Mutation Research, 231 (1990) 177-186 Elsevier
177
MUT 04880
High levels of chromosome instability in polyploids of Saccharomyces cerevisiae V e r n o n W. M a y e r a n d A n d r e s A g u i l e r a a Genetic Toxicology Branch, Division of Toxicological Studies, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC 20204 and a Biochemistry Department, New York University Medical Center, New York, N Y 10016 (U.S.A.) (Received 21 February 1990) (Accepted 27 February 1990)
Keywords: Chromosome loss; Polyploid yeast; Saccharomyces cerevisiae
Summary The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leul-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome I,'11's during mitosis. We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid. Loss of two or three copies occurred at an even greater frequency. These findings suggest either that aneuploidy (3N-l, 3N-2, 4 N - l , 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency. Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation. The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.
Although haploidy and diploidy are the more usual chromosomal constitutions found in nature, diploids bearing extra chromosomes (Muntzing, 1948) and polyploids are not uncommon. Differences in number of chromosomes among cells of an individual plant (Janiki-Ammal, 1940; Snoad, 1955; Frost, 1958; Ostergen and Frost, 1962; Lewis et al., 1971) and among different individual plants of the same species have been reported (Einset, 1948; Dowrick, 1952; Bosemark,
Correspondence: Dr. Vernon W. Mayer, Genetic Toxicology Branch, HFF-166, Food and Drug Administration, 200 C Street S.W., Washington, DC 20204 (U.S.A.).
1956; Sharma and Aiyanger, 1961). Sachs (1952) reported that two species of hamsters were diploid and a third species was apparently tetraploid. Polyploidy has been found among tumor cells (Nowel, 1976; Danes, 1986; Rodenburg et al., 1987) as well as in subpopulations of apparently normal cells in intact animals (Hultin et al., 1970; Goldberg et al., 1984; Bohman et al., 1985). Animals in which polyploidy has been reported include earthworms (Muldal, 1952), salamanders (Humphrey and Fankhauser, 1949; Fankhauser and Humphrey, 1959; Schmid et al., 1985; Bogart and Licht, 1986), frogs (Becak et al., 1966, 1967), lizards (Pennock, 1965) and salmonid fishes (Wolf et al., 1969; Johnson et al., 1987). Polyploid
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178
variants of unicellular uninuclear organisms have been reported in the yeast Saccharomyces (Roman et al., 1951; Pomper et al., 1954) and the pathogenic yeast Candida albicans (Hubbard et al., 1985; Suzuki et al., 1986). The finding of polyploid plants (Einset, 1948), animals (Darlington, 1953) and yeast (Roman and Sands, 1953) and the lack of constancy in chromosome number among such polyploids (JanikiAmmal, 1940; Snoad, 1955; Lewis et al., 1971) suggest that polyploidy might not be a genetically stable combination. These exceptions in nature may, however, play an important role in evolution and the development of new species. We have used the yeast Saccharomyces cerevisiae to study the genetic consequences of polyploidy in the asexual (mitotic) life cycle of a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII were constructed and used to follow the loss of one, two or
three chromosomes. With an increase in ploidy number, an increase in chromosome instability was found. Materials and methods
Yeast strains The diploid Saccharornyces cerevisiae strain D61.M and two haploid mating-type tester strains carrying an adel mutant allele were obtained from Dr. F.K. Zimmermann, Technische Hochschule, Darmstadt (F.R.G.). Strain D61.M, as described previously (Zimmermann and Scheel, 1984; Zimmermann et al., 1985; Mayer et al., 1986), was used as the parent for construction of the triploid and tetraploid strains. Genotypes of the parent strain and the different constructs are given in Table 1. Strain D61.M and all derivatives require adenine because they are homozygous for the ADE2 mutation on chromosome XV. Since strain D61.M is ADE6/ade6 heterozygous, derivatives
TABLE1 G E N O T Y P E O F STRAINS Strain Diploid D61.M (+ / - ) Tfiploid VM1 (+/-/-)
Chromosome VII + cyh2
Chromosome XV
+ leul
-0 0
+ ade6
ade2 ade2
+
+
cyh2 cyh2
leul leul
-0 0 0
+ ade6 ade6
ade2 ade2 ade2
+
+
-0
+
cyh2
leul
+
o 0
ade6
ade2 ade2 ade2
Tetraploid VM3 (+/-/-/-)
+ cyh2 cyh2 cyh2
+ leul leul leul
0 0 0 0
+ ade6 ade6 ade6
ade2 ade2 ade2 ade2
Tetraploid VM4
+
+
0
+
ade2 ade2 ade2 ade2
Triploid VM2
(+/+/-)
(+/+/-/-)
Tetraploid VM5
(+/+/+/-)
+
+
+
cyh2 cyh2
leul leul
+
0 0 0
ade6 ade6
+
+
0
+
+ +
+ +
0
+ +
leul
0 0
ade6
cyh2 0, position of centromere on chromosome VII.
+
ade2 ade2 ade2 ade2
179 containing the wild-type allele of ADE6 produce red colonies because the cells accumulate a redpigmented precursor of adenine. If only the mutant allele of ADE6 is present, the red pigment is not formed and colonies are white. Two other genes located on chromosome VII are also of interest in testing for chromosome loss; CYH2 and LEU1 mutant alleles are recessive and produce cycloheximide-resistant and leucine auxotrophic phenotypes. The recessive mutant alleles of these three genes are situated on one copy of chromosome VII; ADE6 is located on one side of the centromere and CYH2 and LEU1 are located on the other side of the centromere. Because the homologous copy of chromosome VII carries dominant wild-type alleles of these 3 genes, diploid strain D61.M cells are cycloheximide-sensitive, form red colonies, and are leucine-independent. Loss of the copy of chromosome VII carrying the 3 wild-type alleles permits expression of the recessive alleles; such cells are cycloheximideresistant, form white colonies and are leucine-dependent. Simultaneous expression of the 3 recessive alleles in the same clone is necessary and sufficient evidence to conclude chromosome loss in strain D61.M (Mayer and Goin, 1989). The two haploid mating-type tester strains both have a mutant ADEI allele that confers a requirement of adenine for growth and red colonies. Because ADE1 mutants complement ADE2 and ADE6 mutants, mating with these test strains can be detected by clones that form white colonies that are adenine-independent.
Media and chemicals The standard 1% yeast extract, 2% peptone, 2% glucose medium (YEPD) was used for propagation of the cultures. YEPD medium containing 1.5 ppm cycloheximide (U.S. Biochemicals, Cleveland, OH) was used to detect resistance to this drug. The synthetic complete medium (SC) used was previously described (Zimmermann, 1984). Omission medium, used to detect the auxotrophic phenotypes of ADE6 and LEU1, consisted of SC medium from which adenine or leucine, respectively, was omitted. Sporulation medium was previously described (Sherman et al., 1986). All media were solidified as necessary by using 2% agar.
