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A test for uniparental disomy in Saccharomyces cerevisiae
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation Research 306 (1994) 187-196
A test for uniparental disomy in Saccharomyces cerevisiae Friedrich K. Z i m m e r m a n n * Institut fiir Mikrobiologie, TechnischeHochschule Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany (Received 2 September 1993; revision received 17 November 1993; accepted 19 November 1993)
Abstract Uniparental disomy is a condition in a diploid organisms where one parental chromosome is absent and its homolog from the other parent duplicated. It can be a cause of genetic somatic disease in mammals because of imprinting. Imprinting creates a sex-specific pattern of epigenetic gene inactivation at least in mammals and, consequently, a complete set of both maternal and paternal chromosomes is required for normal development. Moreover, it has been shown for several types of tumors that recessive tumor alleles originally present in a heterozygous condition in normal somatic tissue have become homozygous in the tumor cells. Homozygosity is frequently caused by uniparental disomy. A similar situation is found in Saccharomyces cerevisiae where the spontaneous or induced expression of linked recessive alleles flanking a common centromere is preponderantly due to isodisomy where one of the homologs is lost and the retained homolog duplicated. In contrast to the situation in Aspergillus nidulans, isodisomy does not appear to be caused by two sequential and independent events of malsegregation resulting first in an unstable trisomic condition from which a normal disomic condition is restored through segregational loss of one supernumerary chromosome. Rather, an as yet unknown mechanism seems to directly generate isodisomy and thus Saccharomyces cerevisiae could provide a short-term test for the detection of this type of genetic change.
Key words: Chromosomal malsegregation; Imprinting; Uniparental disomy; Isodisomy; Saccharomyces cerevisiae
1. Introduction It has b e e n tacitly a s s u m e d for m a n y d e c a d e s t h a t t h e c o n t r i b u t i o n s o f a u t o s o m a l g e n e s by t h e m a t e r n a l a n d t h e p a t e r n a l g a m e t e s a r e functionally equivalent. H o w e v e r , e v i d e n c e a c c u m u l a t e d d u r i n g t h e 1980s i n d i c a t e d t h a t - at least in
* Corresponding author. Tel. +49 6151 16 28 55; Fax +49 6151 16 48 08.
m a m m a l s - this n e e d n o t b e so. M o u s e p r o n u c l e i can b e m a n i p u l a t e d to p r o d u c e zygotes with two f e m a l e o r two m a l e p r o n u c l e i . T h e r e s u l t i n g d i p l o i d g y n o g e n e t i c a n d a n d r o g e n e t i c eggs d o n o t d e v e l o p to t e r m b u t result in typically m a l f o r m e d structures. T h e g y n o g e n e t i c eggs form n o r m a l e m b r y o s b u t t h e yolk sacs a n d the p l a c e n t a s a r e u n d e r d e v e l o p e d . A n d r o g e n e t i c eggs d e v e l o p n o r mal p l a c e n t a s b u t p o o r l y d i f f e r e n t i a t e d e m b r y o s ( r e v i e w e d by Solter, 1988). E q u i v a l e n t e m b r y o n i c m a l f o r m a t i o n s w e r e f o u n d in m a n . O v a r i a n tera t o m a s which c o n t a i n all t h r e e e m b r y o n i c layers
0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(93)E0214-B
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F.K. Zimmermann /Mutation Research 306 (1994) 187-196
but no placental tissues were shown to contain two maternal genomes with no paternal contribution whereas hydatiform moles contained only two paternal but no maternal genomes. The analysis of human triploid fetuses showed that those with two maternal and one paternal genome form only small placentas and abort early while those with two paternal and one maternal genome consist mainly of placental tissue (see review by Hall, 1990). In conlusion, both parents must contribute their genomes to the zygote for normal development in mammals. Experiments with transgenic mice showed that the expression of several transgenes differed depending on whether they were transmitted through the father or the mother and this difference could be attributed to sex-specific methylation (Surani et al., 1988). These authors concluded that about one quarter of the mouse genome showed this sex-specific expression or imprinting (for a review of the early work see Hall, 1991). Imprinting - sex-specific expression of certain genes - means that both maternal and paternal genes are required for proper function. Indeed, Spence et al. (1988) and Voss et al. (1988) could show that two patients with the recessive genetic disease cystic fibrosis carried two copies of the maternal chromosome 7, a condition called maternal disomy. Mutations affecting a gene subjected to imprinting show remarkable patterns of inheritance. In heterozygotes, such mutations are only expressed when their corresponding wild type alleles derive from the parent which shows imprinting. Formally speaking, the mutant allele appears to be dominant. However, the same mutant allele appears to be recessive when it is contributed by the parent where it is imprinted because then it is the wild type allele from the non-imprinting parent which is fully active and the mutant allele is imprinted (for review, see Wilson, 1992). Consequences of genomic imprinting are best illustrated by a pair of different genetic defects. The Prader-Willi syndrome is characterized by certain anatomical and behavioral disorders like mild to moderate mental retardation and morbid obesity whereas the Angelman syndrome im-
presses by severe mental retardation, poor coordination, a happy disposition and inappropriate laughter. Both syndromes occur sporadically but occasionally they have been observed in one and the same family. Cytogenetic studies have yielded a surprising result. About 70-75% of the PraderWilli patients and 70-80% of the Angelman patients carry the same deletion of the 1 5 q l l - q 1 3 region. Interestingly enough, the Prader-Willi patients inherit the deletion from their fathers and the Angelman patients from their mothers. Another 20-25% of the Prader-Willi patients show a maternal and 2 - 5 % of the Angelman patients a paternal disomy of chromosome 15. Consequently, the same defects can be caused by a deletion in the presence of the imprinted wild type allele and also by the presence of two wild type alleles provided they are contributed by the imprinting parent. This implies that the 1 5 q l l q13 region comprises genes which show complementary imprinting in females and males (for review, see Nicholls, 1993). Henry et al. (1991) reported on uniparental disomy in patients with the Beckwith-Wiedemann syndrome which is chracterized by numerous growth anomalies and a predisposition to several childhood malignancies such as Wilms' tumor. Actually, the same syndrome can be caused by either uniparental disomy or deletions in the region 1 lp15.5 when it is transmitted by the mother indicating that the paternal region is imprinted. Moreover, investigations of the Beckwith-Wiedemann syndrome in monozygotic twins has shown a considerable discordance in tumor incidences suggesting that post-zygotic events can lead to somatic uniparental disomic mosaics (for review, see Junien, 1992). Caron et al. (1993) showed that a preferential loss of the maternal chromosome lp36 can lead to neuroblastoma, a childhood neural crest tumor. In this report, loss of heterozygosity or paternal disomy had occurred between the normal somatic and the tumor cells implying that the tumor suppressor gene is imprinted during male gametogenesis and only the maternally derived gene is active. In addition to that, it could be demonstrated that there are also neuroblastoma patients carrying germline mutations.
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
The mechanisms leading to uniparental disomy or loss of heterozygosity have been investigated in the case of retinoblastoma by Cavenee et al. (1983, 1986, 1991). Retinoblastoma is a "dominant" genetic disease with the defect frequently inherited from one affected parent. Cavenee and co-workers used restriction fragment length polymorphism to identify the origin of chromosome 13 which carries the retinoblastoma rb gene in tumor in comparison to normal somatic cells and the parents of the patient. There were 33 informative cases of which 19 showed uniparental disomy or loss of heterozygosity with the retained entire chromosome deriving from the affected parent. The "constitutional" genotype of the normal cells was retained in nine cases indicating that the homozygous condition was limited to a narrow region. Homozygosity extending from the rb region to the telomere suggested somatic reciprocal recombination as the causative mechanism in four patients. Chromosome loss leading to a monosomi¢ condition for chromosome 13 was found in only one patient. These data suggest an unusual type of chromosomal malsegregation resulting in uniparental disomy. Actually, there are two possible types of uniparental disomy. Isodisomy refers to a pair of chromosomes which are completely identical whereas heterodisomy means that both homologs in a diploid cell are from one and the same parent but not necessarily identical or isodisomic (Engel, 1980). Interestingly enough, isodisomy but not heterodisomy is the usual condition of uniparental disomy. Formally, isodisomy can be explained by a first malsegregational event leading to a trisomic condition which, being unstable, leads to a subsequent chromosome loss restoring a complete diploid heterozygous or isodisomic condition. Such a mechanism of sequential malsegregational events has been established for Aspergillus nidulans (see K~ifer, 1988). However, Resnick and Zimmermann (1987) postulated for Saccharomyces cerevisiae that occasionally anaphase starts before the kinetochores have divided so that replicated chromatids are held together and migrate to one spindle pole. Isodisomy will result if this happens to both members of a homologous
189
pair and the undivided pairs of chromatids move to opposite spindle poles. In this communication we report on the preponderance of spontaneous and induced isodisomy in Saccharomyces cerevisiae.
