapping
chromosomes. Therefore, we focused on the devel-
opment of a better separation technique, allowing the
number and size of chromosomes to be determined
more precisely.
METHODS
Yeast strains.
The yeast species investigated in the present
study were mostly type strains obtained from the Agri-
cultural Research Service (NRRL), National Center for
Agricultural Utilization Research,
US
Department of Agri-
culture, Peoria, IL,
USA: S.
cerevisiae NRRL Y-12632T,
S.
castellii NRRL Y-12630T,
S.
dairenensis NRRL Y-12639T,
S.
exiguus NRRL Y-12640T,
S.
servazzii NRRL Y-12661T,
S.
unisporus NRRL Y-1556' and
S.
kluyveri NRRL Y-12651T.
Non-type strains included in the study were
S.
cerevisiae
YPH755, which is an ade- derivative of YPH149 (Gerring et
al., 1991),
S.
unisporus CBS 2422 from the culture collection
of the Centraalbureau voor Schimmelcultures (CBS), Delft,
The Netherlands, and
S.
kluyveri
XM
8-5 from L. Marsh,
Albert Einstein College of Medicine, Bronx, NY,
USA.
Preparationof chromosome-sized
DNA.
Chromosome plugs
were prepared following a modified protocol of Gerring et
al. (1991).Yeast cells were grown to stationary phase (OD,,,
10-14) in rich medium [YPD; 10 g yeast extract (1
YO
w/v),
20 g peptone (2
YO
w/v) and 40 ml 50
YO
glucose (2
O/O
w/v)
1-9. Samples (1 ml) were spun down and washed twice in 1ml
EDTA/Tris solution (50 mM EDTA, 10 mM Tris, pH 7.5).
Protoplasts were prepared with zymolyase [O. 15 ml EDTA/
Tris solution plus 1 pl zymolyase (20 mg 100 T ml-' 10 mM
sodium phosphate buffer, pH 7.5)] and placed at
42
"C for 30
s. Low-melting-point agarose (0.25ml of a 1
%,
w/v, solution
in 125 mM EDTA, pH 7.5) was added at 42 "C and mixed
with cells. The cell suspension was gently pipetted into the
plug holders and placed on ice. After the plugs had hardened
they were transferred to a 12
x
75 mm Falcon (glass) tube
and washed as described previously (Gerring et al., 1991).
Plugs were stored at
4
"C in EDTA/Tris solution. Plugs
stored in this fashion are stable for several years.
Pulsed-field gel electrophoresis.
Yeast chromosomes were
separated by pulsed-field gel electrophoresis using either
Bio-Rad CHEF-DRII or Bio-Rad Chef-Mapper. Gels were
prepared as 1
YO
ultra-pure agarose (KILOrose; Clontech)
(1.1
g agarose in 0-5
x
TBE). Plugs were inserted in the gel
and fixed with agarose. Gels were run in 0.5
x
TBE at 14 "C.
After electrophoresis, the gels were stained in electrophoresis
buffer containing 0.5 pg ethidium bromide ml-' for 424h.
Separation program for
Saccharomyces sensu lato
chro-
mosomes on Bio-Rad CHEF-DRII.
The CHEF-DRII ap-
paratus (Bio-Rad) allows programming of two different
blocks. In order to get optimal separation of all Saccharo-
myces sensu lato chromosomes, a gel must be subjected to
two CHEF-DRII runs. Long pulse-times allow separation
of large chromosomes while shorter pulse-times give better
separation of smaller chromosomes. The best separation of
all chromosomes was obtained when the large chromosomes
were separated first followed by separation of smaller
RESULTS
Molecular karyotypes of
Saccharomyces sensu lato
species
Several different separation programs were tested on
chromosomes of Saccharomyces sensu lato type strains.
The results of the optimized four-step pulsed-field
electrophoresis separation are shown in Fig. 1. The
number and sizes of chromosomal bands together with
the total sizes of the genomes were calculated on the
basis of the migration of the YPH755 chromosomes.
The sizes of individual chromosomes and genome sizes
are depicted in Table 1.
The separation of chromosomes was not perfectly
linear. Lack of linearity was particularly true for
chromosomes larger than 1000
kb.
The chromosome
sizes reported here are still therefore to be regarded as
approximate. In addition, the intensity of single
chromosomes within the same preparation varied (Fig.
1). Even though the chromosomes were separated
using different programs, it is not possible to exclude
completely the possibility that some single bands were
actually composed of two chromosomes. This should
be taken into account when the genome sizes are
compared.
The four-step program generally allowed separation of
all Saccharomyces sensu lato chromosomal bands.
However, the resolution of bands varied slightly
among different runs and different plugs and occasion-
ally particular bands could
not
be distinguished.
