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?????
???. 14: 723?731 (1998)
Comparison of the Structural Characteristics of
Chromosome VI in Saccharomyces Sensu Stricto:
The Divergence, Species-Dependent Features and
Uniqueness of Sake? Yeasts
ATSUMI NAKAZATO1, TOSHIMORI KADOKURA1, MAKOTO AMANO1, TADASHI HARAYAMA1,
YASUFUMI MURAKAMI2, MASAHISA TAKEDA1, MORIYA OHKUMA3, TOSHIAKI KUDO3 AND
TAKICHI KANEKO1*
1
Department of Fermentation and Brewing, Tokyo University of Agriculture, Sakuragaoka 1-Chome, Setagayaku,
Tokyo 156?0054, Japan
2
Division of Human Genome Research and Gene Bank, Tsukuba Life Science Center, The Institute of Physical and
Chemical Research (RIKEN), Koyadai, Tsukubashi, Ibarakiken 305?0074, Japan
3
Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), Wakoshi,
Saitamaken 351?0198, Japan
Received 29 August 1997; accepted 10 November 1997
Previous studies have revealed that chromosome VI of sake? yeasts is much larger than that of the other strains of
Saccharomyces cerevisiae. Southern analysis using segments of chromosome VI of a laboratory strain as probes
suggested that the nucleotide sequence of a major portion of this chromosome is conserved, but considerable
diversity was found in the distal parts in the other strains. Physical maps also indicated that differences in length of
chromosome VI were mainly due to differences in its ends. NotI was found to generate 9 kb and/or 16 kb fragments
from the left telomere of chromosome VI in most sake? yeasts, but no fragment in the case of AB972. SfiI produced
one or two 30?50 kb fragments from the right end of this chromosome in all sake? yeasts tested, but produced a 20 kb
fragment in the case of AB972. All S. cerevisiae strains not employed in sake? brewing were the same as AB972 in
these respects. S. paradoxus had one NotI site in chromosome VI, while S. bayanus had two, one of which is possibly
common to both species. The SfiI site mentioned above was present in chromosome VI of all species, while that of
S. bayanus and S. paradoxus each had a second site distinct from the other. Chromosome VI of S. pastorianus was
not distinguishable from that of S. bayanus. 1998 John Wiley & Sons, Ltd.
Yeast 14: 723?731, 1998.
??? ????? ? Chromosome length; chromosome VI; electrophoretic karyotype; RFLP; Saccharomyces; sake? yeast;
taxonomy
INTRODUCTION
Saccharomyces cerevisiae and related yeasts have
been employed in a variety of industries and,
hence, are the most extensively studied group of
yeasts. A number of reports had been published
concerning their taxonomy prior to the time when
van der Walt (1970) divided them into only 17
species on the basis of phenotypical data. Since
then, molecular biological considerations have
*Correspondence to: T. Kaneko, 3?10?2, Shakujiidai,
Nerimaku, Tokyo 177?0045, Japan. Fax: (+81) 3 3996 8386.
CCC 0749?503X/98/080723?09 $17.50
1998 John Wiley & Sons, Ltd.
become more and more important in this field.
Yarrow (1984) categorized these yeasts as a single
species based chiefly on DNA base composition
and sexual behavior. However, Martini and
Martini (1987) conducted homology studies and
classified them into four species: S. cerevisiae, S.
bayanus, S. paradoxus and S. pastorianus; although
S. pastorianus was considered to be a hybrid
between S. cerevisiae and S. bayanus. Yamada
et al. (1993) were able to clearly re-identify almost
all 121 strains of Saccharomyces sensu Yarrow
maintained in their culture collection, the Institute
?. ???????? ?? ??.
724
for Fermentation Osaka (IFO). Accordingly, in
our opinion, a reasonable taxonomic system has
been established for these yeasts.
On the other hand, the categorization of industrial strains is dependent on their usage, not only
for convenience but because biologically trivial
characters are often very important for specific
purposes. Among the industrial strains, sake? yeasts
are distinct: they grow in the absence of vitamins,
produce more than 20% ethanol, form a high foam
layer during active growth, and are heterothallic.