2. Material and methods
2.1. The test system and evaluation of results Diploid yeast strain D61.M was constructed by Zimmermann et al. (1985) to monitor loss of chromosome VII by the expression of three recessive markers present in a heterozygous condition: cyh2 is a recessive resistance marker which on a medium with 1.7 ppm cycloheximide allows selection of homo- or hemizygous cells arising in a culture of heterozygous sensitive cells; ade6 located on the other side of the centromere is a recessive mutant allele which prevents the accumulation of a red pigment in cells homozygous for ade2. leul is a recessive centromere marker which causes a requirement for leucine and is located on the same chromosome arm as cyh2. D61.M is also homozygous for ilvl-92 which causes a requirement for isoleucine. Consequently, requirements for adenine (because of the homozygous condition of ade2) and isoleucine can be used to identify D61.M cells. Three types of colonies are obtained after plating on a selective cycloheximide medium. Red colonies are the most frequent class of resistant colonies of which usually fewer than 10% express centromere marker leul. White colonies on this medium can express leul and they can result from loss of the chromosome carrying the dominant wild type alleles or alternatively from two events of mitotic recombination between the centromere and ade6 on one side and leul and the centromere on the other. This is rare because of the close centromere linkage of leul. White colonies not expressing leul cannot be caused by chromosome loss. Induction of chromosome loss is indicated when at least 30% of the white resistant colonies express leul (Zimmermann and Rohlfs, 1991; Zimmermann and Mohr, 1992). The criteria for the evaluation of the experimental
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data have been defined by Albertini and Zimmermann (1991) based on the analysis of tests with 111 chemicals.
mermann et al. (1985) even though many aprotic polar solvents induce chromosome loss with a continuous incubation at temperatures below the standard 28°C (Zimmermann et al., 1988).
2.2. Genetic analysis Presumptive chromosome loss segregants were analyzed by tetrad analysis using a de Fonbrune micromanipulator. All spore colonies were tested for the three diagnostic markers, trp5 a recessive marker causing a tryptophan requirement and located in repulsion to the markers ade6-1eul and cyh2. Additional heterozygous markers are recessive hisl causing a histidine requirement, MAL2 a dominant heterozygous marker for maltose utilization on chromosome III and on the same chromosome the two mating type alleles MATa and MATa. A strain carrying both these alleles cannot mate but does sporulate. All strains expressing only one of these alleles cannot sporulate but mate irrespective of their ploidy. Mating types were assayed by crossing spore clones with MATa and MATa type tester strains. These mating type alleles segregate regularly so that a sporal clone shows either a MATa or a MATa reaction. On the other hand, haploid strains carrying both alleles cannot mate but initiate sporulation. This combination of properties is diagnostic for a sporal clone carrying both alleles due to an original tri- or tetrasomy of the sporulated isolate. Cells trisomic for chromosome III are either M A T a / M A T a / M A T a or M A T a / MATa/MATa. They form spores which are MATa/MATa in addition to MATa/MATa or M A T a / M A T a and the normal type with MATa and MATa. The MATa/MATa spores cannot mate but can be induced to sporulate. Even though they do not form viable spores an unambiguous cleavage of the cells is indicative of attempted sporulation and thus of a usually trisomic condition of the original cell.
2.3. Mutagen-induced induction of chromosome loss Chromosome loss was induced according to the cold shock protocol of 4 h at 28°C, followed by an overnight storage in an ice bath and a final incubation period at 28°C as described by Zim-
2. 4. Media used The media used and preparation of cultures have been described by Zimmermann et al. (1985).
3. Results
Chromosomal malsegregation in diploid cells is considered to result in aneuploidy based on the observation of monosomics (chromosome loss) and trisomics (chromosome gain) in cytological preparations. Based on this common knowledge, the first genetic analysis of the "monosomic" segregants obtained by Parry and Zimmermann (1976) in strain D6 and in strain D61.M yielded an unexpected result (Zimmermann et al., 1985). The majority of the isolates, expressing the three diagnostic recessive markers cyh2, leul and ade6, obtained in the controls and after induction with bavistan, ethyl acetate and methyl ethyl ketone were perfectly diploid. The expected monosomics for chromosome VII should result in tetrads containing two dead spores, those lacking chromosome VII, and two viable spores with a complete haploid set of chromosomes. The results of a genetic analysis of the expected monosomics is shown in Table 1. Only about 10% of the isolates showed this pattern. However, about 10% of those isolates which were perfectly diploid segregated spores which could not mate with MATa or MATa tester strains but could be induced to sporulate. This indicated that the observed "chromosome loss" was associated with a high frequency of chromosome gain resulting in a trisomic condition of chromosome III. Non-mating spore cultures derived from diploid strains are extremely rare and occur definitely at frequencies below 1 in 1000. Consequently, selecting for cells expressing the three diagnostic markers in fact selects for isodisomy frequently associated with malsegregation for additional chromosomes.
F.K~ Zimmermann / Mutation Research 306 (1994) 187-196
In order to identify the spontaneous genetic events leading to the simultaneous expression of the three diagnostic markers cyh2, leul and ade6, 5-ml volumes of a rich yeast extract-peptone-glucose medium were inoculated with approximately 1000 cells of strain D61.M. This inoculum is much smaller than the critical population size expected to contain cells with chromosome loss. The spontaneous frequencies of such cells have varied in control platings on the selective cycloheximide medium between 0.09 5< 10 -6 and 3.01 × 10 -6 (Zimmermann and Mohr, 1992). These 5-ml cultures were grown to the late exponential growth phase to a total population size of about 5 × 108 each and samples were plated on the selective cycloheximide medium. Clones derived from only one colony expressing the three diagnostic markers per culture were sporulated and tetrad analysis performed. The results obtained with 51 isolates are shown in Table 2. Only one isolate showed the expected two dead to two viable spores in all of nine tetrads. Non-mating spores were observed in four additional cases indicating that the selected colony was trisomic for chromosome III. Tetrads with fewer than four viable spores occur at low freTable 1 Genetic analysis of Saccharomyces cerevisiae segregants showing chromosome loss Origin of segregants
Number analyzed
Control 66 Control a 8 Bavistan b 12 Ethyl acetate b 20 Methyl ethyl ketone b 14 Propionitrile 20 min 24 Propionitrile 660 min 26 Total 170
Monosomics Trisomy-3 3 0 3 1 2 3 1 13
5 0 1 2 2 4 4 18
Number analyzed, total number of chromosome loss isolates tested; monosomics, isolates where all tetrads showed a two viable/two dead spore segregation; trisomy-3, isolates which segregated non-mating sporal clones. Control, colonies from various control platings including those from Table 2. Propionitrile at 17.91 mg/ml in the cold shock experiment reported in Table 3:20 min, sample plated at 20 min; 660 rain, sample plated at 660 min after transfer from ice bath to 28°C. a Data from Parry and Zimmermann (1976). b Data from Zimmermann et al. (1985).