Chromosomal bands were numbered from the bottom
of the gel.
1926
In
term
iiona
I
Journa
I
of Systernatic Bacteriology
49
Yeast karyotypes
Fig. 1.
Karyotypes of
Saccharomyces sensu lato
species. (a) Chromosome gel. (b) Schematic illustration of the separated
chromosomes based on many independent experiments. Chromosome-sized DNA from
5.
cerevisiae
YPH755,
5.
cerevisiae
Y-12632T,
5.
dairenensis
Y-1263gT,
5.
castellii
Y-12630T,
5.
servazzii
Y-12661T,
5.
unisporus
Y-1 556T,
5.
unisporus
CBS
2422,
5.
exiguus
Y-12640T,
5. kluyveri
Y-12651T and
5. kluyveri
XM 8-5 was separated by pulsed-field electrophoresis. The
chromosomes were separated using a four-step program: step 1, pulse 240 s, 8 h; step 2, pulse 160 s, 10 h; step 3, pulse
90 s, 14 h; step
4,
pulse 60 s, 8 h.
*,
A YPH755 chromosome has been fragmented at RAD2 resulting in separation
of
chromosome
VII
and
XV.
Note also that the two
5.
unisporus
strains exhibited slightly different patterns.
Table
f.
Numbers of chromosomes and chromosome and genome sizes of
Saccharomycessensu
lato
yeasts
,
.
,
..
,
.
,
.
,
.
,
.
,,,,,,,,
.
,
.
, ,,,,,
.
,
.
,
.
,,,,
.
, ,
.
,
.
,,,,,,
.
,
.
,,
...
,
.
,,
.
,
.
,
.
,,
.
,
.
,,
....
,
.
,,
.
,
.
,,
.
,
.
,,,,,,
......
,
.
,
...
.
,
.
,
.
,
.
,
..........
,
.
,,
.
,
........
,
.... .............................
.
. ....... ..................... .... ...........................
,
. ..... ..
.
. ..
,
..
,
.
,
.
,
. .
,
...
.
. .
,
.
,,
. .
,
....
,
....
.
.
,
.
..
..
.
Chromosomes are numbered by size from the bottom
of
the gel (see Fig. 1). The numbers presented are in kb and are means,
obtained by compiling the results from various pulsed-field gels.
All
strains are type strains except S. cerevisiae, which is
represented by
YPH149/YPH755
(Gerring
et al., 1991).
Note that the size
of
the sequenced strains of S.
cerevisiae
is
approximately 13400 kb (Goffeau et al., 1996).
S.
unisporus
S.
dairenensis
S.
castellii
S.
servazzii
S.
exiguus
S.
kluyveri
S.
cerevisiae
No.
of
chromosomes
9
9
12
12
16
7
16
Sizes
of
individual chromosomes
:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
5 50
660
820
860
890
570
5
60
395
970
110
300
410
510
750
950
-
-
-
-
-
-
-
-
-
220
280
360
445
555
610
690
760
800
830
920
960
1010
1100
1600
1900
730
800
060
200
300
510
600
800
920
-
-
-
-
-
-
-
620
630
470
660
720
800
840
960
1020
1080
1110
1300
660
740
780
880
970
020
080
250
300
470
510
780
950
100
690
720
740
870
960
010
100
250
850
11920
9720
12330
11560
18355
10000
13040
Genome size
1927
lnternational Journal
of
Systematic Bacteriology
49
R.
F.
Petersen, T. Nilsson-Tillgren and
J.
Piikur
Table
2.
Chromosome numbers and estimated genome sizes compared with previously published results
Data were obtained from Naumov
et
al. (1995), Naumova
et
al. (1996), Jager
&
Philippsen (1989) and Vaughan-Martini
et
al.
(1993). The sequenced strain of S.
cerevisiae
contains 16 chromosomes and its genome size, including repetitive sequences, is
approximately 13400 kb (Goffeau
et
al.,
1996).
Estimated genome size (kb)
Chromosome number
Species
Present
Vaughan-
Vaughan-
Naumovl
Jager
&
Present
Martini
Naumova
Philippsen*
study
Martini
study
6
7
8
11920
7920
9
9
12
12
16
7
8
9720
9415
-
-
9
9-1 3t
107
12330
10430
10
11
560
11375
S. unisporus
S.
exiguus
S.
kluyveri
11
9-1 37
11
14-16$
117
18
355
10
800
7
5
10
000
9550
-
*
Only a few species were represented by type strains in this study.
7
The chromosome number for the type strain was not stated specifically.
1
Data from Naumova et
al.
(1996); other data in this column are from Naumov et
al.
(1995).