Although it has been confirmed that these yeasts
should be taxonomically placed in S. cerevisiae
(Yamada et al., 1990), we thought that some
differences may be detectable at the molecular level
in accordance with their phenotypes.
Pulsed-field gel electrophoresis (PFGE) has revealed that chromosome lengths are so variable
that the electrophoretic pattern is even strainspecific (Querol et al., 1992). Nevertheless, the
patterns are often locally species-specific (Yamada
et al., 1993), and Goto et al. (1990) have already
suggested that chromosome VI in sake? yeasts is
longer than that in the other groups.
In a previous study (Nakazato et al., 1998), we
examined the chromosomes of various yeast
groups by PFGE, estimated their lengths as
accurately as possible, and compared mainly the
small ones. Chromosome VI in sake? yeasts was
28715 kb in size, whereas in wine yeasts it
was 25614 kb. Shochu, awamori, brewers? and
bakers? yeasts were between these extremes. The
lengths of chromosomes I and IX showed a very
similar tendency.
In the present study, we have compared the
structural characteristics of chromosome VI in
sake? yeasts and other Saccharomyces strains. The
sequence of a major portion of this chromosome
was found to be well conserved but the distal parts
were highly variable. The sake? yeasts were found
to have characteristic sequences at the left end of
the chromosome. Further studies revealed that
these yeasts can be classified into four groups
based on restriction fragment length patterns: sake?
yeasts, the other S. cerevisiae, S. bayanus and
S. paradoxus. On this basis, S. pastorianus was
indistinguishable from S. bayanus.
MATERIALS AND METHODS
Yeast strains
The sake? yeasts were selected from several
groups. Strains K7, K9 and K10 were typical
1998 John Wiley & Sons, Ltd.
Kyokai yeasts, which have been developed in the
National Research Institute for Brewing and are
most widely used today. The second group comprised historical strains IFO 0244, IFO 0249, IFO
0309 and another Kyokai yeast, K5, which were
isolated 70?100 years ago. ATCC 32696 through
32699 were selected from ten ATCC strains, which
were originally isolated in this laboratory from
sake? mashes collected at various brewers? facilities
in the 1960s. We recently isolated some new strains
from nature and one firm used them for brewing
commercial sake?. Two such strains were also employed; strain FM was isolated from soil collected
at the top of Mount Fuji, while NR was isolated
from the tundra in Finmark, Norway.
The laboratory strain AB972 was employed as a
reference. The complete nucleotide sequence of
chromosome VI has been determined for its isogenic strain, S288C (Murakami et al., 1995). Some
typical strains of the other industrial groups were
included for comparison.
For the comparison between species, not only
type strains but some reference strains were also
used. These were selected from among recently
re-identified IFO strains (Yamada et al., 1993).
Throughout the present work, we examined only
those strains in which chromosome VI forms a
single and well-isolated band.
Electrophoresis of chromosomes
Each yeast was grown in a medium containing
2% peptone, 1% yeast extract and 2% glucose at
30C for 40 h without shaking. Cells were washed
in 10 m?-Tris, pH 7� containing 5 m?-EDTA,
embedded in a block of 1% agarose gel that
contained Zymolyase-100T at 0�mg/ml and 5%
2-mercaptoethanol, and incubated in 10 m?-Tris,
pH 7� containing 0�?-EDTA at 37C for 24 h.
The block was washed and incubated in a mixture
of the same Tris?EDTA, 2 mg of Proteinase K and
10 mg of N-lauroylsarcosine per ml at 50C for
48 h. It was then cut into pieces and subjected
to electrophoresis. Each piece thus prepared
contained 12?15 OD660 of cells.
PFGE was carried out in CHEF-D?II (BioRad
Labs) using a 1% low melting point agarose gel at
200 V and 12C. The buffer, pH 8� contained
45 m?-Tris?HCl, 45 m?-boric acid and 1 m?EDTA. To clearly separate several smaller chromosomes, a 25?40 s pulse was applied for 18 h.