191
Table 2 Genetic analysis of presumptive chromosome loss colonies of yeast strain D61.M. Segregation pattern of viable versus dead spores Segregation pattern
Number of isolates
1. Asci containing only mating spores 4:0 4:0 4:0 4:0 3:1 2:2
3:1 3:1 2:2
2:2
2:2
5 24 4 13 1 1
2. Asci containing non-mating spores 4:0 3:1 4:0 3:1 Total
2:2
2 2 51
These isolates arose in independent cultures which were started with about 1000 cells. An average of 10 asci was dissected per isolate. 4:0, four viable spores per ascus; 3: 1, three viable and one dead spore; 2 : 2, two viable and two dead spores; non-mating spore colonies could be induced to initiate incomplete sporulation.
quencies after sporulation of normal diploid strains. However, an increase in dead spores indicates the presence of genetic damage or chromosomal rearrangements especially if there is more than one dead spore per tetrad. There were 12 isolates which yielded a few tetrads with only two viable spores. This could indicate that the selected event, loss of one of the chromosome VII homologs, was associated with additional genetic damage. However, it was perfectly obvious that chromosome loss leading to a monosomic condition was rare. If one of the chromosome VII homologs was lost, this loss was compensated by a duplication of the retained homolog. Induction of chromosome loss in diploid nuclei has been extensively studied in Aspergillus nidulans. As shown by K~ifer (1988), trisomics grow poorly and are unstable. One of the supernumerary chromosomes is lost and this can result in isodisomic diploids. Consequently in this organism, isodisomy definitely results from a first event of chromosome gain resulting in a trisomic condition followed by a random loss of one the the three homologs reconstituting a diploid constitution. Chromosomal malsegregation in Aspergillus is so frequent that non-selective plating is possible.
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Chromosomal malsegregation in yeast is a rare event. The frequency of spontaneous loss of chromosome VII detected on a non-selective medium w a s 4 . 8 5 X 10 - 6 ( Z i m m e r m a n n and Mohr, 1992) and the highest induced frequency detected by selective plating was 1.7 x 10 -3 (Mayer and Goin, 19871. Cells of strain D61.M divide once or rarely twice when plated on the selective cycloheximide medium unless they express recessive cyh2. Nevertheless, chromosome loss can be induced by chemicals at concentrations which inhibit cell division or even kill cells. However, chromosome loss cannot be induced when cells are incubated in a medium without adenine and isoleucine which does not support growth (Table 5 in Zimm e r m a n n et al., 1988). The "cold shock" protocol - including an incubation period in a growth medium at 28°C, followed by an incubation in an ice bath and a subsequent incubation period again at 28°C - was calibrated by Z i m m e r m a n n et al. (1985, 1988). The inducing chemical has to be present during all three incubation phases. Cycloheximide resistant white colonies requiring leucine - the presumptive chromosome loss types - appeared rapidly after the cultures previously kept in an ice bath had been warmed up to 28°C. This and the lack of growth between addition of the chemical and the time of plating demonstrated that no net growth occurred during the entire incubation period and the limited residual growth on the selective medium suggested that induction of chromosome loss requires only one or a few rounds of cell division and segregation of daughter nuclei. Table 3 shows an experiment with propionitrile which was performed in order to test whether the resistant colonies appearing upon plating at 20 min after storage in the ice bath, no cell divisions up to the time of plating, and at 660 min, cell titer increased 2.2-fold, differed in their content of resistant isolates with a two v i a b l e / t w o dead cell segregation. Only three of the 24 isolates from the 20-min plating and only one of the 660-min plating showed this type of segregation, but at both sampling times four colonies showed the additional chromosome III trisomy signalled by non-mating spore progeny (Table 1).
Table 3 The appearence of propionitrile-induced chromosome loss segregants during incubation at 28°C following overnight cold shock in Saccharomyces cereuisiae Sample
Titer
Control Start 5.7 Before ice 42.9 After ice 40.7 200 min 74.4
x 1.0 7.5 7.1 13.0
Propionitrile 17. 9 m g / ml Start 6.6 Before ice 7.5 After ice 8.9 1.0 20 min 40 min 60 min 7.9 0.9 80 min 100 min 15(1 min 10.7 1.2 300 rain 14.4 1.6 420 rain 19.6 2.2 660 rain 20.0 2.2
Colonies 3 10 2 30 1 9 2 383 409 440 563 589 820 1 212 1392 1 618
X 1.0 3.3 0.7 10.0
1.(1 192 204 22(1 282 295 410 607 696 809
Frequency
x
1.72 0.78 0.33 1.34
1.0 0.5 0.2 0.8
0.51 4.01 0.75
1.0
186.44
246
255.45 281.02 243.36 269.67
341 375 324 360
Titer, colony-forming u n i t s x l 0 ¢ ' ; Colonies, white colonies (test for expression of leul only in controls); Frequency, frequency of white colonies per 106 colony-forming units; x , factor increase; Start, determinations at the beginning of the experiment; Before ice, after 4 h at 28°C; After ice, before transfer to 28°C. 20 min up to 660 min refers to time of incubation at 28°C after incubation in ice.
Cell titers in these experiments were determined as colony-forming units which is not necessarily the same as an actual hemocytometer count. Separation of the mother and newly budded daughter cell does not immediately follow the completion of cytokinesis so that a growing diploid yeast culture contains in addition to single cells also doublets of a mother and her completed daughter cell. They all, upon dilution and plating, form a single colony. As shown in Table 4, in untreated controls the titers determined by hemocytometer counts are about 1.4 times higher than the ones based on colony-forming units. The titer of colony-forming units at the dose with the highest induction of chromosome loss was the same as in the control at the start of the experiment. However, the hemocytometer count showed that the cell titer had increased threefold. This indicated that at least one cell division had oc-
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
curred during the course of the experiment, and in a few cases one or very few more. Expression of a recessive resistance by a malsegregational event requires residual growth and division. Therefore, cells were treated with 18.8 m g / m l propionitrile in a cold shock experiment. Part of the ceils were plated directly on the selective medium after the usual 4 h incubation at 28°C. The rest of the culture was centrifuged and the sedimented cells resuspended in fresh medium and incubated for various lenghts of time up to 8 h before plating. Table 5 shows that the cell titer increased 10-fold and the frequency of the isodisomics decreased by about one third. This demonstrated clearly that additional cell divisions before selecting for resistance did not enhance the frequency of isodisomics. The observed decline in the frequency of isodisomics could be caused by a reduced growth of these variants. C h r o m o s o m e loss frequencies in Saccharomyces cerevisiae are too rare to be accurately studied without selective plating. However, pooled data from non-selective plating from control and experimental cultures showed that 79 of 409 white colonies expressed leul and cyh2 (19.3%) and 14 white sectors of 79 red-white sectored colonies (17.7%) simultaneously expressed these markers. In only one of the red-white colonies did the red
Table 4 Cell titers determined by hemocytometer cell counts and as colony-forming units in control and propionitrile-treated cultures of Saccharomycescerevisiae Dose
CFU
Hemocyt
Ratio
Frequency
Control 15.1 mg/ml 1.70 mg/ml 1.88 mg/ml 2.07 mg/ml 2.44 mg/ml
45.7 41.7 18.9 7.4 7.9 5.3
88.5 92.2 55.6 24.9 22.8 16.1
0.52 0.45 0.34 0.30 0.35 0.33
1.00 13.19 125.40 318.02 140.02 64.5
The cold shock protocol was followed. Starting titer in colony-forming units 7.2×106. CFU, colony-forming units;
Hemocyt, microscopic count, a bud of about three quarters the size of the mother cell was considered to be a functionally separated daughter cell; Ratio, CFU titer divided by hemocytometer titer; Frequency, white resistant colonies (propionitrile) and white resistant colonies expressing leul (control) per 10 6 colony-formingunits, mg/ml refers to propionitrile concentration.
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Table 5 The effect of non-selective post-treatment growth on the recovery of chromosome loss in Saccharomycescerevisiae Growth (h)
Titer ( x l 0 6)
x
Colonies
x
Frequency (×10 -6)
×
Control 0
78.2
1.0
5
1.0
1.07
1.0
8
203.0
2.6
30
6.0
0.49
0.5
1.0 555 1.2 705 1.9 795 2.4 1130 5.2 1911 10.1 3705
1.0 1.3 1.4 2.1 3.4 6.7
304.95 329.44 232.46 255.66 203.51 200.81
1.0 1.1 0.8 0.8 0.7 0.7
Propionitrile 18.8 mg / ml 0 1 2 3 5 8
6.1 7.1 11.4 14.7 31.5 61.5
The cold shock protocol was followed. Growth hours, hours in medium without cycloheximide; Colonies, absolute numbers of white colonies in experimental samples and of white colonies expressing leul in control samples, x, factor of increase over 0-h platings.
sector express the recessive marker trp5 diagnostic for the other homolog and the white sector leul and cyh2. This again indicated that the generation of induced chromosome loss does not require many division cycles.