Thin agar slabs used for ascus dissections were made with 4% agar in water. Isolation of mating diploids Mating isolates from strain D61.M were identified by the method of marker recovery (Hawthorne, 1963; Gerlach, 1974; Hooper and Hall, 1975; Paquin and Adams, 1982). Suspensions of D61.M cells were diluted and plated on YEPD to produce approximately 100 colonies per plate 2 days after incubation at 28 ° C. Such plates were used as master plates for replica-plating onto YEPD plates containing a lawn of one or the other mating-type tester strain. Approximately 100 of these plates, containing the test colonies and lawn of mating-type tester strain, were incubated overnight and replica-plated onto SC medium lacking adenine. Only those cells that mated could grow into colonies on adenine omission medium as a result of complementation of the ADE1 wild-type alleles in strain D61.M and the ADE2 wild-type allele carried in the haploid M A T tester strains. Such colonies, identified by their ability to mate and grow on the adenine omission medium, were taken from their position on the original master plates, streaked on YEPD and retested for their mating response and for their inability to sporulate. Strain D61.M is a heterothallic diploid and therefore mates only rarely but sporulates with very high efficiency. Several isolates, obtained in the manner described, mated as either MATa or MATa. They were seen, upon careful inspection of suspensions, to sporulate at less than 1% efficiency, consistent with previous observations by others (Zakarov and Kozina, 1967; Hooper et al., 1974). Since these isolates were obtained without treatments which might enhance the frequency of mating-type switching, it was assumed at this point that all markers of interest on chromosome VII were in the same configuration as in the parent D61.M strain. Construction of tetraploids and triploids All matings, ascus dissections and spore clone analyses were carded out by standard techniques (Sherman et al., 1986). A tetraploid, with two chromosome VII's carrying wild-type dominant alleles of CYH2, LEU1 and ADE6 and two chromosome VII's carrying mutant recessive alleles of
180 these 3 genes, was made by crossing diploids of opposite mating type. Sporulation and dissection of this tetraploid resulted in diploid spore progeny, some of which were capable of mating and carried the desired configuration of markers on chromosome VII. Selected diploids were then crossed to create the desired tetraploid strains VM3, VM4 and VM5 (Table 1). Strain VM3 ( + / - / - / - ) contained one chromosome VII carrying wild-type alleles and three chromosome V I I ' s carrying recessive alleles for all marker genes. Strain VM4 ( + / + / - / - ) contained two wild-type and two recessive chromosome VII's, and VM5 ( + / + / + / - ) had three wild-type and one recessive chromosome VII's. These tetraploid strains were sporulated and asci dissected to show that all markers on chromosome VII segregated as expected for tetraploids containing the introduced marker configuration (Roman et al., 1955). Additional sporulation and ascus dissections of tetraploid spore clones, in which all 4 spore clones sporulated and did not mate, were conducted to further verify the desired marker configuration. The triploids VM1 and VM2 (Table 1) were constructed in a similar manner by crossing mating diploids with haploids obtained from ascus dissections of strain D61.M. Thus strain VM1 (+/-/-) contained one wild-type and two recessive chromosome V I I ' s and VM2 ( + / + / - ) contained two wild-type and one recessive chromosome VII's. Sporulation and ascus dissections of these triploids resulted in high spore lethality typical of triploids (Pomper et al., 1954; Campbell et al., 1981) and segregation of the markers in viable spores consistent with the desired marker configuration of each strain. Determination of spontaneous chromosome loss frequencies Yeast cultures used to determine spontaneous chromosome VII loss were grown in Y E P D medium and then suspended in sterile water at appropriate cell concentrations. Aliquots of the cell suspensions were plated on YEPD medium containing 1.5 ppm cycloheximide and were incubated for 5-7 days; only red or white cycloheximide-resistant colonies grew on these plates. Cycloheximide-resistant white colonies were counted, streaked onto YEPD master plates, in-
cubated overnight, and the master plates were replica-plated onto SC and SC without leucine for identification and enumeration of resistant white colonies expressing leul. Colonies growing on cycloheximide-YEPD that were white due to petite mutation rather than expression of ade6 were identified by observing whether they turned red or pink on SC medium. The majority of those colonies that turn red or pink on SC medium fail to grow on a yeast extract peptone glycerol medium (Mayer et al., 1986). The viable cell population plated on the selective cycloheximideYEPD medium was determined from colony counts on Y E P D plates seeded with dilutions of the cell suspension. Results
The frequencies of observed chromosome loss in the diploid, two triploid and three tetraploid strains are shown in Table 2. The range is quite broad among the strains, encompassing four orders of magnitude from 10 - 4 to 10 - 8 , and appears to be related to the genetic constitution of the particular strain. Among the tetraploid series, the highest frequency (3.99 X 10 - 4 ) is seen in the strain (VM3) in which the loss of only one wildtype chromosome VII is detected. The next highest frequency (1.52 × 10 - 6 ) is seen in that tetraploid, VM4, requiring the loss of at least the two wildtype chromosome VII's. The lowest frequency in this group is seen in VM5 (3.70 x 10-8), in which three wild-type chromosome V I I ' s must be lost to be recorded as a loss event. A similar relationship is also apparent for the two triploid strains. Triploid VM1, which must lose one wild-type chromosome VII, shows a frequency of 1.42 x 10 -5 and triploid VM2, which must lose two wild-type chromosome VII's, is detected at a frequency of 2.15 × 10 -8 There are, of course, chromosome VII loss combinations in each strain that will not be detected by our selection protocol, which restricts detection to loss of at least all chromosome V I I ' s bearing wild-type alleles. If a chromosome VII carrying the recessive alleles is lost, it will not be detected; neither will loss of fewer than the requisite number of wild-type chromosomes be detected. We can, however, calculate the theoretical
181 TABLE 2 OBSERVED F R E Q U E N C I E S OF EXPRESSION OF CHROM O S O M E VII LOSS IN A D I P L O I D , TWO T R I P L O I D A N D T H R E E T E T R A P L O I D STRAINS OF Saccharornyces
cerevisiae Ploidy (genotype)
Population screened
Number of cyh2,
leul, ade6
Frequency of chromosome loss
colonies Diploid D61.M a
9.55 X 108
724
7.58 X 1 0 - 7
6.07 × 106
86
1.42 x 10 - 5
2.00 x 108
43
2.15 x 1 0 - s
2.28 X 105
909
3.99 X 1 0 - 4
7.41 x 10 s
1128
1.52 x 10 - 6
6.27 × 109
232
3.70 × 1 0 - 8
(+/-) Triploid VM1 b
(+/-/-) Triploid VM2 c
(+/+/-) Tetraploid VM3 d
(+/-/-/-) Tetraploid VM4 e
(+/+/-/-) Tetraploid VM5 f
(+/+/+/-) + = Chromosome VII carrying wild-type dominant alleles; - = chromosome VII carrying mutant recessive alleles. a Arithmetic mean of more than 100 determinations. b Arithmetic mean of 9 determination. c Arithmetic mean of 15 determinations. d Arithmetic mean of 7 determinations. e Arithmetic mean of 21 determinations. r Arithmetic mean of 8 determinations.
frequency of loss of any combination of chromosome V I I 's, based upon the observed frequency of such events (fi in Table 3). In the diploid D61.M strain there are two copies of chromosome VII, either one of which could be lost (but not both). However, only loss of the chromosome carrying the three wild-type alleles will be scored as chromosome loss. Therefore the actual frequency of loss is twice the observed frequency (2 × 7.58 × 10-7 = f l = 1.52 × 10-6). Similarly, in triploid VM1 only one-third of the possible loss of a single chromosome can be observed, so the theoretical frequency of loss is 3 × 1.42 × 10 -5 = f l = 4.26 × 10 -5. In triploid strain VM1, however, a second chromosome V I I could be lost, yet the phenotype would be the same as for the loss of the single wild-type chromosome VII. We can calculate the expected frequency of this event by considering the behavior of strains in which loss of two copies of chromosome V I I can be detected. In triploid strain VM2 there are three possible ways in which two copies of chromosome VII can be lost, only one of which can be detected. Thus the theoretical frequency for the loss of any two copies is three times the observed frequency: 3 × 2.15 × 10 -8 = f2 = 6.45 × 10 -8. Using this expected frequency (6.45 x 10 -8) as the value for undetected loss of two copies in triploid VM1, we can conclude that the contribution of this event is minimal. As stated above, in the case of triploid VM2, only one of
TABLE 3 C A L C U L A T I O N S O F S I N G L E C H R O M O S O M E VII LOSS EVENTS F R O M T H E O B S E R V E D F R E Q U E N C I E S IN A D I P L O I D , T WO T R I P L O I D A N D T H R E E T E T R A P L O I D STR A IN S O F Saccharomyces cereoisiae Diploid
Triploid VM1
Triploid VM2
Tetraploid VM3
Tetraploid VM4
Tetraploid VM5
(+/-)
(+/-/-)
(+/+/-)
(+/-/-/-)
(+/+/-/-)
(+/+/+/-)
Observed
7.85 x 10 -7
1.42×10 - s
2 . 1 5 x 1 0 -8
3.99x10 -4
1 . 5 2 x 1 0 -6
3.70x10 -s
fi a 1 2
1 . 5 2 × 1 0 -6 -
4.26X10 -5 -
6.45 X 10 - s
1 . 6 0 × 1 0 -3
9.12 X 10 -6
-
3
.
.
.
.
.
1.48 X 10-
7
F b
1.52X 1 0 - 6
4.26 X 10-5
2.54X 1 0 - 4
1.60 X 10-3
3.02X 1 0 - 3
5.29 X 10_3
Fold increase
1X
28 ×
167 x
1050 X
1987 X
3480 x
+ = Chromosome VII carrying wild-type dominant alleles; - = chromosome VII carrying m u t a n t recessive alleles. a f i = Calculated frequency of loss of i chromosome V I l ' s (i =1, 2, 3). b F = Calculated frequency of loss of a single chromosome VII.
