INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY,
OCt. 1986, p. 569-572
0020-7713/86/040569-04$02.00/0
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 4
Electrophoretic Karyotyping of Laboratory and Commercial Strains
of Saccharomyces and Other Yeasts
J. R. JOHNSTON? AND R. K. MORTIMER*
Department of Biophysics and Medical Physics, University of California, Berkeley, California 94720
Of 29 strains of various Saccharomyces spp., 24 gave generally similar chromosomal band patterns (14 to 17
bands in the size range from 200 to 2,000 kilobase pairs), as determined by orthogonal field alternation gel
electrophoresis. However, most of these stains showed unique band patterns due to chromosome polymorphisms. Strains of Saccharomyces kluyveri, Candida albicans, Candida utilis, Kluy veromyces luctis, Pichia
canadensis, and Schwanniomyces occidentalis gave bands that were indicative of small numbers of larger
chromosomes (>1,000 kilobase pairs). These results suggest that Saccharomyces kluyveri should be included in
another genus and that Saccharomyces spp. may be different from other yeasts in having a large number of
chromosomes, the majority of which are smaller than 1,000 kilobase pairs.
The technique of pulsed field gradient gel electrophoresis
or orthogonal field alternation gel electrophoresis (OFAGE)
has been used to separate and characterize intact chromosomal deoxyribonucleic acid (DNA) molecules of the yeast
Saccharomyces cerevisiae (2, 3, 13). By using hybridization
probes of genes located on chromosomes I to XVI of this
organism, Carle and Olson (3) identified discrete singlet
bands for 15 of the 17 chromosomes identified genetically
(11). It was necessary to use four different laboratory strains
with chromosome length polymorphisms to resolve all 15
chromosomal bands. Chromosome XII, which is the largest
chromosome in this organism, did not consistently enter the
gel, and chromosome XVII, which is identified only with a
single centromere-linked gene, was not detected in these
studies. Carle and Olson (3) showed that the smallest six
chromosomes (chromosomes I, VI, 111, IX, VIII, and V)
have the same order of increasing size as is seen genetically.
In fact, the entire genome of Saccharomyces cerevisiae
shows an approximately linear relationship between physical
size as determined by OFAGE and genetic map length (11).
Thus, OFAGE appears to offer a reliable means of characterizing the chromosomal sets of various yeasts and possibly
other lower eucaryotic organisms; for such characterizations
the term “electrophoretic karyotyping” has been suggested
by Carle and Olson (3). In this paper, we describe variations
in the karyotypes of several strains of Saccharomyces species, including strains used commercially in baking, brewing,
distilling, and winemaking, as well as the yeasts Candida
albicans, Candida utilis, Kluyveromyces lactis, Pichia
(Hansenula)canadensis, and Schwanniomyces occidentalis.
With certain modifications, the procedures described by
Carle and Olson (2, 3) and by Olson (personal communication) were followed. These include a 50-s pulse time, 300 V
between electrodes, a 1.5% agarose gel, 18-h runs, and a
12°C operating temperature. In some experiments, the resolution of bands corresponding to the larger DNA molecules
was improved by using 1.0% agarose gels. The cells were
prepared as described by Carle and Olson (3) with the
modification that the mixture of cells, low-melting-point
agarose, and Zymolyase was first cast in plastic spectropho-
* Corresponding author.
t Present address: Department of Bioscience and Biotechnology,
University of Strathclyde, Glasgow, G1 lXW, Scotland.
tometer cuvettes with their bottoms removed by milling and
then sealed with Parafilm. The strains which we used are
listed in Table 1.
Throughout these experiments, the strain used as a standard was strain X2180-1A (Yeast Genetic Stock Center,
University of California, Berkeley). This strain gave essentially the same pattern of bands as that found for strain
AB972 (3), namely, singlet bands for chromosomes 1
through IV, VI, IX through XI, and XIV and doublet bands
for chromosomes V and VIII, XI11 and XVI, and VII and
XV. The other six Yeast Genetic Stock Center strains used,
four of which are progenitor strains of X2180-1A (10) and
two of which (strains ABXL-1D and BX24-2B) carry the
FLOl gene (3,gave patterns broadly similar to those of
X2180-1A (Fig. 1A). Nevertheless, there were some minor
polymorphisms among these strains, and, in particular,
strain YO2587 showed many of these size differences.