For separation of all chromosomes, the conditions
were modified to a 100?45 s pulse for 24 h followed
by a 130 s pulse for another 16 h.
?????
???. 14: 723?731 (1998)
?????????? ?? ?? ?. ??????????
Restriction map of chromosome VI
Because it was difficult to recover intact chromosomes from the gel after the first PFGE, enzyme
treatment of a chromosome was conducted in situ.
A small block was cut out from the gel with
chromosome VI in it, washed with the electrophoresis buffer, placed in a solution containing 20
U of restriction enzyme in 250 靗, and incubated
under conditions suggested for the enzyme for at
least 20 h.
The block was then directly loaded into a new
gel for the second electrophoresis, which was carried out under the same conditions as for the first
one. In this case, however, a 20?25 s pulse was
applied for 18 h, followed by a 0�2�s pulse
for 6 h.
Since the amount of DNA in a single band was
limited, the gel was stained with SYBR Green
(Molecular Probes, Inc.) at a 5103 dilution for
2?3 h.
Segments of chromosome VI
We have a library of chromosome VI of strain
S288C used for the sequencing studies (Murakami
et al., 1995). We selected appropriate clones from
it and amplified the segments, which have been
inserted into the SmaI site of plasmid pUC19,
using the PCR method.
Southern hybridization
The agarose gel with DNA bands in it was
immersed in 0� ?-HCl for 20 min, washed with
distilled water, and then treated with 0�?-NaOH
containing 1�?-NaCl. DNA, thus denatured, was
transferred to a Hybond-N + filter (Amersham).
Hybridization was carried out in the presence of
50% formamide, 0� ?-NaCl and 75 m?trisodium citrate at 42C for 20 h. DigoxigeninELISA was performed to detect hybrids using a
DNA labeling and detection system of Boehringer
Mannheim.
RESULTS
Distribution of sequences homologous to
chromosome VI segments from S288C in
representative strains
In order to find clues for understanding the
nature of the differences in chromosome length, we
first tested whether different strains share the same
sequences with the laboratory strain. From among
1998 John Wiley & Sons, Ltd.
725
Table 1. Hybridization of segments of S288C chromosome VI with the same chromosome of the other strains.
Strain
Probe Position (kb)
no.
of sequence AB972 K7 IFO 2260 IFO 2011
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0�1?0�3
9�
19�
29�
39�
50�
59�
69�
79�
89�
100�
109�
120�
130�
141�
151�
160�
170�
179�
190�
200�
209�
219�
231�
240�
249�
260�
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Each probe had the sequence at the indicated position in
chromosome VI of S288C. Except for probe 1, position
numbers are rounded and sizes of the probes were 1�2�kb.
a series of segments of chromosome VI in S288C,
27 segments were chosen at intervals of about
10 kb in terms of their positions in the original
chromosome. Using these as probes, we performed
Southern analysis of the chromosomes in a few
representative strains: a sake? yeast K7, a wine
yeast IFO 2260 and a brewer?s yeast IFO 2011.
The probes used and their hybridization with
chromosome VI of these strains are summarized in
Table 1. Most probes hybridized with the chromosome of all strains tested. However, probes 1 to 4,
which had partial sequences in the left distal part
of S288C chromosome VI and probe 15, which
represents a segment beginning at about the 140 kb
position, did not hybridize with chromosome VI of
some strains.
?????
???. 14: 723?731 (1998)
?. ???????? ?? ??.
726
Figure 1. Southern hybridization with probes 2 and 3. Left,
chromosome patterns; middle, hybridization with probe 2;
right, hybridization with probe 3. Lane 1, K7; lane 2, AB972;
lane 3, IFO 2260; lane 4, IFO 2011.