4. Discussion Selection for chromosome loss in diploid mitotic cells in Saccharomyces cerevisiae showed uniparental disomy or isodisomy to be 10 times more frequent than monosomy. As pointed out in the Introduction, uniparental dis0my contributes considerably to h u m a n genetic disease. The potential importance of fungal systems for the detection of chromosomal malsegregation is that they are based on the expression of recessive diagnostic markers and thus they signal not only aneuploidy but also isodisomy. This aspect was not considered in the European Community Aneuploidy Project (Parry and Sors, 1993) which included an evaluation of Aspergillus nidulans and Saccharomyces cerevisiae fungal systems (Parry, 1993). There is a strong correlation between the abilities of an agent to interfere with mammalian tubulin aggregation in vitro and to induce mitotic
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chromosome loss in Saccharomyces cerevisiae strain D61.M as summarized by Albertini and Zimmermann (1991). Moreover, the fact that cold shock and incubation at lower temperature enhances induction of chromosome loss by aprotic polar solvents (Zimmermann et al., 1988) strongly indicates that microtubules a n d / o r tubulins are the primary cellular targets for induction. The mechanistic question is whether isodisomy results from two sequential reactions as shown for Aspergillus nidulans (see K~ifer, 1988) or whether there is a special and as yet unidentified novel mechanism as suggested by Resnick and Zimmermann (1987). A sequential mechanism with a primary event resulting in a trisomic condition followed by a gradual selection of secondary isodisomic segregants is not very likely in Saccharomyces cereuisiae because chromosome loss can be induced at high frequencies with doses which completely inhibit growth. Morever, cells are always plated out of the treatment medium onto the selective cycloheximide medium with no or only very limited intermediate growth in non-selective media. It is obvious that at least one error-struck mitosis must be completed for chromosome loss to occur. This is documented by the fact that chromosome loss cannot be induced in a medium without isoleucine and adenine which are required for growth of strain D61.M (Table 5 in Zimmermann et al., 1988). Direct plating on the selective medium blocks growth of sensitive cells after one or at most two rounds of cell division. Only those which express cyh2 continue to grow. A two step process of the induction of isodisomy via a trisomic intermediate state and a final accidental and random chromosome loss resulting in an isodisomic diploid could be accommodated with a first round of nuclear division before plating and a second round on the selective medium. However, if loss of a homolog in a trisomic cell is accidental it would not only occur in the first division cycle after the formation of the trisomic state but also in later divisions. Non-selective growth after the induction period should then increase the incidence of cycloheximide resistant isodisomics. As shown in Table 5, this is definitely not the case. Therefore,
it is likely that the postulated two sequential reactions of chromosome gain, causing a trisomic condition, and chromosome loss, restoring a disomic condition, are tightly linked. This view is also supported by the results obtained by plating on non-selective media. There, presumptive chromosome loss segregants expressing the three diagnostic markers can be found at equal frequencies among completely white and also red-white sectored colonies. This shows that a second chromosomal malsegregation event leading to a final isodisomy must occur very early or concomitantly with the first event. Otherwise, the frequency of chromosome loss would be higher among sectored colonies on a non-selective medium. On the other hand, the malsegregational mechanism proposed by Resnick and Zimmermann (1987) based on a lack of kinetochore division before the start of anaphase requires only one nuclear division to create an isodisomic state. Mayer and Aguilera (1990) constructed a series of triploid and tetraploid derivatives of D61.M with variable numbers of chromosome VII recessive, ade6-leul-cyh2, homologs. They found that spontaneous loss of the hornolog with the dominant markers in the normal diploid averaged 7.58 x 10 -7, loss of the only dominant homolog from the triploid 1.42 × 10 -5 and in the tetraploid 3.99 X 10 -4. This shows that the rate of chromosome loss increases with ploidy. Moreover, chromosome loss could even be detected in a tetraploid carrying three dominant homologs and the computed frequency in this case was 3480 times higher than in the heterozygous diploid. This assumption is based on the classical chromosome loss concept. Extrapolating from the simple chromosome loss frequencies in the diploid would predict chromosome loss to be virtually undetectably low in the tetraploid with three "dominant" homologs. It is conceivable that occasional spontaneous or induced defects in the assembly a n d / o r function of the spindle fiber apparatus not only lead to the "classical" types of chromosome loss or gain. A spontaneous or induced retardation of kinetochore separation before anaphase could not only explain the data presented here but also
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
provide a plausible explanation for those reported by Mayer and Aguilera (1990). Isodisomy may be caused by a novel type of failure of the system for chromosome segregation and cause a re-assortment of chromosomes without an intermediate state of cytologically detectable aneuploidy. The final result will be the elimination of one of the two homologs with a concomitant duplication of the retained homolog. The test system for chromosome loss in Saccharomyces cerevisiae allows for a ready detection of such events.
5. Acknowledgements I would like to thank M.A. Resnick, NIEHS, Research Triangle Park, NC, for many stimulating discussions which led to the experiments reported in this publication, and also F.E. Wiirgler, Institut fiir Toxikologie, Eidgen6ssische Technische Hochschule und Universit~it Ziirich, Switzerland, for drawing my attention to the importance of uniparental disomy.
6. References Albertini, S., and F.K. Zimmermann (1991) The detection of chemically induced chromosomal malsegregation in Saccharomyces cerevisiae D61.M: a literature survey (19841990), Mutation Res., 258, 237-258. Caron, H., P. van Sluis, M. van Hoeve, J. de Kraker, J. Bras, R. Slater, M. Mannens, P.A. Vofite, A. Westerveld and R. Versteeg (1993) Allelic loss of chromosome lp36 in neuroblastoma is of preferential maternal origin and correlates with N-myc amplification, Nature Genet., 4, 187-190. Cavenee, W.K., T.P. Dryja, R.A. Philips, W.F. Benedict, R. Godbout, L. Galli, A.L. Murphree, L.C. Strong and R.L. White (1983) Expression of recessive alleles by chromosomal mechanisms in retinoblastoma, Nature, 305, 779-784. Cavenee, W.K., A. Koufos and M.F. Hansen (1986) Recessive mutant genes predisposing to human cancer, Mutation Res., 168, 3-14. Cavanee, W.K., H.J. Scrable and C.D. James (1991) Molecular genetics of human cancer predisposition and progression, Mutation Res., 247, 199-202. Engel, E. (1980) A new genetic concept: Uniparental disomy and its potential effect, isodisomy, Am. J. Med. Genet., 6, 137-143.