The Northern Regional Research Laboratory type strains
of Saccharomyces species all showed differences, varying
from minor to major, from each other. For example, the type
strains of Saccharomyces carlsbergensis, Saccharomyces
uvarum, Saccharomyces bayanus, and Saccharomyces
kluyveri gave 14, 14, 17, and 3 chromosomal bands, respectively. The bands for the first three species were distributed
over the same approximate size range and showed the same
general pattern as strain X2180-1A (Fig. 1A and B). However, the three bands observed for Saccharomyces kluyveri
were all in the size range of the largest Saccharomyces
cerevisiae chromosomes. These variations in OFAGE patterns are generally consistent with the differences in percentage of DNA reassociation found between these species by
Martini and Kurtzman (8). The percentages of DNA reassociation between the type strain of Saccharomyces cerevisiae and the type strains of Saccharomyces carlsbergensis,
Saccharomyces uvarum, Saccharomyces bayanus, and Saccharomyces kluyveri were 57, 14, 5, and 0%, respectively.
Between the type strain of Saccharomyces bayanus and the
type strains of Saccharomyces uvarurn, Saccharomyces
carlsbergensis, and Saccharomyces kluyveri the corresponding values were 98, 72, and 24%, respectively. Even though
Saccharomyces uvarum and Saccharomyces bayanus
showed a high degree of DNA homology as determined by
this test, they showed differences in the numbers and positions of OFAGE bands. Given the common occurrence of
chromosomal polymorphisms among various strains of Sac-
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INT. J. SYST.BACTERIOL.
NOTES
TABLE 1. Strains of yeast examined by OFAGE
Strain
X2180-1A
EM93
99R
YO2022
YO2587
ABXL-1D
BX24-2B
Y-12632Tb
Y-12663T
Y-12624T
Y-12693T
Y-12651T
57-79
(= CBS 6546)
61-234
68-912
67-588
Fleischman
Red Star
Budweiser
Sierra Nevada
Thousand Oaks
Bass
Species or type
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
bergensis
Saccharomyces
Saccharomyces
Origin
cerevisiae
cerevisiae
cerevisiae
cerevisiae
cerevisiae
cerevisiae
cerevisiae
cerevisiae
uvarum
bayanus
carls-
YGSC"
YGSC
YGSC
YGSC
YGSC
YGSC
YGSC
C. P. Kurtzman
C. P. Kurtzman
C. P. Kurtzman
C. P. Kurtzman
kluyveri
kluyveri
C. P. Kurtzman
H. J. Phaff
Champagne
Saccharomyces kluyveri
Saccharomyces kluyveri
Saccharomyces kluyveri
Bakers' yeast, United States
Bakers' yeast, United States
Brewers' yeast, United States
Brewers' yeast, United States
Brewers' yeast, United States
Brewers' yeast, United
Kingdom
Brewers' yeast, United
Kingdom
Brewers' yeast, United
Kingdom
Distillers' yeast, Scotland
Distillers' yeast, Scotland
Distillers' yeast, United
States
Wine yeast
Montrachet
Wine yeast
Y- 12983T
Y-7586T
Y-8279T
NCYC 982'
Y-1888=
Candida albicans
Candida utilis
Kluyveromyces lactis
Lipomyces lipofer
Pichia (Hansenula)
canadensis
Pichia canadensis
(Hansenula wingei)
Schizosaccharomyces pombe
Schwanniomyces occidentalis
Schwanniomyces occidentalis
Lorimer
'I'ennent
ID1
D6
ID513
Y-2340T
!372hY-lOT
NCYC 953
a
H. J. PhaB
H. J. Phaff
H. J. PhaB
Retail outlet
Retail outlet
R. Yocum (beer)
Beer isolate
Beer isolate
Beer isolate
Beer isolate
Beer isolate
D. C. Watson
D. C. Watson
S. Fogel,
J. Welch
S . Fogel,
J. Welch
S. Fogel,
J. Welch
C. P. Kurtzman
C. P. Kurtzman
C. P. Kurtzman
D. C. Watson
C. P. Kurtzman
C. P. Kurtzman
J. Carbon
C. P. Kurtzman
D. C. Watson
YGSC, Yeast Genetic Stock Center, University of California, Berkeley.