Figure 1 shows the hybridization patterns obtained using probes 2 and 3. Probe 2 hybridized
not only with chromosome VI but also with chromosome XIV of strain AB972. In the case of K7, it
did not react with chromosome VI but hybridized
with three other chromosomes. Results obtained
with probe 3 were similar to these, but differed in
that it hybridized with chromosomes X and IV of
AB972, and chromosome VI of K7, too. Thus, the
same probes hybridized to different chromosomes
depending on the test strains. Patterns found with
probes 1 and 4 were also very similar to these.
These results indicate that some sequences
within about 30 kb from the left end need not be
present in chromosome VI, whether or not they are
dispensable for these yeasts. In addition, similarity
between patterns obtained with different probes
suggests that some of the relevant regions have
been transferred or copied together from one
chromosome to another.
The sequence data (Murakami et al., 1995)
suggest that the sequence of probe 15 was a part
of transposon Ty1?17. We therefore examined
its distribution among small chromosomes of a
variety of strains.
The results are shown in Figure 2, where lanes 1
to 10 are sake? yeasts, 11 to 13 are brewers? yeasts,
14 is a bakers? yeast, and the others are wine
yeasts, respectively. Although most strains have
been placed in S. cerevisiae, IFO 2003 belongs to
S. pastorianus, and taxonomic positions of IFO
2000, IFO 2015 and IFO 2220 can be questionable.
1998 John Wiley & Sons, Ltd.
Figure 2. Distribution of the transposon among small chromosomes of various industrial strains. Top, chromosome patterns; bottom, hybridization with probe 15, which has a partial
sequence of transposon Ty1?17.
Nevertheless, the transposon was detected in all
strains, suggesting that its distribution is not limited to the species S. cerevisiae. Also, their hybridization patterns tended to differ from strain to
strain, while the intensity of the signal was highly
variable probably depending on the copy number
and the degree of sequence homology of the transposon. Sake? yeasts generally gave relatively weak
signals, while each of multiple bands of the
homologous chromosomes in some brewers? and
bakers? yeasts appeared to have the transposon.
On the other hand, the other 22 probes
showed uniform hybridization patterns; they all
hybridized with chromosome VI of all strains but
never hybridized with any other chromosome of
any of the strains tested. Thus, the sequences
present in probes 5 through 27, except for the
transposon, appear to be essential and specific for
chromosome VI.
Physical map of K7 chromosome VI
As the second step, we compared the physical
map of chromosome VI of strain K7 with that of
?????
???. 14: 723?731 (1998)
?????????? ?? ?? ?. ??????????
727
Figure 3. Restriction maps of chromosome VI of strains
AB972 and K7.
AB972. It has been reported that the latter has a
site for restriction enzyme SfiI about 20 kb away
from the right end, but no site for NotI (Link and
Olson, 1991).
SfiI excised a 30 kb fragment from the K7
chromosome, while a 20 kb fragment was
generated as expected in the case of AB972. On the
other hand, NotI produced two fragments, 9 and
16 kb in size, from K7 chromosome, and a double
digestion experiment revealed that these were
derived from its left end. As for their positions in
the chromosome, however, there seem to be many
possibilities, because K7 is diploid. If the two
fragments are located side by side in the same
chromosome, a 25 kb fragment should be produced as an intermediate. But we did not detect
such a fragment. Therefore, we consider that the
9 kb segment is derived from one of the two
homologous chromosomes and the 16 kb segment
is derived from the other.
To outline the physical maps, we selected AscI
and PmeI as the most useful enzymes, using a
computer and the sequence data (Murakami et al.,
1995). The results obtained are summarized in
Figure 3. Three AscI sites were present at essentially the same locations within chromosome VI in
the two strains. On the other hand, AB972 had two
sites, while K7 had three for PmeI. However, this
difference does not seem very important, since the
nucleotide sequences are generally variable to
some extent; in fact, Wicksteed et al. (1994) found
considerable variations in sequences even among
closely related strains.