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Hall, J.G. (1990) Genomic imprinting: review and relevance to human disease, Am. J. Hum. Genet., 46, 857-873. Hall, J.G. (1991) Genomic imprinting, Curt. Biol., 1, 34-39. Henry, L., C. Bonaiti-Pelli6, V. Chehensse, C. Beldjord, C. Schwartz, G. Utermann and C. Junien (1991) Uniparental disomy in a genetic cancer-predisposing syndrome, Nature, 351, 665-667. Junien, C. (1992) Beckwith-Wiedemann syndrome, tumorigenesis and imprinting, Curr. Opin. Genet. Dev., 2, 431-438. K~fer, E. (1988) MMS-induced primary aneuploidy and other genotoxic effects in mitotic cells of Aspergillus, Mutation Res., 201, 385-399. Mayer, V.W., and A. Aguilera (1990) High levels of chromosome instability in polypioids of Saccharomyces cerevisiae, Mutation Res., 231, 177-186. Mayer, V.W., and C.J. Goin (1987) Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae, Mutation Res., 187, 21-30. Nicholls, R.D. (1993) Genomic imprinting and candidate genes in the Prader-Willi and Angelman syndromes, Curr. Opin. Genet. Dev., 3, 445-456. Parry, J.M. (1993) An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential aneugens, Mutation Res., 287, 23-28. Parry, J.M., and A. Sots (1993) The detection and assessment of the aneugenic potential of environmental chemicals: the European Community Aneuploidy Project, Mutation Res., 287, 3-16. Parry, J.M., and F.K. Zimmermann (1976) The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae, Mutation Res., 36, 49-66. Resnick, M.A., and F.K. Zimmermann (1987) The genetic detection, mechanisms and relevance of chemically induced chromosome aneuploidy in yeast, in: S.S. Sandhu, D,M. DeMarini, M.J. Mass, M.M. Moore and J.L. Mumford (Eds.), Short-term Bioassays in the Analysis of Complex Environmental Mixtures V, Plenum, New York, pp. 321-329. Solter, D. (1988) Differential imprinting and expression of maternal and paternal genomes, Annu. Rev. Genet., 22, 282-303. Spence, J.E., R.G. Perciaccante, G.M. Greig, H.F. Willard, D.H. Ledbetter, J.F. Hejtmancik, M.S. Pollack, W.E. O'Brien and A.L. Beaudet (1988) Uniparental disomy as a mechanism for human genetic disease, Am. J. Hum. Genet., 4, 59-62 Surani, M.A., W. Reik and N.D. Allen (1988) Transgenes as molecular probes for genomic imprinting, Trends Genet., 4, 59-62. Voss, R., E. Ben-Simon, A. Avital, S. Godfrey, J. Zlotogota, J. Dagan, Y. Tikochinski and J. Hillel (1989) Isodisomy of chromosome 7 in a patient with cystic fibrosis: could uniparental disomy be common in humans?, Am. J. Hum. Genet., 45, 373-380.
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Wilson, G.N. (1992) Mutational risk in females: genomic imprinting and maternal molecules, Mutation Res., 296, 157-165. Zimmermann, F.K., and A. Mohr (1992) Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae, Mutation Res., 270, 151-166. Zimmermann, F.K., and A. Rohlfs (1991) The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae, Mutation Res., 250, 239-249.
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. Zimmermann, F.K., U.L.I. Holzwarth, I. Scheel and M.A. Resnick (1988) Aprotic polar solvents that affect porcine brain tubulin aggregation in vitro induce aneuploidy in yeast cells growing at low temperatures, Mutation Res., 201,431-442.
ELSEVIER
Mutation Research 306 (1994) 187-196
A test for uniparental disomy in Saccharomyces cerevisiae Friedrich K. Z i m m e r m a n n * Institut fiir Mikrobiologie, TechnischeHochschule Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany (Received 2 September 1993; revision received 17 November 1993; accepted 19 November 1993)
Abstract Uniparental disomy is a condition in a diploid organisms where one parental chromosome is absent and its homolog from the other parent duplicated. It can be a cause of genetic somatic disease in mammals because of imprinting. Imprinting creates a sex-specific pattern of epigenetic gene inactivation at least in mammals and, consequently, a complete set of both maternal and paternal chromosomes is required for normal development. Moreover, it has been shown for several types of tumors that recessive tumor alleles originally present in a heterozygous condition in normal somatic tissue have become homozygous in the tumor cells. Homozygosity is frequently caused by uniparental disomy. A similar situation is found in Saccharomyces cerevisiae where the spontaneous or induced expression of linked recessive alleles flanking a common centromere is preponderantly due to isodisomy where one of the homologs is lost and the retained homolog duplicated. In contrast to the situation in Aspergillus nidulans, isodisomy does not appear to be caused by two sequential and independent events of malsegregation resulting first in an unstable trisomic condition from which a normal disomic condition is restored through segregational loss of one supernumerary chromosome. Rather, an as yet unknown mechanism seems to directly generate isodisomy and thus Saccharomyces cerevisiae could provide a short-term test for the detection of this type of genetic change.
Key words: Chromosomal malsegregation; Imprinting; Uniparental disomy; Isodisomy; Saccharomyces cerevisiae
1. Introduction It has b e e n tacitly a s s u m e d for m a n y d e c a d e s t h a t t h e c o n t r i b u t i o n s o f a u t o s o m a l g e n e s by t h e m a t e r n a l a n d t h e p a t e r n a l g a m e t e s a r e functionally equivalent. H o w e v e r , e v i d e n c e a c c u m u l a t e d d u r i n g t h e 1980s i n d i c a t e d t h a t - at least in
* Corresponding author. Tel. +49 6151 16 28 55; Fax +49 6151 16 48 08.
m a m m a l s - this n e e d n o t b e so. M o u s e p r o n u c l e i can b e m a n i p u l a t e d to p r o d u c e zygotes with two f e m a l e o r two m a l e p r o n u c l e i . T h e r e s u l t i n g d i p l o i d g y n o g e n e t i c a n d a n d r o g e n e t i c eggs d o n o t d e v e l o p to t e r m b u t result in typically m a l f o r m e d structures. T h e g y n o g e n e t i c eggs form n o r m a l e m b r y o s b u t t h e yolk sacs a n d the p l a c e n t a s a r e u n d e r d e v e l o p e d . A n d r o g e n e t i c eggs d e v e l o p n o r mal p l a c e n t a s b u t p o o r l y d i f f e r e n t i a t e d e m b r y o s ( r e v i e w e d by Solter, 1988). E q u i v a l e n t e m b r y o n i c m a l f o r m a t i o n s w e r e f o u n d in m a n . O v a r i a n tera t o m a s which c o n t a i n all t h r e e e m b r y o n i c layers
0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(93)E0214-B
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but no placental tissues were shown to contain two maternal genomes with no paternal contribution whereas hydatiform moles contained only two paternal but no maternal genomes. The analysis of human triploid fetuses showed that those with two maternal and one paternal genome form only small placentas and abort early while those with two paternal and one maternal genome consist mainly of placental tissue (see review by Hall, 1990). In conlusion, both parents must contribute their genomes to the zygote for normal development in mammals. Experiments with transgenic mice showed that the expression of several transgenes differed depending on whether they were transmitted through the father or the mother and this difference could be attributed to sex-specific methylation (Surani et al., 1988). These authors concluded that about one quarter of the mouse genome showed this sex-specific expression or imprinting (for a review of the early work see Hall, 1991). Imprinting - sex-specific expression of certain genes - means that both maternal and paternal genes are required for proper function. Indeed, Spence et al. (1988) and Voss et al. (1988) could show that two patients with the recessive genetic disease cystic fibrosis carried two copies of the maternal chromosome 7, a condition called maternal disomy. Mutations affecting a gene subjected to imprinting show remarkable patterns of inheritance. In heterozygotes, such mutations are only expressed when their corresponding wild type alleles derive from the parent which shows imprinting. Formally speaking, the mutant allele appears to be dominant. However, the same mutant allele appears to be recessive when it is contributed by the parent where it is imprinted because then it is the wild type allele from the non-imprinting parent which is fully active and the mutant allele is imprinted (for review, see Wilson, 1992). Consequences of genomic imprinting are best illustrated by a pair of different genetic defects. The Prader-Willi syndrome is characterized by certain anatomical and behavioral disorders like mild to moderate mental retardation and morbid obesity whereas the Angelman syndrome im-
presses by severe mental retardation, poor coordination, a happy disposition and inappropriate laughter. Both syndromes occur sporadically but occasionally they have been observed in one and the same family. Cytogenetic studies have yielded a surprising result. About 70-75% of the PraderWilli patients and 70-80% of the Angelman patients carry the same deletion of the 1 5 q l l - q 1 3 region. Interestingly enough, the Prader-Willi patients inherit the deletion from their fathers and the Angelman patients from their mothers. Another 20-25% of the Prader-Willi patients show a maternal and 2 - 5 % of the Angelman patients a paternal disomy of chromosome 15. Consequently, the same defects can be caused by a deletion in the presence of the imprinted wild type allele and also by the presence of two wild type alleles provided they are contributed by the imprinting parent. This implies that the 1 5 q l l q13 region comprises genes which show complementary imprinting in females and males (for review, see Nicholls, 1993). Henry et al. (1991) reported on uniparental disomy in patients with the Beckwith-Wiedemann syndrome which is chracterized by numerous growth anomalies and a predisposition to several childhood malignancies such as Wilms' tumor. Actually, the same syndrome can be caused by either uniparental disomy or deletions in the region 1 lp15.5 when it is transmitted by the mother indicating that the paternal region is imprinted. Moreover, investigations of the Beckwith-Wiedemann syndrome in monozygotic twins has shown a considerable discordance in tumor incidences suggesting that post-zygotic events can lead to somatic uniparental disomic mosaics (for review, see Junien, 1992). Caron et al. (1993) showed that a preferential loss of the maternal chromosome lp36 can lead to neuroblastoma, a childhood neural crest tumor. In this report, loss of heterozygosity or paternal disomy had occurred between the normal somatic and the tumor cells implying that the tumor suppressor gene is imprinted during male gametogenesis and only the maternally derived gene is active. In addition to that, it could be demonstrated that there are also neuroblastoma patients carrying germline mutations.
