differential mitotic stability yeast disomes derived … · 2003-07-30 · differential mitotic...

DIFFERENTIAL MITOTIC STABILITY OF YEAST DISOMES DERIVED FROM TRIPLOID MEIOSIS

DOUGLAS CAMPBELL, JOHN S. DOCTOR, JEANE H. FEUERSANGER AND

MARK M. DOOLITTLE

Department of Biology, Holy Cross College, Worcester, Massachusetts 01610

Manuscript received November 3, 1980 Revised copy received April 27,1981

ABSTRACT

The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies em- ployed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses ( N f l + N ) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability.

ISOMIC haploids ( N f 1) of Saccharomyces cerevisiae have proved useful Din genetic mapping (MORTIMER and HAWTHORNE 1973; WICKNER 1979; HILGER and MORTIMER 1980) , in the isolation of mitotic and meiotic recombination-defective mutants (ROTH and FOGEL 1971; RODARTE-RAMON 1972; RODARTE- RAMON and MORTIMER 1972; FOGEL and ROTH 1974; SANFILIPPO 1976; MALONEY and FOGEL 1980) and in a variety of meiotic segregational analyses (SHAFFER et al. 1971; CULBERTSON and HENRY 1973). During vegetative growth, disomes lose at low frequency one of the two homologous chromosomes (N+l+N); yet, with one exception, the mitotic stability of single disomes has not been determined (CAMPBELL, FOGEL and LUSNAK 1975).

Disomes can be readily recovered among the meiotic segregants of triploids (PARRY and Cox 1970). Since segregation of the three homologs at meiosis I is expected to be two-by-one, half of the meiotic products should be disomic (N4-1) for any given chromosome and half should be haploid. The expected independent segregation of each homologous triad means that meiotic segregants can have a chromosome constitution ranging from completely haploid to completely diploid. Genetics 98: 039-255 June, 1981.

240 D. CAMPBELL et al.

Ordinarily, such segregants will be disomic for one or more chroinosomes ( N + l + l + . . .).

Disomy among the meiotic segregants of triploids can be detected by crossing the segregants to haploid testers carrying recessive indicator markers. The resultant hybrids will be diploid and trisomic for one or more chromosomes ( 2 N + l + l + . . .). If the segregants are wild type for the indicator markers, the diploids will have the genetic configuration +/m, if the segregant is haploid for the marked chromosome, or f /+ /m, if the segregant is disomic for the marked chromosome. These two possibilities can be distinguished by tetrad analysis of the hybrid diploid. Segregation of the indicator will be 2+:2m in the first case; frequent 4+:0m and 3+: Im segregations will be found in the second case.

This paper presents a comparative analysis of disome recovery and stability within the yeast genome, using the method outlined above. Previous work (PARRY and Cox 1970; MORTIMER and HAWTHORNE 1973; MORTIMER, personal communication) has suggested that the incidence of disomy among the meiotic segregants of triploids may vary from one chromosome to another. Differential disome recovery could be due to departures from the expectcd two-by-one meiotic segregation of homologs in the triploid or (more likely) to differences in the post-meiotic mitotic stability of disomy among the several yeast chromosomes.

Comparisons of disome recovery and stability for all 17 yeast chromosomes (HILGER and MORTIMER 1980) may not be wholly meaningful if several haploid tester strains o r triploids have to be used to cover the entire genome. Ideally, disomy should be isolated and identified among the segregants of a single triploid using a single multiply marked haploid tester. This would assure that disome recovery after triploid meiosis occurs in a highly uniform genetic background. We have therefore constructed inbred triploids in which all homologous centromeres are identical by descent. Since a separate recessive marker is needed to identify disomy for each chromosome, we have also constructed single haploid testers carrying indicator markers for all 17 chromosomes. The results establish significant differences in disome recovery frequencies among the yeast chromosomes and suggest that these differences are wholly attributable to variations in individual disome mitotic stability.*