Because the former experiments suggested that
the sequence is variable in the terminal part of the
left arm, we employed Sse837I, which has a unique
recognition site in S288C chromosome VI at about
1998 John Wiley & Sons, Ltd.
Figure 4. Proposed structure of chromosome VI of various
sake? yeasts. For instance, a reverse triangle shows that probe 2,
which has a partial sequence around position 10 kb of chromosome VI of strain S288C, hybridized with the indicated
restriction fragment.
the 29 kb position. Although the chromosome in
AB972 had a site at the expected locus, no site was
found in K7.
These results, in accordance with the hybridization data described above, suggest that the difference between the chromosomes in the two strains
is limited essentially to their distal parts.
NotI and SfiI sites in chromosome VI of various
sake? yeasts
Because the above results stimulated our interest
in examining other sake? yeasts, chromosome VI
was analysed in 11 strains using NotI and SfiI.
?????
???. 14: 723?731 (1998)
?. ???????? ?? ??.
728
Upon treatment with NotI, no fragment was
produced from chromosome VI in the case of four
strains, which are all of the old isolates. By contrast, either the 9 kb or the 16 kb fragment, or
both, were liberated in all the other strains. These
results are shown in Figure 4.
The results obtained using SfiI were rather uniform; one or two short fragments were generated
in all the strains used. Their lengths, however,
ranged from 30 to 50 kb. Further, four strains were
found to have two different fragments. Similar to
the two NotI fragments described above, these
fragments were confirmed to be separately located,
or one on each of the two homologous chromosomes. It is of interest that the SfiI fragment
obtained from sake? yeasts was always longer than
that of the laboratory strain.
probes 3 and 27 have partial sequences located
near positions 20 and 260, respectively, of chromosome VI of the laboratory strain, we suppose that
a very long sequence between these positions is
basically conserved in all sake? yeasts. If so, differences in the right arm between strains may be
limited to the region between position 260 kb and
the end.
Incidentally, the sake? yeasts are generally diploid, although it is almost impossible to isolate a
haploid strain. Therefore, in the case of IFO 0249,
for instance, we could not determine whether
probe 2 hybridized with only one of the homologous chromosomes, and, if so, which one it was.
Because of these problems, details of the schemes
shown in Figure 4 should be taken to be rather
tentative.
Distribution of sequences corresponding to those
from a laboratory strain in sake? yeasts
In the hybridization experiments described
above, we used only one strain of sake? yeast and
its whole genome. To examine the details, we
carried out Southern hybridization of the S288C
chromosome segments with restriction fragments of chromosome VI from the above 11 sake?
yeast strains. The results are summarized in
Figure 4.
Probe 1, whose sequence (Table 1) comprises
the subtelomeric repeats Y (Murakami et al.,
1995), hybridized with all the 16 kb NotI fragments, and also the major fragments of some
strains, but did not react with any other fragments. It is of interest that no probe hybridized
with the 9 kb NotI fragments. These findings
suggest that most sake? yeasts share closely related
9 kb and/or 16 kb fragments. Sequences homologous to probe 2 were found only in the major
fragments derived from five strains.
As already shown in Figure 2, probe 15 hybridized with chromosome VI of three out of ten sake?
yeasts tested. It did not react with that of another
strain, K7 (Table 1).
With this exception, probes 3 to 26 all hybridized with the major fragments derived from all
strains tested. It is interesting that all sake? yeasts
displayed a sequence homologous to probes 3
and 4, though some S. cerevisiae strains did not
(Table 1).
Probe 27 hybridized with all the small fragments
excised with SfiI. Thus, probes 3 through 27 hybridized with all the chromosomes tested. Because
NotI and SfiI sites in chromosome VI of various
S. cerevisiae strains
So far we have concentrated on sake? yeasts and
examined a few reference strains for comparison.
Because we have been interested in the differences
between industrial strains of S. cerevisiae, their
restriction patterns were compared and the results
are shown in Table 2.
NotI generated a 16 kb fragment from chromosome VI of one of the wild sake? yeasts. A 9 kb
fragment was generated from chromosome VI of a
shochu yeast, but no fragment from that of an
awamori yeast. This is in accordance with the
finding that chromosome VI of shochu yeasts is a
little smaller than that of sake? yeasts, but that of
awamori yeasts is significantly smaller (Nakazato
et al., 1998), even though similar materials and
methods are used for the production of sake?,
shochu and awamori.