182 three possible two-copy losses can be detected. The other two events as well as the loss of one copy cannot be detected. Similar relationships can be calculated for each of the tetraploid strains. In tetraploid VM3 there are 4 possible ways to lose one copy of chromosome VII, only one of which can be detected. As in the case of triploid VM1, loss of additional chromosome V I I ' s in strain VM3 occurs at expected frequencies that would not contribute measurably to the total frequency. A similar consideration of the observed frequencies of chromosome VII loss in tetraploid strains VM4 and VM5 results in expected frequencies of loss of 6 x 1.52 x 10 - 6 = f 2 = 9.12 x 10 - 6 and 4 x 3.70 x 10 -8 =f3 = 1.48 x 10 -7. By using the expected values obtained for f~ as described above, the probability of losing a single copy of chromosome VII can now be calculated ( F in Table 3) for each of the strains. For the strains that are designed to detect the loss of only one chromosome VII (D61.M, VM1, VM3), the expected values ( F ) are the same as for fi. In the cases of strains VM2 and VM4, in which the loss of two chromosome V I I ' s is detected, the probability of losing one copy ( F ) is the square root of the frequency calculated for fi, assuming that the loss of any particular pair is random. In the case of tetraploid VM5, which detects the loss of three chromosome VII's, the probability of losing one copy ( F ) is the cube root of the value for fi- From observation of the values calculated for F in Table 3 for single chromosome VII loss in strains D61.M (diploid), VM1 (triploid) and VM3 (tetraploid), it becomes immediately apparent that the expected frequency of loss increases with ploidy. The frequency for loss in triploid VM1 is approximately 30-fold higher and in tetraploid VM3 is over 1000-fold higher than in the diploid. A second set of observations can be made if we assume that the frequency at which the first chromosome VII is lost is consistent for strains of the same ploidy. Loss of the first chromosome therefore would be at frequencies of 4.26 x 10 -5 for both triploids and 1.60 x 10-3 for the three tetraploids. According to this assumption of consistency, the probability at which a second chromosome VII is lost can be calculated from the fi values obtained from strains VM2 and VM4 since fl x f of the second lost chromosome = f z- The
calculated value for the second chromosome VII loss in the triploid VM2 is therefore f z / f l = 6.45 x 1 0 - 8 / 4 . 2 6 x 1 0 - 5 = 1 . 5 1 x 1 0 -3 , a value approximately 35-fold higher than the calculated frequency for loss of the first chromosome VII. Calculation of the frequency of loss of a second chromosome VII in tetraploid VM4 gives 5.70 x 10 3, a value that is nearly 4-fold higher than the calculated frequency for loss of the first chromosome VII in strain VM3. Similar consideration of the calculated frequency for a single chromosome VII loss in tetraploid strain VM5 indicates an approximate 10-fold increase in frequency of loss of the third copy of chromosome VII over the loss of the first. These results and calculations indicate that when a single chromosome is lost there is an increased probability that additional chromosomes will be lost, whether or not additional chromosomes are lost as a result of independent events or a single multiple chromosome loss event. The involvement of multiple chromosomes in genome instability is further suggested by our observations of M A T locus (chromosome I I I ) expression. A m o n g 302 chromosome VII-loss clones from diploid D61.M, 11 mated (3.6%), implying that these clones had also lost one of the two copies of chromosome III. In contrast we found, among 229 chromosome VIIloss clones from tetraploid VM4, 60 that mated (26%). Discussion In this study we have shown that chromosome loss in Saccharomyces cerevisiae increases dramatically as ploidy increases. Although our strains are designed specifically to detect loss of chromosome VII, we believe that this phenomenon should also apply to other chromosomes. A direct demonstration in support of this conclusion, however, awaits further experimentation. We have determined the frequency at which a genetically marked chromosome VII is lost from diploid, triploid and tetraploid strains. Since we determined chromosome loss by screening for colonies that expressed the recessive mutant alleles of ADE6, CYH2 and LEU1, it was important to show that mitotic recombination did not significantly contribute to the origin of this phenotype,
183 especially since coincident gene conversion covering large stretches of DNA occurs in this region of chromosome VII during mitosis (Golin and Esposito, 1984; Golin and Tampe, 1988). The contribution of mitotic recombination to simultaneous expression of all three recessive alleles was shown to be less than 5% of the diploid colonies screened (Mayer and Goin, 1989) and considerably less than that in triploids and tetraploids (V.W. Mayer, unpublished data). We therefore feel reasonably certain that the phenotype scored represents chromosome loss, even at higher ploidy. Our observations on genome instability reported in this communication are restricted to mitotic cells; however, there is reason to believe that polyploid meiotic cells might yield similar results. It is well documented that meiosis in triploids leads to high levels of aneuploid ascospores (Parry and Cox, 1970; Campbell et al., 1981). Aberrant tetrads have also been reported to be present among the meiotic products of tetraploid yeast strains (Pomper et al., 1954; Roman et al., 1955). Among 1000 ascospore clones produced by sporulation of tetraploid strains that were triplex at several loci, 64 suspected monosomics were found (Bruenn and Mortimer, 1970). Monosomy could be verified for some of the clones. Malsegregation of chromosome 111 is suggested by our own data for ascospore clones from tetraploid meiosis, which showed elevated numbers of asci that deviated from the expected pattern at the M A T locus. Among 394 four-spore viable diploid asci, only two spore clones failed to mate (0.5%); all others showed the expected 2 : 2 segregation for M A T . Among 884 four-spore viable tetraploid asci, 34 (3.9%) presumed diploid ascospore clones deviated from the expected 4 : 0 , 0 : 4 or 2 : 2 (mating:nonmating) phenotypes. These results suggest that aneuploidy may not be uncommon during polyploid meiosis. Our observation that if one chromosome is lost, additional chromosome V I I ' s are lost at increasingly higher frequencies may be interpreted in two ways: (1) Either various aneuploidy states (3N - 1, 3 N - 2, 4 N - 1, 4 N - 2, 4 N - 3) lead to greater genome instability, (2) or multiple chromosome loss events occur at high frequency. At this point we cannot distinguish between these possibilities, but we believe that greater genome instability is
conferred not only to the homologs of chromosome VII that we can measure but also to all chromosomes. If chromosome loss in the tetraploid were independently affecting each set of four homologs, then once two chromosomes had been lost, the frequency of further loss of chromosome VII would be expected to be close to the value found for the diploid strain. Some evidence is available that supports our conclusion that chromosome loss is not specifically restricted to a single chromosome or its homologs. Parry and Cox (1970) found a high tolerance of aneuploidy among ascospores from triploids and that disomy of only a single chromosome was rare. In other studies (Campbell et al., 1981), multiple disomy was found among ascospore clones from triploids. Deviations from a random distribution among the involved chromosomes were thought to be the result of differential ability to proliferate because of their variable burdens of multiple disomy. The aneuploidy observed in a diploid homozygous for a mutant RAD52 gene was not restricted to any particular chromosome pair (Mortimer et al., 1981). Diploid strains treated with methyl benzimidazol-2-yl-carbamate (MBC) lost different chromosomes with similar frequencies (Wood, 1982), and multiple chromosome loss occurred at frequencies higher than expected. Zimmermann et al. (1985) reported evidence for multiple aneuploidy in diploid D61.M cells treated with ethyl acetate. The causes for chromosome instability in polyploid yeast are unknown. Overexpression of tubulin genes leads to aneuploidy (Burke et al., 1989). Agents such as MBC (Quinlan et al., 1980; Kilmartin, 1981; Wood and Hartwell, 1982) or nocodazole (Hoebeke et al., 1976) that interact with tubulin or microtubules to block the cell cycle at a critical point before cytokinesis also induce aneuploidy (Wood, 1982; Zimmermann et al., 1984). Very little is known about higher ploidy with regard to the effects of malfunctional genes or failure of structural elements on genome stability. A normal synaptonemal complex has been shown to occur in tetraploid yeast (Byers and Goetsch, 1975). Electron microscopic studies in haploid and diploid yeast cells reveal that a single microtubule is attached to each chromosome (Peterson and Ris, 1976), unlike larger eukaryotic
184
organisms in which many microtubules attach to each chromosome. Perhaps some critical components of the spindle apparatus, which ensure the high segregational fidelity observed in haploids and diploids, become limiting as ploidy increases. Support for this concept has been reported (Futcher and Carbon, 1986) in that excess copies of CEN-bearing plasmids lead to cellular toxicity, destabilization of the plasmids, and an increase in the loss frequency of chromosome III. Recently, similar experiments by M.A. Resnick (personal communication) indicated that haploid yeast tolerated a 30-50% increase in CEN-bearing plasmids, while diploids tolerated only 3-6%. Triploid and tetraploid strains were found to be even less tolerant of extra centromeric plasmids. Other possible reasons for genome instability at higher ploidy include improper chromosome pairing, improper microtubule function, failure to replicate the entire genome or an imbalance of critical gene products. Regrettably there is little direct information bearing on these points. In plants (Huskins, 1934; Janiki-Ammal, 1940; Lewis et al., 1971), animals (Danes, 1986), and the pathogenic yeast Candida albicans (Whelan et al., 1985), subpopulations of cells with a number of chromosomes lower than those of their predecessors have been found. These findings together with our results suggest that polyploidy is unstable in nature. The inference that several salmonid fish species were derived from a tetraploid ancestor suggests that polyploidization may play an important role in evolution (Ohno, 1970; Johnson et al., 1987). Polyploid instabilities, together with chromosome rearrangements (White, 1969), might be an important factor to be considered in the early stages of the origination of new species. The large number of different species described for the imperfect yeast Candida (Kreger-Van Rij, 1984), together with the absence of a sexual cycle and the presence of different chromosome numbers, is consistent with an interpretation that polyploidization could be, in some cases, a transient stage in the generation of diversity among yeasts. Acknowledgement We thank Kimberly Anne Rau for her excellent technical support, especially for her diligence and care in much of the ascus dissection work.