The
S.
cerevisiae type strain was included in the
analysis. Only 12 bands were resolved using the four-
step program, demonstrating that a number of chro-
mosomes ran as non-distinguishable double bands.
Thus, the karyotypes obtained using pulsed-field
electrophoresis may not always express the true num-
ber of chromosomes in a species. In the following
analysis, our results are compared with the previously
published data on Saccharomyces sensu lato type
strains.
The present results show that the S. dairenensis type
strain (Fig. 1) contains nine chromosomes with sizes
ranging from 730 to 1920 kb. The S. dairenensis chro-
mosomal bands were generally well resolved and could
be distinguished using the four-step program. How-
ever, bands 5 and 6, as well as 7 and 8, occasionally ran
close to each other. The total genome size was
calculated to be 11920 kb (Table 2), which is larger
than the size reported by Vaughan-Martini et al.
(1993) (7929 kb). Note that the number of bands
observed by us in this strain was higher than that
reported by Vaughan-Martini et al. (1993), Jager
&
Philippsen (1 989) and Naumov et al. (1995).
S. castellii (Fig. 1) contains nine chromosomes with a
size range similar to that of S. dairenensis. The chro-
mosomes ranged from 550 to 1900 kb. The S. castellii
chromosomal bands were generally well resolved.
Band 4 appeared as one band with a high intensity and
may be a double band. Occasionally, band 9 was not
easily detectable. It either appeared as a very weak
band or it ran as a double band with band 8. The total
genome size was calculated to be 9720 kb, which is
similar to the size reported by Vaughan-Martini et al.
(1993) (9415 kb).
Separation of
S.
servazzii chromosomes (Fig. 1)
identified 12 chromosomal bands. Chromosome sizes
ranged from 570 to 1950 kb. With only two chro-
mosomes belonging to the large size class, most
chromosomes were medium-sized. Bands 1-4 ran in
one group; sizes ranged from 570 to 720 kb. Bands 3
and 4 were sometimes difficult to distinguish. Band 3
was very thick. Bands 8 and 9 appeared with high
intensity on the gel and may each contain two
chromosomes. The genome size was calculated as
12330 kb. Vaughan-Martini et
al.
(1993) reported the
presence
of
11 bands and a genome size of 10430 kb
for the S. servazzii type strain.
As with S. servazzii, 12 bands were visible upon
separation of chromosomes from the S. unisporus type
strain (Fig. 1). Furthermore, the chromosomes were
very much within the same size range, 560-1880 kb.
Bands 2 and 3 were sometimes difficult to distinguish
because they often ran as a double band. Similarly,
bands 9 and 10 were sometimes difficult to distinguish.
The genome size was calculated to be 11
560
kb.
Vaughan-Martini et al. (1993) reported 1 1 bands and a
genome size of 11 375 kb. In the case of
S. unisporus,
two strains were analysed. They exhibited a slightly
different separation pattern of nuclear chromosomes.
A large chromosome of 1400 kb was present in CBS
2422, but it was not present in the type strain. Instead,
the type strain contained two bands of 1110 and
720 kb. Note that the karyotypes of
S.servazzii
and
S.
unisporus
exhibit a similar pattern and many chro-
mosomes may be homologous. This
is
in agreement
with the sequencing data on the rDNA genes which
have been taken to show that these two species are
closely related (James et al., 1997; Kurtzman
&
Robnett, 1991; Oda et
al.,
1997).
Sixteen chromosomal bands were identified in
S.
exiguus (Fig. 1). The chromosome sizes ranged from
395 to 2100 kb, thus belonging to all three size classes.
Two chromosomes were smaller than 500 kb, five
1928
International Journal
of
Systematic Bacteriology
49
Yeast karyotypes
chromosomes were between 500 and 1000 kb and nine
chromosomes were larger than 1000 kb. Chromosomal
bands 10 and 11, 12 and 13 and 15 and 16 often ran as
double bands. The large number of heavy chromo-
somes results in the highest genome size obtained
within the Saccharomyces sensu lato species, 18355 kb.
According to the present results, this species has
the largest genome among Saccharomyces yeasts.
Vaughan-Martini et at. (1993) reported 11 bands and a
total genome size of 10800 kb. Note that this is the
only species among the Saccharomyces sensu lato
species that contains small chromosomes
(<
500 kb).
S.
kluyveri contained seven large chromosomes (Fig.
1). Their sizes ranged from 970 to 1950 kb and the
genome size was 10000 kb. All chromosomes were
resolved as single bands by the four-step program. The
number of chromosomes and the genome size agree
with the results obtained by Vaughan-Martini et al.
(1993).