All the other S. cerevisiae strains including the
type strain had no NotI site in their chromosome
VI.
Chromosomes VI in each of these strains were
found to have a single SfiI site, and the fragment
size ranged from 20 to 30 kb, except in the case of
sake? yeasts, a shochu yeast and an awamori yeast.
The latter two had different-sized fragments as
observed with some sake? yeasts. Both wild sake?
strains had 30 kb SfiI fragments.
Thus it was concluded that, with respect to
chromosome VI, sake? yeasts and related strains
differ from the other groups of S. cerevisiae in
having small NotI fragment(s) and longer SfiI
fragment(s).
1998 John Wiley & Sons, Ltd.
?????
???. 14: 723?731 (1998)
?????????? ?? ?? ?. ??????????
Table 2.
729
Lengths of restriction fragments (kb) of chromosome VI from various strains.
Restriction enzyme
Strain
S. cerevisiae
Laboratory strain
AB972
Wine yeasts
IFO 2252, IFO 2260, IFO 2300
IFO 2363
Bakers? yeast
IFO 2044
Brewers? yeasts
IFO 2000*
IFO 2011, IFO 10217T
Wild yeasts
FM
NR
Awamori yeast
IFO 2110
Shochu yeast
SH-4
Sake? yeasts
IFO 0244
IFO 0249, IFO 0309
K5
K7, K9
K10
ATCC 32696
ATCC 32697, ATCC 32698
ATCC 32699
S. bayanus
IFO 1127T, IFO 1620, IFO 10563
S. pastorianus
Brewers? yeasts
IFO 1167, IFO 1961, IFO 2003, IFO 10010
S. paradoxus
IFO 10609T
IFO 10554, IFO 10695
NotI
SfiI
?
20
?
?
25
30
?
30
?
?
20
30
?
16
30
30
?
30,50
9
40,50
?
?
?
9,16
16
16
16
9
30
30,40
30,35
30
30
30,50
50
35
50,100,125
20,120,135
50,100,125
20,120,135
115,190
110,175
25,70,210
25,55,205
In S. cerevisiae, the largest fragments are omitted. TType strain; *questionable taxonomic status.
NotI and SfiI sites in chromosome VI of various
Saccharomyces species
Finally, we considered the differences between
chromosome VI in various Saccharomyces species.
PFGE patterns of small chromosomes indicated
that chromosome VI in S. paradoxus is around
295 kb in length. On the other hand, this chromosome in S. bayanus and S. pastorianus was about
275 kb long, while that in S. cerevisiae was a little
smaller. Figure 5 shows some typical data on the
action of NotI in cleavage of chromosome VI from
1998 John Wiley & Sons, Ltd.
strains of different species, while the lengths of the
fragments generated are summarized in Table 2. In
contrast to most sake? yeasts, no small fragment
was found after treatment with NotI in the case of
any other strains. NotI cleaved the chromosome VI
of S. bayanus and that of S. pastorianus into three
fragments. Moreover, their common sizes suggest
that they share the same sites. On the other hand,
S. paradoxus was found to have one site only and
the fragments generated were of 115 and 190 kb, or
110 and 175 kb.
?????
???. 14: 723?731 (1998)
?. ???????? ?? ??.
730
Figure 5. NotI digests of chromosome VI of various Saccharomyces species. Lane 0, reference, intact chromosomes of K7;
lane 1, K7; lane 2, IFO 1127; lane 3, IFO 10563; lane 4, IFO
1167; lane 5, IFO 2003; lane 6, IFO 10609; lane 7, IFO 10695.
Whether the NotI site in chromosome VI of
S. paradoxus is located at a position distinct from
the ones in the other two species remains to be
determined. However, we speculate that the 190 or
175 kb fragment in S. paradoxus may correspond
to the 175 kb fragment, which was found as an
intermediate in the digests in the case of the two
species.