References Becak, M.L., W. Becak and M.N. Rabello (1966) Cytological evidence of constant tetraploidy in the bisexual South American frog Odontophrynus americanus, Chromosoma, 19, 188-193. Becak, M.L., W. Becak and M.N. Rabello (1967) Further studies on polyploid amphibians (Ceratophrydidae), I. Mitotic and meiotic aspects, Chromosoma, 22, 192-201. Bogart, J.P., and L.E. Licht (1986) Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma, Can. J. Genet. Cytol., 28, 605-617. Bohman, R., C.T. Tamura, M.H. Doolittle and J. Cascarano (1985) Growth and aging in the rat: changes in total protein, cellularity and polyploidy in various organs, J. Exp. Zool., 233, 385-396. Bosemark, N.O. (1956) On accessory chromosomes in Festuca pratensis, III. Frequency and geographical distribution of plants with accessory chromosomes, Hereditas, 42, 189-210. Bruenn, J., and R.K. Mortimer (1970) Isolation of monosomics in yeast, J. Bacteriol., 102, 548-551. Burke, D., P. Gasdaska and L. Hartwell (1989) Dominant effects of tubulin overexpression in Saccharomyces cerevisiae, Mol. Cell. Biol., 9, 1049-1059. Byers, B., and L. Goetsch (1975) Electron microscopic observations on the meiotic karyotype of diploid and tetraploid Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5056-5060. Campbell, D., J.S. Doctor, J.H. Feuersanger and M.M. Doolittle (1981) Differential mitotic stability of yeast disomes derived from triploid meiosis, Genetics, 98, 239-255. Danes, B.S. (1986) Chromosome instability: a biological significance of in vitro hyperdiploidy in heritable single tumors, Medical Hypothesis, 19, 367-370. Darlington, C.D. (1953) Polyploidy in animals, Origin of polyploidy, Nature (London), 171, 191-194. Dowrick, G.J. (1952) The chromosomes of Chrysanthemum, I. The species, Heredity, 6, 365-375. Einset, J. (1948) The occurrence of spontaneous triploid and tetraploids in apples, Proc. Am. Soc. Hort. Sci., 51, 61-63. Fankhauser, G., and R.R. Humphrey (1959) The origin of spontaneous heteroploids in the progeny of diploid, triploid and tetraploid axolotl females, J. Exp. Zool., 142, 379-422. Frost, S. (1958) Studies of the genetical effects of accessory chromosomes in Centaurea scabiosa, Hereditas, 44, 113-122. Futcher, B., and J. Carbon (1986) Toxic effects of excess cloned centromeres, Mol. Cell. Biol., 6, 2213-2222. Gerlach, W.L. (1974) Sporulation in mating type homozygotes of Saccharomyces cereoisiae, Heredity, 32, 241-249. Goldberg, I.D., E.M. Rosen, H.M. Shapiro, L.C. Zoler, K. Myrick, S.E. Levenson and L. Christenson (1984) Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta, Science, 226, 559-561. Golin, J.E., and M.E. Esposito (1984) Coincident gene conversion during mitosis in Saccharomyces, Genetics, 107. 355365. Golin, J..E., and H. Tampe (1988) Coincident recombination during mitosis in Saccharomyces: distance-dependent and -independent components, Genetics, 119, 541-547.
185 Hawthorne, D.C. (1963) A deletion in yeast and its bearing on the structure of the mating type locus, Genetics, 48, 17271729. Hoebeke, J., G. Van Nijen and M. De Brabander (1976) Interaction of oncodazole (R 17934), a new anti-tumoral drug, with rat brain tubulin, Biochem. Biophys. Res. Commun., 69, 319-324. Hooper, A.K., and B.D. Hall (1975) Mating type and sporulation in yeast, I. Mutations which alter mating-type control over sporulation, Genetics, 80, 41-59. Hooper, A.K., P.T. Magee, S.K. Welch, M. Friedman and B.D. Hall (1974) Macromolecule synthesis and breakdown in relation to sporulation and meiosis in yeast, J. Bacteriol., 119, 619-628. Hubbard, M.J., R.T. Poulter, P.A. Sullivan and M.G. Shepherd (1985) Characterization of a tetraploid derivative of Candida albicans ATCC 10261, J. Bacteriol., 161, 781-783. Huitin, M., A. Karlman, J. Lindsten and L. Tiepolo (1970) Aneuploidy and polyploidy in germ-line cells of the male Chinese hamster (Cricetulus griseus), Hereditas, 65, 197202. Humphrey, R., and G. Fankhauser (1949) Three generations of polyploids in Amblistomid salamanders, J. Hered., 40, 7-12. Huskins, C.L. (1934) Anomalous segregation of triploid tomato, Heredity, 25, 281-286. Janiki-Ammal, E.K. (1940) Chromosome diminution in a plant, Nature (London), 146, 839-840. Johnson, K.R., J.E. Wright Jr. and B. May (1987) Linkage relationships reflecting ancestral tetraploidy in Salmonid fish, Genetics, 116, 579-591. Kilmartin, J.V. (1981) Purification of yeast tubulin by self-assembly in vitro, Biochemistry, 20, 3629-3633. Kreger-Van Rij, N.J.W. (1984) The Yeast, A Taxonomic Study, 3rd Edn., Elsevier, Amsterdam. Lewis, W.H., R.L. Oliver and T.J. Luikart (1971) Multiple genotypes in individuals of Claytonia oirginica, Science, 172, 564-565. Mayer, V.W., and C.J. Goin (1989) Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae, Mutation Res., 224, 471-478. Mayer, V.W., C.J. Goin and F.K. Zimmermann (1986) Anew ploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae, Mutation Res., 160, 19-26. Mortimer, R.K., R. Contopoulou and D. Schild (1981) Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cereoisiae, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5778-5782. Muldal, S. (1952) The chromosomes of earthworms, I. The evolution of polyploidy, Heredity, 6, 55-76. Muntzing, A. (1948) Accessory chromosomes in Poa alpina, Heredity, 2, 49-61. Nowel, P.C. (1976) The clonal evolution of tumor cell populations, Science, 194, 23-28. Ohno, S. (1970) Evolution By Gene Duplication, Springer, New York. Ostergen, G., and S. Frost (1962) Elimination of accessory
chromosomes from the roots in Haplopappus gracillis, Hereditas, 48, 363-366. Paquin, C., and J. Adams (1982) Isolation of sets of a, a, a/a, a / a and a / a isogenic strains in Saccharomyces cerevisiae, Curt. Genet., 6, 21-24. Parry, E.M., and B.S. Cox (1970) The tolerance of anenploidy in yeast, Genet. Res. Camb., 16, 333-340. Pennock, L.A. (1965) Triploidy in parthenogenetic species of the telid lizard, genus Cnemidophorus, Science, 149, 539540. Peterson, J.B., and H. Ris (1976) Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae, J. Cell Sci., 22, 219-242. Pomper, S., K.M. Daniels and D.W. McKee (1954) Genetic analysis of polyploid yeast, Genetics, 39, 343-355. Quinlan, R.A., C.I. Pogson and K. Gull (1980) The influence of the microtubule inhibitor, methyl benzimidazole-2-yl carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae, J. Cell Sci., 46, 341-352. Rodenburg, C.J., C.J. Cornelisse, P.A.M. Heintz, J. Hermans and G.J. Fleuren (1987) Tumor ploidy as a major prognostic factor in advanced ovarian cancer, Cancer, 59, 317-323. Roman, H., and S.M. Sands (1953) Heterogeneity of clones of Saccharomyces derived from haploid ascospores, Proc. Natl. Acad. Sci. (U.S.A.), 39, 171-179. Roman, H., D.C. Hawthorne and H.C. Douglas (1951) Polyploidy in yeast and its beating on the occurrence of irregular genetic ratios, Proc. Natl. Acad. SCI. (U.S.A.), 37, 79-84. Roman, H., M.M. Phillips and S.M. Sands (1955) Studies of polyploid Saccharomyces, I. Tetraploid segregation, Genetics, 40, 546-561. Sachs, L. (1952) Polyploid evolution and mammalian chromosomes, Heredity, 6, 357-364. Schmid, M., T. Haaf and W. Schemp (1985) Chromosome banding in Amphibia, IX. The polyploid karyotypes of Odontophrynus americanus and Ceratophrys ornata (Anura, Leptodactylidae), Chromosoma, 91,172-184. Sharma, A.K., and H.R. Aiyanger (1961) Occurrence of Bchromosomes in diploid Allium stracheyi Baker and their elimination in polyploids, Chromosoma, 12, 310-317. Sherman, F., G.R. Fink and J.B. Hicks (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Snoad, B. (1955) Somatic instability of chromosome number in Himenocallis calathinum, Heredity, 9, 129-134. Suzuki, T., T. Kanbe, T. Kuroiwa and K. Tanaka (1986) Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans, J. Gen. Microbiol., 132, 443-453. Whelan, W.L., D.M. Markie, K.G. Simkin and R.M. Poulter (1985) Instability of Candida albicans hybrids, J. Bacteriol., 161, 1131-1136. White, M.J.D. (1969) Chromosomal rearrangements and speciation in animals, Annu. Rev. Genet., 3, 75-98. Wolf, U., H. Ritter, N.B. Atkin and S. Ohno (1969) Polyploidization in the fish family Cyprinidae, order Cypriniformes, I. DNA-content and chromosome sets in various species of Cyprinidae, Humangenetik, 7, 240-244.
186 Wood, J.S. (1982) Genetic effects of methyl benzimidazole-2-yl carbamate on Saccharomyces cerevisiae, Mol. Cell. Biol., 2, 1064-1078. Wood, J.S., and L.H. Hartwell (1982) A dependent pathway of gene functions leading to chromosome segregation in S. cerevisiae, J. Cell Biol., 94, 718-726. Zakharov, I.A., and T.N. Kozina (1967) The genetics of mating in yeast (Saccharomyces cerevisiae), Genetika, 11,110-115. Zimmermann, F.K. (1984) Basic principles and methods of genotoxicity testing in the yeast Saccharomyces cerevisiae, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, 2nd edn., Elsevier, Amsterdam.
Zimmermann, F.K., and I. Scheel (1984) Genetic effects of 5-azacytidine in Saccharomyces cerevisiae, Mutation Res., 139, 21-24. Zimmermann, F.K., V.W. Mayer and 1. Scheel (1984) Induction of aneuploidy by oncodazole (nocodazole), an antitubulin agent, and acetone, Mutation Res., 141, 15-18. Zimmermann, F.K., V.W. Mayer, I. Scheel and M.A. Resnick (1985) Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae, Mutation Res., 149, 339-351.