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
The mechanisms leading to uniparental disomy or loss of heterozygosity have been investigated in the case of retinoblastoma by Cavenee et al. (1983, 1986, 1991). Retinoblastoma is a "dominant" genetic disease with the defect frequently inherited from one affected parent. Cavenee and co-workers used restriction fragment length polymorphism to identify the origin of chromosome 13 which carries the retinoblastoma rb gene in tumor in comparison to normal somatic cells and the parents of the patient. There were 33 informative cases of which 19 showed uniparental disomy or loss of heterozygosity with the retained entire chromosome deriving from the affected parent. The "constitutional" genotype of the normal cells was retained in nine cases indicating that the homozygous condition was limited to a narrow region. Homozygosity extending from the rb region to the telomere suggested somatic reciprocal recombination as the causative mechanism in four patients. Chromosome loss leading to a monosomi¢ condition for chromosome 13 was found in only one patient. These data suggest an unusual type of chromosomal malsegregation resulting in uniparental disomy. Actually, there are two possible types of uniparental disomy. Isodisomy refers to a pair of chromosomes which are completely identical whereas heterodisomy means that both homologs in a diploid cell are from one and the same parent but not necessarily identical or isodisomic (Engel, 1980). Interestingly enough, isodisomy but not heterodisomy is the usual condition of uniparental disomy. Formally, isodisomy can be explained by a first malsegregational event leading to a trisomic condition which, being unstable, leads to a subsequent chromosome loss restoring a complete diploid heterozygous or isodisomic condition. Such a mechanism of sequential malsegregational events has been established for Aspergillus nidulans (see K~ifer, 1988). However, Resnick and Zimmermann (1987) postulated for Saccharomyces cerevisiae that occasionally anaphase starts before the kinetochores have divided so that replicated chromatids are held together and migrate to one spindle pole. Isodisomy will result if this happens to both members of a homologous
189
pair and the undivided pairs of chromatids move to opposite spindle poles. In this communication we report on the preponderance of spontaneous and induced isodisomy in Saccharomyces cerevisiae.
2. Material and methods
2.1. The test system and evaluation of results Diploid yeast strain D61.M was constructed by Zimmermann et al. (1985) to monitor loss of chromosome VII by the expression of three recessive markers present in a heterozygous condition: cyh2 is a recessive resistance marker which on a medium with 1.7 ppm cycloheximide allows selection of homo- or hemizygous cells arising in a culture of heterozygous sensitive cells; ade6 located on the other side of the centromere is a recessive mutant allele which prevents the accumulation of a red pigment in cells homozygous for ade2. leul is a recessive centromere marker which causes a requirement for leucine and is located on the same chromosome arm as cyh2. D61.M is also homozygous for ilvl-92 which causes a requirement for isoleucine. Consequently, requirements for adenine (because of the homozygous condition of ade2) and isoleucine can be used to identify D61.M cells. Three types of colonies are obtained after plating on a selective cycloheximide medium. Red colonies are the most frequent class of resistant colonies of which usually fewer than 10% express centromere marker leul. White colonies on this medium can express leul and they can result from loss of the chromosome carrying the dominant wild type alleles or alternatively from two events of mitotic recombination between the centromere and ade6 on one side and leul and the centromere on the other. This is rare because of the close centromere linkage of leul. White colonies not expressing leul cannot be caused by chromosome loss. Induction of chromosome loss is indicated when at least 30% of the white resistant colonies express leul (Zimmermann and Rohlfs, 1991; Zimmermann and Mohr, 1992). The criteria for the evaluation of the experimental
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data have been defined by Albertini and Zimmermann (1991) based on the analysis of tests with 111 chemicals.
mermann et al. (1985) even though many aprotic polar solvents induce chromosome loss with a continuous incubation at temperatures below the standard 28°C (Zimmermann et al., 1988).
2.2. Genetic analysis Presumptive chromosome loss segregants were analyzed by tetrad analysis using a de Fonbrune micromanipulator. All spore colonies were tested for the three diagnostic markers, trp5 a recessive marker causing a tryptophan requirement and located in repulsion to the markers ade6-1eul and cyh2. Additional heterozygous markers are recessive hisl causing a histidine requirement, MAL2 a dominant heterozygous marker for maltose utilization on chromosome III and on the same chromosome the two mating type alleles MATa and MATa. A strain carrying both these alleles cannot mate but does sporulate. All strains expressing only one of these alleles cannot sporulate but mate irrespective of their ploidy. Mating types were assayed by crossing spore clones with MATa and MATa type tester strains. These mating type alleles segregate regularly so that a sporal clone shows either a MATa or a MATa reaction. On the other hand, haploid strains carrying both alleles cannot mate but initiate sporulation. This combination of properties is diagnostic for a sporal clone carrying both alleles due to an original tri- or tetrasomy of the sporulated isolate. Cells trisomic for chromosome III are either M A T a / M A T a / M A T a or M A T a / MATa/MATa. They form spores which are MATa/MATa in addition to MATa/MATa or M A T a / M A T a and the normal type with MATa and MATa. The MATa/MATa spores cannot mate but can be induced to sporulate. Even though they do not form viable spores an unambiguous cleavage of the cells is indicative of attempted sporulation and thus of a usually trisomic condition of the original cell.
2.3. Mutagen-induced induction of chromosome loss Chromosome loss was induced according to the cold shock protocol of 4 h at 28°C, followed by an overnight storage in an ice bath and a final incubation period at 28°C as described by Zim-
2. 4. Media used The media used and preparation of cultures have been described by Zimmermann et al. (1985).
3. Results
Chromosomal malsegregation in diploid cells is considered to result in aneuploidy based on the observation of monosomics (chromosome loss) and trisomics (chromosome gain) in cytological preparations. Based on this common knowledge, the first genetic analysis of the "monosomic" segregants obtained by Parry and Zimmermann (1976) in strain D6 and in strain D61.M yielded an unexpected result (Zimmermann et al., 1985). The majority of the isolates, expressing the three diagnostic recessive markers cyh2, leul and ade6, obtained in the controls and after induction with bavistan, ethyl acetate and methyl ethyl ketone were perfectly diploid. The expected monosomics for chromosome VII should result in tetrads containing two dead spores, those lacking chromosome VII, and two viable spores with a complete haploid set of chromosomes. The results of a genetic analysis of the expected monosomics is shown in Table 1. Only about 10% of the isolates showed this pattern. However, about 10% of those isolates which were perfectly diploid segregated spores which could not mate with MATa or MATa tester strains but could be induced to sporulate. This indicated that the observed "chromosome loss" was associated with a high frequency of chromosome gain resulting in a trisomic condition of chromosome III. Non-mating spore cultures derived from diploid strains are extremely rare and occur definitely at frequencies below 1 in 1000. Consequently, selecting for cells expressing the three diagnostic markers in fact selects for isodisomy frequently associated with malsegregation for additional chromosomes.