On the other hand, one site for SfiI, which was
located at a position 20 or 25 kb from the right
end, was common to chromosome VI in all the
strains tested, although its distance from the end
was larger in sake? yeasts. Chromosome VI in
S. cerevisiae had no other site, while that in
S. bayanus and S. paradoxus had the second sites
whose positions were distinct from each other. All
strains of S. pastorianus displayed essentially the
same fragments as those observed in the case of
S. bayanus.
Because S. pastorianus is thought to be a hybrid
(Martini and Martini, 1987), the possible existence
of a S. cerevisiae type chromosome VI in this
species was examined carefully. Such a chromosome was not found in two out of four strains
tested, and, if present in the other two strains, it is
limited to less than one third of all the homologous
chromosomes.
DISCUSSION
Comparison of chromosome VI in different groups
of industrial yeasts suggested that this chomosome
1998 John Wiley & Sons, Ltd.
in S. cerevisiae is apparently composed of two
different parts. If AB972 is taken as the standard,
the sequences in the distal regions of chomosome,
30?40 kb at the left end and 10 kb at the right end,
are highly variable, while those in the major portion are highly conserved. In fact, all segments of
the main part of this chromosome from a laboratory strain hybridized to chromosome VI of all
sake? yeasts and the other S. cerevisiae strains
tested. In contrast, segments from the distal region
hybridized with chromosome VI of some but not
all strains, and also with a few other chromosomes
of all strains tested.
These results are in remarkable agreement
with the observations concerning the nucleotide
sequence of this chromosome. Murakami et al.
(1995) reported that it has a large number of short
ORFs and shares almost identical or homologous
partial sequences with chromosomes II and III in
its left end. Data concerning chromosome VIII
(Johnson et al., 1994), for instance, also suggested
that both its ends were gene-poor and contain
sequences common to the other chromosomes.
As for the right end, the only information obtained from the present work is that the SfiI
fragment produced from it varies in length, and
that a sequence more than 10 kb long is probably
conserved. Thus, variations between strains are
very local and this is why we did not realize its
characteristics. On the other hand, sequence data
suggest that the region contains only a few short
genes (Murakami et al., 1995). Therefore, we suppose that both ends of chromosome VI are similar
in nature.
It has been reported that meiotic recombinations occur very frequently in small chromosomes
(Kaback et al., 1989) and also that chromosome
rearrangements occur during vegetative growth
(Longo and Vezinhet, 1993). If both ends of a
chromosome do not play a genetically important
role, recombination events affecting such regions
can occur without serious effects and, therefore,
rather freely. We think that chromosome length
polymorphism results mostly from such modifications in these regions.
It was revealed that the restriction patterns of
chromosome VI in S. cerevisiae, S. bayanus and S.
paradoxus differ from one another. It is interesting
that these three species share the feature of having
one SfiI site, while S. bayanus and S. paradoxus
probably share the feature of one NotI site in
chromosome VI. Also, all of the test strains of the
same species always had the same site(s), except
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?????????? ?? ?? ?. ??????????
that sake? yeasts were different from the other
S. cerevisiae strains. Another point is that chromosome VI in four strains of S. pastorianus was
indistinguishable from that in S. bayanus. Thus,
we could not confirm the coexistence of an
S. cerevisiae-type chromosome in S. pastorianus.
Although this does not favor the hybrid hypothesis
(Martini and Martini, 1987), more information
will be required before reaching a conclusion,
because chromosome VI accounts for only 2% of
the whole genome.
We found that the left end of chromosome VI in
sake? yeasts is unique in that it has a NotI site at
position 9 or 16 kb, and that the sequence between
a SfiI site and the right end of this chromosome
in sake? yeasts is longer than that in the other
Saccharomyces strains. These results suggest that
sake? yeasts differ, not only with respect to phenotypic characters, but also at the chromosome
level from the other yeasts. Thus, we suppose that
they are a phylogenetically unique subgroup of
S. cerevisiae.
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