177
MUT 04880
High levels of chromosome instability in polyploids of Saccharomyces cerevisiae V e r n o n W. M a y e r a n d A n d r e s A g u i l e r a a Genetic Toxicology Branch, Division of Toxicological Studies, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC 20204 and a Biochemistry Department, New York University Medical Center, New York, N Y 10016 (U.S.A.) (Received 21 February 1990) (Accepted 27 February 1990)
Keywords: Chromosome loss; Polyploid yeast; Saccharomyces cerevisiae
Summary The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leul-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome I,'11's during mitosis. We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid. Loss of two or three copies occurred at an even greater frequency. These findings suggest either that aneuploidy (3N-l, 3N-2, 4 N - l , 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency. Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation. The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.
Although haploidy and diploidy are the more usual chromosomal constitutions found in nature, diploids bearing extra chromosomes (Muntzing, 1948) and polyploids are not uncommon. Differences in number of chromosomes among cells of an individual plant (Janiki-Ammal, 1940; Snoad, 1955; Frost, 1958; Ostergen and Frost, 1962; Lewis et al., 1971) and among different individual plants of the same species have been reported (Einset, 1948; Dowrick, 1952; Bosemark,
Correspondence: Dr. Vernon W. Mayer, Genetic Toxicology Branch, HFF-166, Food and Drug Administration, 200 C Street S.W., Washington, DC 20204 (U.S.A.).
1956; Sharma and Aiyanger, 1961). Sachs (1952) reported that two species of hamsters were diploid and a third species was apparently tetraploid. Polyploidy has been found among tumor cells (Nowel, 1976; Danes, 1986; Rodenburg et al., 1987) as well as in subpopulations of apparently normal cells in intact animals (Hultin et al., 1970; Goldberg et al., 1984; Bohman et al., 1985). Animals in which polyploidy has been reported include earthworms (Muldal, 1952), salamanders (Humphrey and Fankhauser, 1949; Fankhauser and Humphrey, 1959; Schmid et al., 1985; Bogart and Licht, 1986), frogs (Becak et al., 1966, 1967), lizards (Pennock, 1965) and salmonid fishes (Wolf et al., 1969; Johnson et al., 1987). Polyploid
0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
178
variants of unicellular uninuclear organisms have been reported in the yeast Saccharomyces (Roman et al., 1951; Pomper et al., 1954) and the pathogenic yeast Candida albicans (Hubbard et al., 1985; Suzuki et al., 1986). The finding of polyploid plants (Einset, 1948), animals (Darlington, 1953) and yeast (Roman and Sands, 1953) and the lack of constancy in chromosome number among such polyploids (JanikiAmmal, 1940; Snoad, 1955; Lewis et al., 1971) suggest that polyploidy might not be a genetically stable combination. These exceptions in nature may, however, play an important role in evolution and the development of new species. We have used the yeast Saccharomyces cerevisiae to study the genetic consequences of polyploidy in the asexual (mitotic) life cycle of a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII were constructed and used to follow the loss of one, two or
three chromosomes. With an increase in ploidy number, an increase in chromosome instability was found. Materials and methods
Yeast strains The diploid Saccharornyces cerevisiae strain D61.M and two haploid mating-type tester strains carrying an adel mutant allele were obtained from Dr. F.K. Zimmermann, Technische Hochschule, Darmstadt (F.R.G.). Strain D61.M, as described previously (Zimmermann and Scheel, 1984; Zimmermann et al., 1985; Mayer et al., 1986), was used as the parent for construction of the triploid and tetraploid strains. Genotypes of the parent strain and the different constructs are given in Table 1. Strain D61.M and all derivatives require adenine because they are homozygous for the ADE2 mutation on chromosome XV. Since strain D61.M is ADE6/ade6 heterozygous, derivatives
TABLE1 G E N O T Y P E O F STRAINS Strain Diploid D61.M (+ / - ) Tfiploid VM1 (+/-/-)
Chromosome VII + cyh2
Chromosome XV
+ leul
-0 0
+ ade6
ade2 ade2
+
+
cyh2 cyh2
leul leul
-0 0 0
+ ade6 ade6
ade2 ade2 ade2
+
+
-0
+
cyh2
leul
+
o 0
ade6
ade2 ade2 ade2
Tetraploid VM3 (+/-/-/-)
+ cyh2 cyh2 cyh2
+ leul leul leul
0 0 0 0
+ ade6 ade6 ade6
ade2 ade2 ade2 ade2
Tetraploid VM4
+
+
0
+
ade2 ade2 ade2 ade2
Triploid VM2
(+/+/-)
(+/+/-/-)
Tetraploid VM5
(+/+/+/-)
+
+
+
cyh2 cyh2
leul leul
+
0 0 0
ade6 ade6
+
+
0
+
+ +
+ +
0
+ +
leul
0 0
ade6
cyh2 0, position of centromere on chromosome VII.
+
ade2 ade2 ade2 ade2
179 containing the wild-type allele of ADE6 produce red colonies because the cells accumulate a redpigmented precursor of adenine. If only the mutant allele of ADE6 is present, the red pigment is not formed and colonies are white. Two other genes located on chromosome VII are also of interest in testing for chromosome loss; CYH2 and LEU1 mutant alleles are recessive and produce cycloheximide-resistant and leucine auxotrophic phenotypes. The recessive mutant alleles of these three genes are situated on one copy of chromosome VII; ADE6 is located on one side of the centromere and CYH2 and LEU1 are located on the other side of the centromere. Because the homologous copy of chromosome VII carries dominant wild-type alleles of these 3 genes, diploid strain D61.M cells are cycloheximide-sensitive, form red colonies, and are leucine-independent. Loss of the copy of chromosome VII carrying the 3 wild-type alleles permits expression of the recessive alleles; such cells are cycloheximideresistant, form white colonies and are leucine-dependent. Simultaneous expression of the 3 recessive alleles in the same clone is necessary and sufficient evidence to conclude chromosome loss in strain D61.M (Mayer and Goin, 1989). The two haploid mating-type tester strains both have a mutant ADEI allele that confers a requirement of adenine for growth and red colonies. Because ADE1 mutants complement ADE2 and ADE6 mutants, mating with these test strains can be detected by clones that form white colonies that are adenine-independent.
Media and chemicals The standard 1% yeast extract, 2% peptone, 2% glucose medium (YEPD) was used for propagation of the cultures. YEPD medium containing 1.5 ppm cycloheximide (U.S. Biochemicals, Cleveland, OH) was used to detect resistance to this drug. The synthetic complete medium (SC) used was previously described (Zimmermann, 1984). Omission medium, used to detect the auxotrophic phenotypes of ADE6 and LEU1, consisted of SC medium from which adenine or leucine, respectively, was omitted. Sporulation medium was previously described (Sherman et al., 1986). All media were solidified as necessary by using 2% agar.