F.K~ Zimmermann / Mutation Research 306 (1994) 187-196
In order to identify the spontaneous genetic events leading to the simultaneous expression of the three diagnostic markers cyh2, leul and ade6, 5-ml volumes of a rich yeast extract-peptone-glucose medium were inoculated with approximately 1000 cells of strain D61.M. This inoculum is much smaller than the critical population size expected to contain cells with chromosome loss. The spontaneous frequencies of such cells have varied in control platings on the selective cycloheximide medium between 0.09 5< 10 -6 and 3.01 × 10 -6 (Zimmermann and Mohr, 1992). These 5-ml cultures were grown to the late exponential growth phase to a total population size of about 5 × 108 each and samples were plated on the selective cycloheximide medium. Clones derived from only one colony expressing the three diagnostic markers per culture were sporulated and tetrad analysis performed. The results obtained with 51 isolates are shown in Table 2. Only one isolate showed the expected two dead to two viable spores in all of nine tetrads. Non-mating spores were observed in four additional cases indicating that the selected colony was trisomic for chromosome III. Tetrads with fewer than four viable spores occur at low freTable 1 Genetic analysis of Saccharomyces cerevisiae segregants showing chromosome loss Origin of segregants
Number analyzed
Control 66 Control a 8 Bavistan b 12 Ethyl acetate b 20 Methyl ethyl ketone b 14 Propionitrile 20 min 24 Propionitrile 660 min 26 Total 170
Monosomics Trisomy-3 3 0 3 1 2 3 1 13
5 0 1 2 2 4 4 18
Number analyzed, total number of chromosome loss isolates tested; monosomics, isolates where all tetrads showed a two viable/two dead spore segregation; trisomy-3, isolates which segregated non-mating sporal clones. Control, colonies from various control platings including those from Table 2. Propionitrile at 17.91 mg/ml in the cold shock experiment reported in Table 3:20 min, sample plated at 20 min; 660 rain, sample plated at 660 min after transfer from ice bath to 28°C. a Data from Parry and Zimmermann (1976). b Data from Zimmermann et al. (1985).
191
Table 2 Genetic analysis of presumptive chromosome loss colonies of yeast strain D61.M. Segregation pattern of viable versus dead spores Segregation pattern
Number of isolates
1. Asci containing only mating spores 4:0 4:0 4:0 4:0 3:1 2:2
3:1 3:1 2:2
2:2
2:2
5 24 4 13 1 1
2. Asci containing non-mating spores 4:0 3:1 4:0 3:1 Total
2:2
2 2 51
These isolates arose in independent cultures which were started with about 1000 cells. An average of 10 asci was dissected per isolate. 4:0, four viable spores per ascus; 3: 1, three viable and one dead spore; 2 : 2, two viable and two dead spores; non-mating spore colonies could be induced to initiate incomplete sporulation.
quencies after sporulation of normal diploid strains. However, an increase in dead spores indicates the presence of genetic damage or chromosomal rearrangements especially if there is more than one dead spore per tetrad. There were 12 isolates which yielded a few tetrads with only two viable spores. This could indicate that the selected event, loss of one of the chromosome VII homologs, was associated with additional genetic damage. However, it was perfectly obvious that chromosome loss leading to a monosomic condition was rare. If one of the chromosome VII homologs was lost, this loss was compensated by a duplication of the retained homolog. Induction of chromosome loss in diploid nuclei has been extensively studied in Aspergillus nidulans. As shown by K~ifer (1988), trisomics grow poorly and are unstable. One of the supernumerary chromosomes is lost and this can result in isodisomic diploids. Consequently in this organism, isodisomy definitely results from a first event of chromosome gain resulting in a trisomic condition followed by a random loss of one the the three homologs reconstituting a diploid constitution. Chromosomal malsegregation in Aspergillus is so frequent that non-selective plating is possible.
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Chromosomal malsegregation in yeast is a rare event. The frequency of spontaneous loss of chromosome VII detected on a non-selective medium w a s 4 . 8 5 X 10 - 6 ( Z i m m e r m a n n and Mohr, 1992) and the highest induced frequency detected by selective plating was 1.7 x 10 -3 (Mayer and Goin, 19871. Cells of strain D61.M divide once or rarely twice when plated on the selective cycloheximide medium unless they express recessive cyh2. Nevertheless, chromosome loss can be induced by chemicals at concentrations which inhibit cell division or even kill cells. However, chromosome loss cannot be induced when cells are incubated in a medium without adenine and isoleucine which does not support growth (Table 5 in Zimm e r m a n n et al., 1988). The "cold shock" protocol - including an incubation period in a growth medium at 28°C, followed by an incubation in an ice bath and a subsequent incubation period again at 28°C - was calibrated by Z i m m e r m a n n et al. (1985, 1988). The inducing chemical has to be present during all three incubation phases. Cycloheximide resistant white colonies requiring leucine - the presumptive chromosome loss types - appeared rapidly after the cultures previously kept in an ice bath had been warmed up to 28°C. This and the lack of growth between addition of the chemical and the time of plating demonstrated that no net growth occurred during the entire incubation period and the limited residual growth on the selective medium suggested that induction of chromosome loss requires only one or a few rounds of cell division and segregation of daughter nuclei. Table 3 shows an experiment with propionitrile which was performed in order to test whether the resistant colonies appearing upon plating at 20 min after storage in the ice bath, no cell divisions up to the time of plating, and at 660 min, cell titer increased 2.2-fold, differed in their content of resistant isolates with a two v i a b l e / t w o dead cell segregation. Only three of the 24 isolates from the 20-min plating and only one of the 660-min plating showed this type of segregation, but at both sampling times four colonies showed the additional chromosome III trisomy signalled by non-mating spore progeny (Table 1).
Table 3 The appearence of propionitrile-induced chromosome loss segregants during incubation at 28°C following overnight cold shock in Saccharomyces cereuisiae Sample
Titer
Control Start 5.7 Before ice 42.9 After ice 40.7 200 min 74.4
x 1.0 7.5 7.1 13.0
Propionitrile 17. 9 m g / ml Start 6.6 Before ice 7.5 After ice 8.9 1.0 20 min 40 min 60 min 7.9 0.9 80 min 100 min 15(1 min 10.7 1.2 300 rain 14.4 1.6 420 rain 19.6 2.2 660 rain 20.0 2.2
Colonies 3 10 2 30 1 9 2 383 409 440 563 589 820 1 212 1392 1 618
X 1.0 3.3 0.7 10.0
1.(1 192 204 22(1 282 295 410 607 696 809
Frequency
x
1.72 0.78 0.33 1.34
1.0 0.5 0.2 0.8
0.51 4.01 0.75
1.0
186.44
246
255.45 281.02 243.36 269.67
341 375 324 360
Titer, colony-forming u n i t s x l 0 ¢ ' ; Colonies, white colonies (test for expression of leul only in controls); Frequency, frequency of white colonies per 106 colony-forming units; x , factor increase; Start, determinations at the beginning of the experiment; Before ice, after 4 h at 28°C; After ice, before transfer to 28°C. 20 min up to 660 min refers to time of incubation at 28°C after incubation in ice.