Obtained from the Northern Regional Research Laboratory, Peoria, 111.
NCYC, National Collection Yeast Cultures, Norwich, United Kingdom.
charomyces, it seems unlikely that these differences reflect
only species differences. However, the result with Saccharomyces kluyveri suggests that this yeast belongs to a genus
other than Saccharomyces. To confirm this, we tested four
independent isolates of Saccharomyces kluyveri obtained
from H. Phaf€. All four strains gave essentially the same
OFAGE pattern as strain Y-12651T (T = type strain) (Fig.
ID). When Saccharomyces kluyveri was first described (12),
this species was placed in the genus Saccharomyces on the
basis of strong fermentation of several sugars, the formation
of four spherical, smooth ascospores in diploid cells, and the
lack of spore liberation from asci at maturity. Authors in
later taxonomic treatises have retained this species in the
genus Saccharomyces. In light of the considerable differences in the chromosome banding patterns of Saccharomy-
ces kluyveri and Saccharomyces cerevisiae, it is interesting
that the mating pheromones of these two species cross-react.
However, no mating was observed between haploid strains
of these species (9).
The majority of the 13 commercial strains of Saccharomyces examined gave unique OFAGE patterns. For example,
the numbers of bands for distiller and brewer strains D1, D6,
Bass, and Thousand Oaks were, 14, 15, 15, and 16, respectively (Fig. 1A and C). In addition to variations in the
number of bands, differences in the positions and relative
intensities of different bands were also seen. The position
differences reflect size polymorphisms. The intensity of
bands is expected to increase in proportion to chromosome
size, provided DNA molecules of different sizes are
equimolar (as expected for euploid cells). However, an
increase in the intensity of a band caused by an integral
factor may indicate that two different chromosomal bands
are migrating together (as for chromosomes V and VIII of
X2180-1A) or aneuploidy (e.g., extra copies of a particular
chromosome migrating together). This type of variation in
band intensity was observed in several of the commercial
strains and is consistent with proposals that many of these
strains are polyploid or aneuploid (reviewed in reference 6).
One way to represent a particular band pattern is by means
of a densitometer plot along the length of the gel lane.
Because these profiles reflect not only the number of chromosomes of different sizes but also, by band intensities, the
relative numbers of homologs of each chromosome, they
provide a potentially useful means of defining the karyotypes
of commercial strains. Southern blotting with genes known
to be on particular chromosomes will be required to provide
a distinction between overlapping of different chromosomes
and aneuploidy. Determination of ploidy is not possible with
these techniques without knowledge of the total amount of
DNA in the gel lane on a per cell basis. Development of such
a determination in combination with the use of chromosomal
probes would provide a most useful definition of the
karyotype of many commercial yeasts. Experiments to identify individual bands from the various commercial yeasts
with known Saccharomyces chromosomes by using Southern blotting procedures are in progress.
The results obtained with the five strains of Saccharomyces kluyveri were of the same general pattern as the results
obtained with strains of several genera other than Saccharomyces (Fig. 1B through D). The number of OFAGE bands
and the approximate corresponding chromosome sizes for
these yeasts are shown in Table 2. Only a small number (one
to four) of bands corresponding to chromosome sizes between approximately 1,000 and 1,600 kilobase pairs were
observed. The OFAGE results with K. lactis support the
results of a previous genetic study on this organism (14)
which suggested that it possesses only a small number of
chromosomes. Also, an earlier genetic study of Saccharomyces kluyveri suggested that it possessed only a small
number of chromosomes (1).In a related study, J. Bassel
(personal communication) has been able to resolve only two
bands in the hydrocarbon-utilizing yeast Yarrowia lipolytica.