Thin agar slabs used for ascus dissections were made with 4% agar in water. Isolation of mating diploids Mating isolates from strain D61.M were identified by the method of marker recovery (Hawthorne, 1963; Gerlach, 1974; Hooper and Hall, 1975; Paquin and Adams, 1982). Suspensions of D61.M cells were diluted and plated on YEPD to produce approximately 100 colonies per plate 2 days after incubation at 28 ° C. Such plates were used as master plates for replica-plating onto YEPD plates containing a lawn of one or the other mating-type tester strain. Approximately 100 of these plates, containing the test colonies and lawn of mating-type tester strain, were incubated overnight and replica-plated onto SC medium lacking adenine. Only those cells that mated could grow into colonies on adenine omission medium as a result of complementation of the ADE1 wild-type alleles in strain D61.M and the ADE2 wild-type allele carried in the haploid M A T tester strains. Such colonies, identified by their ability to mate and grow on the adenine omission medium, were taken from their position on the original master plates, streaked on YEPD and retested for their mating response and for their inability to sporulate. Strain D61.M is a heterothallic diploid and therefore mates only rarely but sporulates with very high efficiency. Several isolates, obtained in the manner described, mated as either MATa or MATa. They were seen, upon careful inspection of suspensions, to sporulate at less than 1% efficiency, consistent with previous observations by others (Zakarov and Kozina, 1967; Hooper et al., 1974). Since these isolates were obtained without treatments which might enhance the frequency of mating-type switching, it was assumed at this point that all markers of interest on chromosome VII were in the same configuration as in the parent D61.M strain. Construction of tetraploids and triploids All matings, ascus dissections and spore clone analyses were carded out by standard techniques (Sherman et al., 1986). A tetraploid, with two chromosome VII's carrying wild-type dominant alleles of CYH2, LEU1 and ADE6 and two chromosome VII's carrying mutant recessive alleles of
180 these 3 genes, was made by crossing diploids of opposite mating type. Sporulation and dissection of this tetraploid resulted in diploid spore progeny, some of which were capable of mating and carried the desired configuration of markers on chromosome VII. Selected diploids were then crossed to create the desired tetraploid strains VM3, VM4 and VM5 (Table 1). Strain VM3 ( + / - / - / - ) contained one chromosome VII carrying wild-type alleles and three chromosome V I I ' s carrying recessive alleles for all marker genes. Strain VM4 ( + / + / - / - ) contained two wild-type and two recessive chromosome VII's, and VM5 ( + / + / + / - ) had three wild-type and one recessive chromosome VII's. These tetraploid strains were sporulated and asci dissected to show that all markers on chromosome VII segregated as expected for tetraploids containing the introduced marker configuration (Roman et al., 1955). Additional sporulation and ascus dissections of tetraploid spore clones, in which all 4 spore clones sporulated and did not mate, were conducted to further verify the desired marker configuration. The triploids VM1 and VM2 (Table 1) were constructed in a similar manner by crossing mating diploids with haploids obtained from ascus dissections of strain D61.M. Thus strain VM1 (+/-/-) contained one wild-type and two recessive chromosome V I I ' s and VM2 ( + / + / - ) contained two wild-type and one recessive chromosome VII's. Sporulation and ascus dissections of these triploids resulted in high spore lethality typical of triploids (Pomper et al., 1954; Campbell et al., 1981) and segregation of the markers in viable spores consistent with the desired marker configuration of each strain. Determination of spontaneous chromosome loss frequencies Yeast cultures used to determine spontaneous chromosome VII loss were grown in Y E P D medium and then suspended in sterile water at appropriate cell concentrations. Aliquots of the cell suspensions were plated on YEPD medium containing 1.5 ppm cycloheximide and were incubated for 5-7 days; only red or white cycloheximide-resistant colonies grew on these plates. Cycloheximide-resistant white colonies were counted, streaked onto YEPD master plates, in-
cubated overnight, and the master plates were replica-plated onto SC and SC without leucine for identification and enumeration of resistant white colonies expressing leul. Colonies growing on cycloheximide-YEPD that were white due to petite mutation rather than expression of ade6 were identified by observing whether they turned red or pink on SC medium. The majority of those colonies that turn red or pink on SC medium fail to grow on a yeast extract peptone glycerol medium (Mayer et al., 1986). The viable cell population plated on the selective cycloheximideYEPD medium was determined from colony counts on Y E P D plates seeded with dilutions of the cell suspension. Results
The frequencies of observed chromosome loss in the diploid, two triploid and three tetraploid strains are shown in Table 2. The range is quite broad among the strains, encompassing four orders of magnitude from 10 - 4 to 10 - 8 , and appears to be related to the genetic constitution of the particular strain. Among the tetraploid series, the highest frequency (3.99 X 10 - 4 ) is seen in the strain (VM3) in which the loss of only one wildtype chromosome VII is detected. The next highest frequency (1.52 × 10 - 6 ) is seen in that tetraploid, VM4, requiring the loss of at least the two wildtype chromosome VII's. The lowest frequency in this group is seen in VM5 (3.70 x 10-8), in which three wild-type chromosome V I I ' s must be lost to be recorded as a loss event. A similar relationship is also apparent for the two triploid strains. Triploid VM1, which must lose one wild-type chromosome VII, shows a frequency of 1.42 x 10 -5 and triploid VM2, which must lose two wild-type chromosome VII's, is detected at a frequency of 2.15 × 10 -8 There are, of course, chromosome VII loss combinations in each strain that will not be detected by our selection protocol, which restricts detection to loss of at least all chromosome V I I ' s bearing wild-type alleles. If a chromosome VII carrying the recessive alleles is lost, it will not be detected; neither will loss of fewer than the requisite number of wild-type chromosomes be detected. We can, however, calculate the theoretical
181 TABLE 2 OBSERVED F R E Q U E N C I E S OF EXPRESSION OF CHROM O S O M E VII LOSS IN A D I P L O I D , TWO T R I P L O I D A N D T H R E E T E T R A P L O I D STRAINS OF Saccharornyces
cerevisiae Ploidy (genotype)
Population screened
Number of cyh2,
leul, ade6
Frequency of chromosome loss
colonies Diploid D61.M a
9.55 X 108
724
7.58 X 1 0 - 7
6.07 × 106
86
1.42 x 10 - 5
2.00 x 108
43
2.15 x 1 0 - s
2.28 X 105
909
3.99 X 1 0 - 4
7.41 x 10 s
1128
1.52 x 10 - 6
6.27 × 109
232
3.70 × 1 0 - 8
(+/-) Triploid VM1 b
(+/-/-) Triploid VM2 c
(+/+/-) Tetraploid VM3 d
(+/-/-/-) Tetraploid VM4 e
(+/+/-/-) Tetraploid VM5 f
(+/+/+/-) + = Chromosome VII carrying wild-type dominant alleles; - = chromosome VII carrying mutant recessive alleles. a Arithmetic mean of more than 100 determinations. b Arithmetic mean of 9 determination. c Arithmetic mean of 15 determinations. d Arithmetic mean of 7 determinations. e Arithmetic mean of 21 determinations. r Arithmetic mean of 8 determinations.
frequency of loss of any combination of chromosome V I I 's, based upon the observed frequency of such events (fi in Table 3). In the diploid D61.M strain there are two copies of chromosome VII, either one of which could be lost (but not both). However, only loss of the chromosome carrying the three wild-type alleles will be scored as chromosome loss. Therefore the actual frequency of loss is twice the observed frequency (2 × 7.58 × 10-7 = f l = 1.52 × 10-6). Similarly, in triploid VM1 only one-third of the possible loss of a single chromosome can be observed, so the theoretical frequency of loss is 3 × 1.42 × 10 -5 = f l = 4.26 × 10 -5. In triploid strain VM1, however, a second chromosome V I I could be lost, yet the phenotype would be the same as for the loss of the single wild-type chromosome VII. We can calculate the expected frequency of this event by considering the behavior of strains in which loss of two copies of chromosome V I I can be detected. In triploid strain VM2 there are three possible ways in which two copies of chromosome VII can be lost, only one of which can be detected. Thus the theoretical frequency for the loss of any two copies is three times the observed frequency: 3 × 2.15 × 10 -8 = f2 = 6.45 × 10 -8. Using this expected frequency (6.45 x 10 -8) as the value for undetected loss of two copies in triploid VM1, we can conclude that the contribution of this event is minimal. As stated above, in the case of triploid VM2, only one of
TABLE 3 C A L C U L A T I O N S O F S I N G L E C H R O M O S O M E VII LOSS EVENTS F R O M T H E O B S E R V E D F R E Q U E N C I E S IN A D I P L O I D , T WO T R I P L O I D A N D T H R E E T E T R A P L O I D STR A IN S O F Saccharomyces cereoisiae Diploid
Triploid VM1
Triploid VM2
Tetraploid VM3
Tetraploid VM4
Tetraploid VM5
(+/-)
(+/-/-)
(+/+/-)
(+/-/-/-)
(+/+/-/-)
(+/+/+/-)
Observed
7.85 x 10 -7
1.42×10 - s
2 . 1 5 x 1 0 -8
3.99x10 -4
1 . 5 2 x 1 0 -6
3.70x10 -s
fi a 1 2
1 . 5 2 × 1 0 -6 -
4.26X10 -5 -
6.45 X 10 - s
1 . 6 0 × 1 0 -3
9.12 X 10 -6
-
3
.
.
.
.
.
1.48 X 10-
7
F b
1.52X 1 0 - 6
4.26 X 10-5
2.54X 1 0 - 4
1.60 X 10-3
3.02X 1 0 - 3
5.29 X 10_3
Fold increase
1X
28 ×
167 x
1050 X
1987 X
3480 x
+ = Chromosome VII carrying wild-type dominant alleles; - = chromosome VII carrying m u t a n t recessive alleles. a f i = Calculated frequency of loss of i chromosome V I l ' s (i =1, 2, 3). b F = Calculated frequency of loss of a single chromosome VII.