The chromosome number and genome sizes obtained
in the present study were compared with previously
published results (Table 2). A large variation between
the results from the different studies is evident. We
suggest that this is due to poorer resolution obtained
by the pulsed-field programs utilized in the previous
studies. It may be that some chromosomes are more
easily recovered upon extraction than others and that
the extraction procedure is important. The discrepancy
may therefore also be a result of the experimental
extraction procedure (different lysing enzymes, buffers,
etc.) as well as variations in the recovery of specific
chromosomes.
Further discrepancies were observed in the estimated
genome sizes, particularly in the case of S. dairenensis
and S. exiguus. These differences are correlated with
the number of chromosomes observed. In the present
study, we documented a larger number of chromo-
somes and therefore also a larger estimated genome
size for the two species. In general,
S.
kluyveri is
reported to have the fewest chromosomes and to
posses a genome size at the lower end of the range
shown by the Saccharomyces sensu lato group. It is
also interesting to point out that all species except S.
exiguus have a smaller genome than that of the
sequenced strain of
S.
cerevisiae, which is 13400 kb
(Goffeau et al., 1996).
strains of some
Saccharomyces sensu lato
yeasts were
studied in more detail. The number of chromosomes
for some species belonging to the
Saccharomyces sensu
lato group described in this report was higher than
generally reported previously (Jager
&
Philippsen,
1989; Naumov et al., 1995; Naumova et al., 1996;
Vaughan-Martini et al., 1993; Table
2).
In addition,
the genome size estimates varied from the previous
estimates (Table 2). In the case of
S.
exiguus, this
difference was almost 8 Mb, and for S. dairenensis the
difference was 3 Mb. These discrepancies are possibly
due to the fact that the separation of chromosomes in
our electrophoresis experiments was more efficient and
thus several
'
novel
'
distinctive chromosome bands
could be detected. However, the genome sizes shown in
Table 2 should still be considered as approximate for
the following reasons. The lack of perfect linearity in
the separation profile for chromosomes larger that
1000 kb may contribute to errors in the size estimates
of individual chromosomes. The ploidy of several of
the yeast species examined,
S.
dairenensis, S.servazzii,
S.
unisporus and S. exiguus, is not known and these
yeasts are not available as monospore strains. Thus,
these strains, which are likely to be diploid, may
contain homologous chromosome pairs that are poly-
morphic and thereby produce
'
extra
'
chromosome
bands on the gel. In short, a single chromosome could
be represented by two differently sized copies. On the
other hand, the patterns of S. kluyveri and S. castellii
type strains are the same as in the case of monospore or
haploid isolates (data not shown). In addition, some
chromosomes could exhibit similar electrophoretic
mobility and consequently not separate into dis-
tinguishable bands. Despite these uncertainties, several
conclusions can be reached from our results.
On the basis of sequence analysis of nuclear and
mitochondria1 genes (James et al., 1997
;
Kurtzman,
1993; Kurtzman
&
Robnett, 1991; Oda et al.,
1997
;
Petersen, 1998
;
Peterson
&
Kurtzman, 199l),
the Saccharomyces sensu lato yeasts can be separated
into several sub-groups consisting of phylogenetically
closely related species
:
(i) S. servazzii and S.unisporus,
(ii)
S.
castelliiand S.dairenensis, (iii) a group containing
S. exiguus
and (iv) a group containing
S. kluyveri.
It is
apparent from our results (Fig. 1, Table 1) that
S.
servazzii
and
S. unisporus,
two closely related yeasts,
contain the same number
of
chromosomes and have
very similar karyotypes.
S.
castellii and
S.
dairenensis,
which are also relatively closely related, contain the
same number of chromosomes, but only a minority of
them seem to be similar in size. The numbers of
chromosomes and karyotypes of
S.
exiguus and
S.
kluyveri
differ substantially from any other
Saccharo-
myces sensu lato
yeasts.
The numbers and sizes of chromosomes in the Sac-
charomyces sensu lato group show significant het-
erogeneity. The Saccharomyces sensu lato chro-
mosomes are either large or medium in size. Small
chromosomes
(<
500 kb) have been detected only in
S.
exiguus. In other genera closely related to Saccharo-
DISCUSSION
The genus Saccharomyces consists of several species
that exhibit a lot
of
variation within the organization
of their nuclear (Jager
&
Philippsen, 1989; Naumov
et al., 1992, 1995; Naumova et al., 1996; Petersen,
1998; Vaughan-Martini et al., 1993) and mito-
chondrial (Groth, 1998; Petersen, 1998; Pigkur et al.,
1998) genomes. One possible approach to understand-
ing the origin of the present polymorphism is to
characterize and compare the organization
of
the
modern yeast genomes. Therefore, karyotypes of type
1929
International Journal of Systematic Bacteriology
49
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