Cell titers in these experiments were determined as colony-forming units which is not necessarily the same as an actual hemocytometer count. Separation of the mother and newly budded daughter cell does not immediately follow the completion of cytokinesis so that a growing diploid yeast culture contains in addition to single cells also doublets of a mother and her completed daughter cell. They all, upon dilution and plating, form a single colony. As shown in Table 4, in untreated controls the titers determined by hemocytometer counts are about 1.4 times higher than the ones based on colony-forming units. The titer of colony-forming units at the dose with the highest induction of chromosome loss was the same as in the control at the start of the experiment. However, the hemocytometer count showed that the cell titer had increased threefold. This indicated that at least one cell division had oc-
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
curred during the course of the experiment, and in a few cases one or very few more. Expression of a recessive resistance by a malsegregational event requires residual growth and division. Therefore, cells were treated with 18.8 m g / m l propionitrile in a cold shock experiment. Part of the ceils were plated directly on the selective medium after the usual 4 h incubation at 28°C. The rest of the culture was centrifuged and the sedimented cells resuspended in fresh medium and incubated for various lenghts of time up to 8 h before plating. Table 5 shows that the cell titer increased 10-fold and the frequency of the isodisomics decreased by about one third. This demonstrated clearly that additional cell divisions before selecting for resistance did not enhance the frequency of isodisomics. The observed decline in the frequency of isodisomics could be caused by a reduced growth of these variants. C h r o m o s o m e loss frequencies in Saccharomyces cerevisiae are too rare to be accurately studied without selective plating. However, pooled data from non-selective plating from control and experimental cultures showed that 79 of 409 white colonies expressed leul and cyh2 (19.3%) and 14 white sectors of 79 red-white sectored colonies (17.7%) simultaneously expressed these markers. In only one of the red-white colonies did the red
Table 4 Cell titers determined by hemocytometer cell counts and as colony-forming units in control and propionitrile-treated cultures of Saccharomycescerevisiae Dose
CFU
Hemocyt
Ratio
Frequency
Control 15.1 mg/ml 1.70 mg/ml 1.88 mg/ml 2.07 mg/ml 2.44 mg/ml
45.7 41.7 18.9 7.4 7.9 5.3
88.5 92.2 55.6 24.9 22.8 16.1
0.52 0.45 0.34 0.30 0.35 0.33
1.00 13.19 125.40 318.02 140.02 64.5
The cold shock protocol was followed. Starting titer in colony-forming units 7.2×106. CFU, colony-forming units;
Hemocyt, microscopic count, a bud of about three quarters the size of the mother cell was considered to be a functionally separated daughter cell; Ratio, CFU titer divided by hemocytometer titer; Frequency, white resistant colonies (propionitrile) and white resistant colonies expressing leul (control) per 10 6 colony-formingunits, mg/ml refers to propionitrile concentration.
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Table 5 The effect of non-selective post-treatment growth on the recovery of chromosome loss in Saccharomycescerevisiae Growth (h)
Titer ( x l 0 6)
x
Colonies
x
Frequency (×10 -6)
×
Control 0
78.2
1.0
5
1.0
1.07
1.0
8
203.0
2.6
30
6.0
0.49
0.5
1.0 555 1.2 705 1.9 795 2.4 1130 5.2 1911 10.1 3705
1.0 1.3 1.4 2.1 3.4 6.7
304.95 329.44 232.46 255.66 203.51 200.81
1.0 1.1 0.8 0.8 0.7 0.7
Propionitrile 18.8 mg / ml 0 1 2 3 5 8
6.1 7.1 11.4 14.7 31.5 61.5
The cold shock protocol was followed. Growth hours, hours in medium without cycloheximide; Colonies, absolute numbers of white colonies in experimental samples and of white colonies expressing leul in control samples, x, factor of increase over 0-h platings.
sector express the recessive marker trp5 diagnostic for the other homolog and the white sector leul and cyh2. This again indicated that the generation of induced chromosome loss does not require many division cycles.
4. Discussion Selection for chromosome loss in diploid mitotic cells in Saccharomyces cerevisiae showed uniparental disomy or isodisomy to be 10 times more frequent than monosomy. As pointed out in the Introduction, uniparental dis0my contributes considerably to h u m a n genetic disease. The potential importance of fungal systems for the detection of chromosomal malsegregation is that they are based on the expression of recessive diagnostic markers and thus they signal not only aneuploidy but also isodisomy. This aspect was not considered in the European Community Aneuploidy Project (Parry and Sors, 1993) which included an evaluation of Aspergillus nidulans and Saccharomyces cerevisiae fungal systems (Parry, 1993). There is a strong correlation between the abilities of an agent to interfere with mammalian tubulin aggregation in vitro and to induce mitotic
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F.K. Zimrnermann / Mutation Research 306 (1994) 187-196
chromosome loss in Saccharomyces cerevisiae strain D61.M as summarized by Albertini and Zimmermann (1991). Moreover, the fact that cold shock and incubation at lower temperature enhances induction of chromosome loss by aprotic polar solvents (Zimmermann et al., 1988) strongly indicates that microtubules a n d / o r tubulins are the primary cellular targets for induction. The mechanistic question is whether isodisomy results from two sequential reactions as shown for Aspergillus nidulans (see K~ifer, 1988) or whether there is a special and as yet unidentified novel mechanism as suggested by Resnick and Zimmermann (1987). A sequential mechanism with a primary event resulting in a trisomic condition followed by a gradual selection of secondary isodisomic segregants is not very likely in Saccharomyces cereuisiae because chromosome loss can be induced at high frequencies with doses which completely inhibit growth. Morever, cells are always plated out of the treatment medium onto the selective cycloheximide medium with no or only very limited intermediate growth in non-selective media. It is obvious that at least one error-struck mitosis must be completed for chromosome loss to occur. This is documented by the fact that chromosome loss cannot be induced in a medium without isoleucine and adenine which are required for growth of strain D61.M (Table 5 in Zimmermann et al., 1988). Direct plating on the selective medium blocks growth of sensitive cells after one or at most two rounds of cell division. Only those which express cyh2 continue to grow. A two step process of the induction of isodisomy via a trisomic intermediate state and a final accidental and random chromosome loss resulting in an isodisomic diploid could be accommodated with a first round of nuclear division before plating and a second round on the selective medium. However, if loss of a homolog in a trisomic cell is accidental it would not only occur in the first division cycle after the formation of the trisomic state but also in later divisions. Non-selective growth after the induction period should then increase the incidence of cycloheximide resistant isodisomics. As shown in Table 5, this is definitely not the case. Therefore,
it is likely that the postulated two sequential reactions of chromosome gain, causing a trisomic condition, and chromosome loss, restoring a disomic condition, are tightly linked. This view is also supported by the results obtained by plating on non-selective media. There, presumptive chromosome loss segregants expressing the three diagnostic markers can be found at equal frequencies among completely white and also red-white sectored colonies. This shows that a second chromosomal malsegregation event leading to a final isodisomy must occur very early or concomitantly with the first event. Otherwise, the frequency of chromosome loss would be higher among sectored colonies on a non-selective medium. On the other hand, the malsegregational mechanism proposed by Resnick and Zimmermann (1987) based on a lack of kinetochore division before the start of anaphase requires only one nuclear division to create an isodisomic state. Mayer and Aguilera (1990) constructed a series of triploid and tetraploid derivatives of D61.M with variable numbers of chromosome VII recessive, ade6-leul-cyh2, homologs. They found that spontaneous loss of the hornolog with the dominant markers in the normal diploid averaged 7.58 x 10 -7, loss of the only dominant homolog from the triploid 1.42 × 10 -5 and in the tetraploid 3.99 X 10 -4. This shows that the rate of chromosome loss increases with ploidy. Moreover, chromosome loss could even be detected in a tetraploid carrying three dominant homologs and the computed frequency in this case was 3480 times higher than in the heterozygous diploid. This assumption is based on the classical chromosome loss concept. Extrapolating from the simple chromosome loss frequencies in the diploid would predict chromosome loss to be virtually undetectably low in the tetraploid with three "dominant" homologs. It is conceivable that occasional spontaneous or induced defects in the assembly a n d / o r function of the spindle fiber apparatus not only lead to the "classical" types of chromosome loss or gain. A spontaneous or induced retardation of kinetochore separation before anaphase could not only explain the data presented here but also
F.K. Zimmermann / Mutation Research 306 (1994) 187-196
provide a plausible explanation for those reported by Mayer and Aguilera (1990). Isodisomy may be caused by a novel type of failure of the system for chromosome segregation and cause a re-assortment of chromosomes without an intermediate state of cytologically detectable aneuploidy. The final result will be the elimination of one of the two homologs with a concomitant duplication of the retained homolog. The test system for chromosome loss in Saccharomyces cerevisiae allows for a ready detection of such events.
5. Acknowledgements I would like to thank M.A. Resnick, NIEHS, Research Triangle Park, NC, for many stimulating discussions which led to the experiments reported in this publication, and also F.E. Wiirgler, Institut fiir Toxikologie, Eidgen6ssische Technische Hochschule und Universit~it Ziirich, Switzerland, for drawing my attention to the importance of uniparental disomy.
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