182 three possible two-copy losses can be detected. The other two events as well as the loss of one copy cannot be detected. Similar relationships can be calculated for each of the tetraploid strains. In tetraploid VM3 there are 4 possible ways to lose one copy of chromosome VII, only one of which can be detected. As in the case of triploid VM1, loss of additional chromosome V I I ' s in strain VM3 occurs at expected frequencies that would not contribute measurably to the total frequency. A similar consideration of the observed frequencies of chromosome VII loss in tetraploid strains VM4 and VM5 results in expected frequencies of loss of 6 x 1.52 x 10 - 6 = f 2 = 9.12 x 10 - 6 and 4 x 3.70 x 10 -8 =f3 = 1.48 x 10 -7. By using the expected values obtained for f~ as described above, the probability of losing a single copy of chromosome VII can now be calculated ( F in Table 3) for each of the strains. For the strains that are designed to detect the loss of only one chromosome VII (D61.M, VM1, VM3), the expected values ( F ) are the same as for fi. In the cases of strains VM2 and VM4, in which the loss of two chromosome V I I ' s is detected, the probability of losing one copy ( F ) is the square root of the frequency calculated for fi, assuming that the loss of any particular pair is random. In the case of tetraploid VM5, which detects the loss of three chromosome VII's, the probability of losing one copy ( F ) is the cube root of the value for fi- From observation of the values calculated for F in Table 3 for single chromosome VII loss in strains D61.M (diploid), VM1 (triploid) and VM3 (tetraploid), it becomes immediately apparent that the expected frequency of loss increases with ploidy. The frequency for loss in triploid VM1 is approximately 30-fold higher and in tetraploid VM3 is over 1000-fold higher than in the diploid. A second set of observations can be made if we assume that the frequency at which the first chromosome VII is lost is consistent for strains of the same ploidy. Loss of the first chromosome therefore would be at frequencies of 4.26 x 10 -5 for both triploids and 1.60 x 10-3 for the three tetraploids. According to this assumption of consistency, the probability at which a second chromosome VII is lost can be calculated from the fi values obtained from strains VM2 and VM4 since fl x f of the second lost chromosome = f z- The
calculated value for the second chromosome VII loss in the triploid VM2 is therefore f z / f l = 6.45 x 1 0 - 8 / 4 . 2 6 x 1 0 - 5 = 1 . 5 1 x 1 0 -3 , a value approximately 35-fold higher than the calculated frequency for loss of the first chromosome VII. Calculation of the frequency of loss of a second chromosome VII in tetraploid VM4 gives 5.70 x 10 3, a value that is nearly 4-fold higher than the calculated frequency for loss of the first chromosome VII in strain VM3. Similar consideration of the calculated frequency for a single chromosome VII loss in tetraploid strain VM5 indicates an approximate 10-fold increase in frequency of loss of the third copy of chromosome VII over the loss of the first. These results and calculations indicate that when a single chromosome is lost there is an increased probability that additional chromosomes will be lost, whether or not additional chromosomes are lost as a result of independent events or a single multiple chromosome loss event. The involvement of multiple chromosomes in genome instability is further suggested by our observations of M A T locus (chromosome I I I ) expression. A m o n g 302 chromosome VII-loss clones from diploid D61.M, 11 mated (3.6%), implying that these clones had also lost one of the two copies of chromosome III. In contrast we found, among 229 chromosome VIIloss clones from tetraploid VM4, 60 that mated (26%). Discussion In this study we have shown that chromosome loss in Saccharomyces cerevisiae increases dramatically as ploidy increases. Although our strains are designed specifically to detect loss of chromosome VII, we believe that this phenomenon should also apply to other chromosomes. A direct demonstration in support of this conclusion, however, awaits further experimentation. We have determined the frequency at which a genetically marked chromosome VII is lost from diploid, triploid and tetraploid strains. Since we determined chromosome loss by screening for colonies that expressed the recessive mutant alleles of ADE6, CYH2 and LEU1, it was important to show that mitotic recombination did not significantly contribute to the origin of this phenotype,
183 especially since coincident gene conversion covering large stretches of DNA occurs in this region of chromosome VII during mitosis (Golin and Esposito, 1984; Golin and Tampe, 1988). The contribution of mitotic recombination to simultaneous expression of all three recessive alleles was shown to be less than 5% of the diploid colonies screened (Mayer and Goin, 1989) and considerably less than that in triploids and tetraploids (V.W. Mayer, unpublished data). We therefore feel reasonably certain that the phenotype scored represents chromosome loss, even at higher ploidy. Our observations on genome instability reported in this communication are restricted to mitotic cells; however, there is reason to believe that polyploid meiotic cells might yield similar results. It is well documented that meiosis in triploids leads to high levels of aneuploid ascospores (Parry and Cox, 1970; Campbell et al., 1981). Aberrant tetrads have also been reported to be present among the meiotic products of tetraploid yeast strains (Pomper et al., 1954; Roman et al., 1955). Among 1000 ascospore clones produced by sporulation of tetraploid strains that were triplex at several loci, 64 suspected monosomics were found (Bruenn and Mortimer, 1970). Monosomy could be verified for some of the clones. Malsegregation of chromosome 111 is suggested by our own data for ascospore clones from tetraploid meiosis, which showed elevated numbers of asci that deviated from the expected pattern at the M A T locus. Among 394 four-spore viable diploid asci, only two spore clones failed to mate (0.5%); all others showed the expected 2 : 2 segregation for M A T . Among 884 four-spore viable tetraploid asci, 34 (3.9%) presumed diploid ascospore clones deviated from the expected 4 : 0 , 0 : 4 or 2 : 2 (mating:nonmating) phenotypes. These results suggest that aneuploidy may not be uncommon during polyploid meiosis. Our observation that if one chromosome is lost, additional chromosome V I I ' s are lost at increasingly higher frequencies may be interpreted in two ways: (1) Either various aneuploidy states (3N - 1, 3 N - 2, 4 N - 1, 4 N - 2, 4 N - 3) lead to greater genome instability, (2) or multiple chromosome loss events occur at high frequency. At this point we cannot distinguish between these possibilities, but we believe that greater genome instability is
conferred not only to the homologs of chromosome VII that we can measure but also to all chromosomes. If chromosome loss in the tetraploid were independently affecting each set of four homologs, then once two chromosomes had been lost, the frequency of further loss of chromosome VII would be expected to be close to the value found for the diploid strain. Some evidence is available that supports our conclusion that chromosome loss is not specifically restricted to a single chromosome or its homologs. Parry and Cox (1970) found a high tolerance of aneuploidy among ascospores from triploids and that disomy of only a single chromosome was rare. In other studies (Campbell et al., 1981), multiple disomy was found among ascospore clones from triploids. Deviations from a random distribution among the involved chromosomes were thought to be the result of differential ability to proliferate because of their variable burdens of multiple disomy. The aneuploidy observed in a diploid homozygous for a mutant RAD52 gene was not restricted to any particular chromosome pair (Mortimer et al., 1981). Diploid strains treated with methyl benzimidazol-2-yl-carbamate (MBC) lost different chromosomes with similar frequencies (Wood, 1982), and multiple chromosome loss occurred at frequencies higher than expected. Zimmermann et al. (1985) reported evidence for multiple aneuploidy in diploid D61.M cells treated with ethyl acetate. The causes for chromosome instability in polyploid yeast are unknown. Overexpression of tubulin genes leads to aneuploidy (Burke et al., 1989). Agents such as MBC (Quinlan et al., 1980; Kilmartin, 1981; Wood and Hartwell, 1982) or nocodazole (Hoebeke et al., 1976) that interact with tubulin or microtubules to block the cell cycle at a critical point before cytokinesis also induce aneuploidy (Wood, 1982; Zimmermann et al., 1984). Very little is known about higher ploidy with regard to the effects of malfunctional genes or failure of structural elements on genome stability. A normal synaptonemal complex has been shown to occur in tetraploid yeast (Byers and Goetsch, 1975). Electron microscopic studies in haploid and diploid yeast cells reveal that a single microtubule is attached to each chromosome (Peterson and Ris, 1976), unlike larger eukaryotic
184
organisms in which many microtubules attach to each chromosome. Perhaps some critical components of the spindle apparatus, which ensure the high segregational fidelity observed in haploids and diploids, become limiting as ploidy increases. Support for this concept has been reported (Futcher and Carbon, 1986) in that excess copies of CEN-bearing plasmids lead to cellular toxicity, destabilization of the plasmids, and an increase in the loss frequency of chromosome III. Recently, similar experiments by M.A. Resnick (personal communication) indicated that haploid yeast tolerated a 30-50% increase in CEN-bearing plasmids, while diploids tolerated only 3-6%. Triploid and tetraploid strains were found to be even less tolerant of extra centromeric plasmids. Other possible reasons for genome instability at higher ploidy include improper chromosome pairing, improper microtubule function, failure to replicate the entire genome or an imbalance of critical gene products. Regrettably there is little direct information bearing on these points. In plants (Huskins, 1934; Janiki-Ammal, 1940; Lewis et al., 1971), animals (Danes, 1986), and the pathogenic yeast Candida albicans (Whelan et al., 1985), subpopulations of cells with a number of chromosomes lower than those of their predecessors have been found. These findings together with our results suggest that polyploidy is unstable in nature. The inference that several salmonid fish species were derived from a tetraploid ancestor suggests that polyploidization may play an important role in evolution (Ohno, 1970; Johnson et al., 1987). Polyploid instabilities, together with chromosome rearrangements (White, 1969), might be an important factor to be considered in the early stages of the origination of new species. The large number of different species described for the imperfect yeast Candida (Kreger-Van Rij, 1984), together with the absence of a sexual cycle and the presence of different chromosome numbers, is consistent with an interpretation that polyploidization could be, in some cases, a transient stage in the generation of diversity among yeasts. Acknowledgement We thank Kimberly Anne Rau for her excellent technical support, especially for her diligence and care in much of the ascus dissection work.