We were unable to see any bands from the yeast Lipomyces
lipofer. The yeast Schizosaccharomyces pombe, which has
been shown genetically to have three chromosomes (7), also
did not yield any bands that entered the gel. In a comparison
with the seven strains of other genera (assuming Saccharomyces kluyveri should be reclassified) described in Table 2,
in addition to Schizosaccharomyces pombe, Y. lipolytica,
and L . lipofer, only yeasts of the genus Saccharomyces were
shown to possess a larger number of chromosomes,--t_he
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VOL. 36, 1986
NOTES
571
FIG. 1. (A) Lane 1, Saccharomyces uvarum Y-12663T;lane 2, Saccharomyces cerevisiae Y-12632T;lane 3 , X2180-1A; lane 4, Bass; lane
5, Tennent. (B) Lane 1, C. utilis Y-7586T;lane 2, Saccharomyces kluyveri Y-12651T;lane 3, X2180-1A; lane 4, P . canadensis Y-1888T;lane
5 , P. canadensis Y-2340T.(C) Lane 1, X2180-1A; lane 2, D6 (distillers' yeast); lane 3, D1 (distillers' yeast); lane 4, K. laciis Y-8279T.(D) Lane
1, X2180-1A; lane 2, Saccharomyces kluyveri 68-192; lane 3, Saccharomyces kluyveri 61-234; lane 4, Saccharomyces kluyveri 67-588; lane 5 ,
Saccharomyces kluyveri 57-79.
majority of which were smaller (<1,000 kilobase pairs). This
unique chromosomal banding pattern of Saccharomyces
spp. may be the result of thousands of years of domestication of this yeast by humans, a domestication that was
probably associated with continuous selection for stronger
fermentation abilities. We propose that this selection resulted in the conversion of a progenitor yeast, such as
Saccharomyces kluyveri, into a yeast with a larger number of
smaller chromosomes. In Saccharomyces cerevisiae, all
fermentation genes identified so far map on the ends of
chromosomes (ll),and there is good evidence that these
polymeric sets of genes arose by duplication of progenitor
genes and associated telomeric sequences, followed by
rearrangements of these sequences to new or existing chromosomal ends (4). Thus, there could have been selection for
chromosomal structural changes (i.e., chromosome breaks)
that would provide new ends. Stabilization of the resulting
centric and acentric fragments would require rearrangements of telomeric sequences, an event that could increase
TABLE 2. Numbers of OFAGE bands and estimated
corresponding DNA sizes for a strain of Saccharomyces kluyveri
and strains of other yeast genera
OFAGE bands
Species
Strain
C . albicans
C. utilis
K . lactis
P . canadensis
P . canadensis
Schwanniomyces
occidentalis
Saccharomyces
kluyveri
Y-12983T
Y-7586T
Y-8279T
Y-2340T"
Y-1888T
Y-10T
4
1
3
2
3
4
1,100,
1,200
1,200,
1,100,
1,000,
1,100,
Y-12651T
3
1,000, 1,300, 1,500
No.
Size (kilobase pairs)
1,300, 1,500, 1,600
1,500, 1,600
1,500
1,400, 1,500
1,200, 1,500, 1,600
a Type strain of Hansenula wingei, which is now a synonym of Pichia
(Hansenula) canadensis.
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572
NOTES
INT. J. SYST.BACTERIOL.
the number of fermentation genes. The acentric fragment
would, in addition, need to acquire centromere function by
an as-yet-undefined mechanism.
Our results suggest that the OFAGE technique could be a
very useful taxonomic tool. From a taxonomic and evolutionary point of view it should be of interest to examine more
yeasts of different genera by this method.
This work was supported by a grant from the Office of Health and
Environmental Research of the U.S. Department of Energy under
contract DE-AC03-76SF00098, by Public Health Service grant
GM30900 from the National Institutes of Health, and by travel grant
D24692 to J.R.J. from S.E.R.C. (United Kingdom).
We thank C. Rebecca Contopoulou for assistance with several of
the runs and the scientists listed in Table 1 yvho provided strains
essential for this study.
3.
4.
5.
6.
7.
8.
ADDENDUM IN PROOF
We recently applied the technique of field inversion gel
electrophoresis (G. F. Carle, M. Frank, apd M. V. Olson,
Science 232:6548, 1986) to strains of Saccharomyces cerevisiae, Saccharomyces kluyveri, and several yeasts of other
genera (R. Contopoulou, J. R. Johnston, R. Mortimer,
unpublished data). We obtained improved resolution of the
large chromosomes, and the numbers of chromosomal bands
for some species are thus revised as follows: Saccharomyces
kluyveri; 5 ; Pichia canadensis Y1888, 5 ; Candida albicans,
5 ; Kluyveromyces lactis, 4; Lipomyces lipofer, 5 ; and
Schwanniomyces occidentalis NCYC 953, 5 .
9.
10.
11.
12.
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