References Becak, M.L., W. Becak and M.N. Rabello (1966) Cytological evidence of constant tetraploidy in the bisexual South American frog Odontophrynus americanus, Chromosoma, 19, 188-193. Becak, M.L., W. Becak and M.N. Rabello (1967) Further studies on polyploid amphibians (Ceratophrydidae), I. Mitotic and meiotic aspects, Chromosoma, 22, 192-201. Bogart, J.P., and L.E. Licht (1986) Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma, Can. J. Genet. Cytol., 28, 605-617. Bohman, R., C.T. Tamura, M.H. Doolittle and J. Cascarano (1985) Growth and aging in the rat: changes in total protein, cellularity and polyploidy in various organs, J. Exp. Zool., 233, 385-396. Bosemark, N.O. (1956) On accessory chromosomes in Festuca pratensis, III. Frequency and geographical distribution of plants with accessory chromosomes, Hereditas, 42, 189-210. Bruenn, J., and R.K. Mortimer (1970) Isolation of monosomics in yeast, J. Bacteriol., 102, 548-551. Burke, D., P. Gasdaska and L. Hartwell (1989) Dominant effects of tubulin overexpression in Saccharomyces cerevisiae, Mol. Cell. Biol., 9, 1049-1059. Byers, B., and L. Goetsch (1975) Electron microscopic observations on the meiotic karyotype of diploid and tetraploid Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5056-5060. Campbell, D., J.S. Doctor, J.H. Feuersanger and M.M. Doolittle (1981) Differential mitotic stability of yeast disomes derived from triploid meiosis, Genetics, 98, 239-255. Danes, B.S. (1986) Chromosome instability: a biological significance of in vitro hyperdiploidy in heritable single tumors, Medical Hypothesis, 19, 367-370. Darlington, C.D. (1953) Polyploidy in animals, Origin of polyploidy, Nature (London), 171, 191-194. Dowrick, G.J. (1952) The chromosomes of Chrysanthemum, I. The species, Heredity, 6, 365-375. Einset, J. (1948) The occurrence of spontaneous triploid and tetraploids in apples, Proc. Am. Soc. Hort. Sci., 51, 61-63. Fankhauser, G., and R.R. Humphrey (1959) The origin of spontaneous heteroploids in the progeny of diploid, triploid and tetraploid axolotl females, J. Exp. Zool., 142, 379-422. Frost, S. (1958) Studies of the genetical effects of accessory chromosomes in Centaurea scabiosa, Hereditas, 44, 113-122. Futcher, B., and J. Carbon (1986) Toxic effects of excess cloned centromeres, Mol. Cell. Biol., 6, 2213-2222. Gerlach, W.L. (1974) Sporulation in mating type homozygotes of Saccharomyces cereoisiae, Heredity, 32, 241-249. Goldberg, I.D., E.M. Rosen, H.M. Shapiro, L.C. Zoler, K. Myrick, S.E. Levenson and L. Christenson (1984) Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta, Science, 226, 559-561. Golin, J.E., and M.E. Esposito (1984) Coincident gene conversion during mitosis in Saccharomyces, Genetics, 107. 355365. Golin, J..E., and H. Tampe (1988) Coincident recombination during mitosis in Saccharomyces: distance-dependent and -independent components, Genetics, 119, 541-547.
185 Hawthorne, D.C. (1963) A deletion in yeast and its bearing on the structure of the mating type locus, Genetics, 48, 17271729. Hoebeke, J., G. Van Nijen and M. De Brabander (1976) Interaction of oncodazole (R 17934), a new anti-tumoral drug, with rat brain tubulin, Biochem. Biophys. Res. Commun., 69, 319-324. Hooper, A.K., and B.D. Hall (1975) Mating type and sporulation in yeast, I. Mutations which alter mating-type control over sporulation, Genetics, 80, 41-59. Hooper, A.K., P.T. Magee, S.K. Welch, M. Friedman and B.D. Hall (1974) Macromolecule synthesis and breakdown in relation to sporulation and meiosis in yeast, J. Bacteriol., 119, 619-628. Hubbard, M.J., R.T. Poulter, P.A. Sullivan and M.G. Shepherd (1985) Characterization of a tetraploid derivative of Candida albicans ATCC 10261, J. Bacteriol., 161, 781-783. Huitin, M., A. Karlman, J. Lindsten and L. Tiepolo (1970) Aneuploidy and polyploidy in germ-line cells of the male Chinese hamster (Cricetulus griseus), Hereditas, 65, 197202. Humphrey, R., and G. Fankhauser (1949) Three generations of polyploids in Amblistomid salamanders, J. Hered., 40, 7-12. Huskins, C.L. (1934) Anomalous segregation of triploid tomato, Heredity, 25, 281-286. Janiki-Ammal, E.K. (1940) Chromosome diminution in a plant, Nature (London), 146, 839-840. Johnson, K.R., J.E. Wright Jr. and B. May (1987) Linkage relationships reflecting ancestral tetraploidy in Salmonid fish, Genetics, 116, 579-591. Kilmartin, J.V. (1981) Purification of yeast tubulin by self-assembly in vitro, Biochemistry, 20, 3629-3633. Kreger-Van Rij, N.J.W. (1984) The Yeast, A Taxonomic Study, 3rd Edn., Elsevier, Amsterdam. Lewis, W.H., R.L. Oliver and T.J. Luikart (1971) Multiple genotypes in individuals of Claytonia oirginica, Science, 172, 564-565. Mayer, V.W., and C.J. Goin (1989) Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae, Mutation Res., 224, 471-478. Mayer, V.W., C.J. Goin and F.K. Zimmermann (1986) Anew ploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae, Mutation Res., 160, 19-26. Mortimer, R.K., R. Contopoulou and D. Schild (1981) Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cereoisiae, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5778-5782. Muldal, S. (1952) The chromosomes of earthworms, I. The evolution of polyploidy, Heredity, 6, 55-76. Muntzing, A. (1948) Accessory chromosomes in Poa alpina, Heredity, 2, 49-61. Nowel, P.C. (1976) The clonal evolution of tumor cell populations, Science, 194, 23-28. Ohno, S. (1970) Evolution By Gene Duplication, Springer, New York. Ostergen, G., and S. Frost (1962) Elimination of accessory
chromosomes from the roots in Haplopappus gracillis, Hereditas, 48, 363-366. Paquin, C., and J. Adams (1982) Isolation of sets of a, a, a/a, a / a and a / a isogenic strains in Saccharomyces cerevisiae, Curt. Genet., 6, 21-24. Parry, E.M., and B.S. Cox (1970) The tolerance of anenploidy in yeast, Genet. Res. Camb., 16, 333-340. Pennock, L.A. (1965) Triploidy in parthenogenetic species of the telid lizard, genus Cnemidophorus, Science, 149, 539540. Peterson, J.B., and H. Ris (1976) Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae, J. Cell Sci., 22, 219-242. Pomper, S., K.M. Daniels and D.W. McKee (1954) Genetic analysis of polyploid yeast, Genetics, 39, 343-355. Quinlan, R.A., C.I. Pogson and K. Gull (1980) The influence of the microtubule inhibitor, methyl benzimidazole-2-yl carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae, J. Cell Sci., 46, 341-352. Rodenburg, C.J., C.J. Cornelisse, P.A.M. Heintz, J. Hermans and G.J. Fleuren (1987) Tumor ploidy as a major prognostic factor in advanced ovarian cancer, Cancer, 59, 317-323. Roman, H., and S.M. Sands (1953) Heterogeneity of clones of Saccharomyces derived from haploid ascospores, Proc. Natl. Acad. Sci. (U.S.A.), 39, 171-179. Roman, H., D.C. Hawthorne and H.C. Douglas (1951) Polyploidy in yeast and its beating on the occurrence of irregular genetic ratios, Proc. Natl. Acad. SCI. (U.S.A.), 37, 79-84. Roman, H., M.M. Phillips and S.M. Sands (1955) Studies of polyploid Saccharomyces, I. Tetraploid segregation, Genetics, 40, 546-561. Sachs, L. (1952) Polyploid evolution and mammalian chromosomes, Heredity, 6, 357-364. Schmid, M., T. Haaf and W. Schemp (1985) Chromosome banding in Amphibia, IX. The polyploid karyotypes of Odontophrynus americanus and Ceratophrys ornata (Anura, Leptodactylidae), Chromosoma, 91,172-184. Sharma, A.K., and H.R. Aiyanger (1961) Occurrence of Bchromosomes in diploid Allium stracheyi Baker and their elimination in polyploids, Chromosoma, 12, 310-317. Sherman, F., G.R. Fink and J.B. Hicks (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Snoad, B. (1955) Somatic instability of chromosome number in Himenocallis calathinum, Heredity, 9, 129-134. Suzuki, T., T. Kanbe, T. Kuroiwa and K. Tanaka (1986) Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans, J. Gen. Microbiol., 132, 443-453. Whelan, W.L., D.M. Markie, K.G. Simkin and R.M. Poulter (1985) Instability of Candida albicans hybrids, J. Bacteriol., 161, 1131-1136. White, M.J.D. (1969) Chromosomal rearrangements and speciation in animals, Annu. Rev. Genet., 3, 75-98. Wolf, U., H. Ritter, N.B. Atkin and S. Ohno (1969) Polyploidization in the fish family Cyprinidae, order Cypriniformes, I. DNA-content and chromosome sets in various species of Cyprinidae, Humangenetik, 7, 240-244.
186 Wood, J.S. (1982) Genetic effects of methyl benzimidazole-2-yl carbamate on Saccharomyces cerevisiae, Mol. Cell. Biol., 2, 1064-1078. Wood, J.S., and L.H. Hartwell (1982) A dependent pathway of gene functions leading to chromosome segregation in S. cerevisiae, J. Cell Biol., 94, 718-726. Zakharov, I.A., and T.N. Kozina (1967) The genetics of mating in yeast (Saccharomyces cerevisiae), Genetika, 11,110-115. Zimmermann, F.K. (1984) Basic principles and methods of genotoxicity testing in the yeast Saccharomyces cerevisiae, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, 2nd edn., Elsevier, Amsterdam.
Zimmermann, F.K., and I. Scheel (1984) Genetic effects of 5-azacytidine in Saccharomyces cerevisiae, Mutation Res., 139, 21-24. Zimmermann, F.K., V.W. Mayer and 1. Scheel (1984) Induction of aneuploidy by oncodazole (nocodazole), an antitubulin agent, and acetone, Mutation Res., 141, 15-18. Zimmermann, F.K., V.W. Mayer, I. Scheel and M.A. Resnick (1985) Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae, Mutation Res., 149, 339-351.