MATERIALS A N D METHODS

Media: Ingredients are given in amounts per liter of distilled water. Media were solidified with 15 g/l agar (YEPD, YPGAL, sporulation medium), U) g/l (synthetic media) or 22.5 g/l (GNA). Recipes for YEPD, GNA and sporulation medium have been described by CAMP- BELL (1980). YPGAL contained 6 g Difco Bacto-peptone, 12 g Difco yeast extract, 20 g D-galactose (autoclaved separately), complete supplement as listed for synthetic complete medium; brom- thymol blue was added as a pH indicator to the agar after autoclaving (2.7 mg/l) as an un- sterilized ethanol (95%) solution (1% wJv). Synthetic complete medium (SC) contained 20 g

Note on chromosome nomenclature: Heretofore, the genetic linkage groups of Saccharomyces cerevisiae have been designated by Roman numerals (“chromosome I to chromosome XVZI”). When only a few chromosomes are under consideration. this format is efficient and unambiguous. In our judgment, however, when all 17 yeast chromosomes tre simultaneously under consideration, as in the present case, the Roman numeral format becomes cumbersome and difficult to follow. We have therefore adopted for this paper a modified nonienclature based on Arabic numerals as follows: ( 1 ) for sentence initiation, “Chrommome 14”; (2) for internal sentence placement, the shorter form “chr. 14”; and (3) for lists, “chromosomes I , 5, 12 and 27.”

DISOME MITOTIC STABILITIES 241 D-glucose, 1.7 g Difco yeast nitrogen base without amino acids or ammonium sulfate, 5 g ammonium sulfate, plus a nutrient supplement as follows (mgJ1 final concentrations) : adenine sulfate 20, L-aspartic acid 250, L-arginine-HC150, L-histidine-HC120, L-isoleucine 50, L-leucine 50, L-lysine-HC1 50, L-methionine 20, L-phenylalanine 50, L-threonine 300, L-tryptophan 50, L-tyrosine 50, uracil 20, L-valine 50. Nutrient supplement was added to the minimal agar after autoclaving as a filter-sterilized aqueous solution. Dropout media were synthetic complete medium lacking single nutrilite supplements. Asp5 was detected on synthetic medium lacking both aspartic acid and threonine (DE ROBICHON-SZULMAJSTER, SURDIN and MORTIMER 1966). Aro7 was detected on synthetic medium lacking tyrosine. All nutrient supplement solutions were adjusted to pH 5.2 to 5.4 with KOH. GNA medium was used solely for ascus dissections.

Yeast strains: Genotypes of the triploid, diploid and haploid strains of Saccharomyces cerevisiae used in this work are listed in Table 1.

Construction of 17M: The 17M(a) and 17M(a) haploid testers are meiotic segregants of diploid strain GT153, heterozygous for 5 and homozygous for 12 of the 17 markers (Table 1). GT153 was constructed by standard methods from haploid strains obtained from the Yeast Genetic Stock Center, Berkeley, CA, and from strains in our collection. Sources of the several genetic markers in GT153 [and in 17M(a) and 17M(4a)] are listed in Table 2. These markers are not a random selection of those available. They were chosen specifically in order to reduce the physiological burden that any multiply marked strain is sure to suffer. By choosing markers with common phenotypes, the number of different nutritional requirements is kept purposely low. An adel ade2 double mutant strain, for example, requires no more adenine for growth than either single mutant alone. Hence, a potential technical difficulty-that the many nutritional requirements of a multiply-marked haploid strain cannot all be alleviated simultaneously by nutrient supplements to a defined, synthetic growth medium-is significantly reduced. Among the 17 chosen markers, there are 11 different nutritional requirements. Except for ade2 and met2, all the markers are linked to their centromeres.

All accessions were crossed to haploid reference strains SI81 or SI85 (Table 1). These strains (from THOMAS MANNEY via SEYMOUR FOGEL) confer good sporulation and spore germination properties upon diploids of which they are a part. This outcrossing procedure was necessary to obtain the desired markers free of undesired genetic and phenotypic properties. In some cases, meiotic segregants of these diploids were again outcrossed to the reference strains. Among the undesired properties we encountered and selected against were: (1) structural heterozygosity

TABLE 1

List of strains

Ploidy Strain

3N am (wya

2N GT153

N GT153-6A (“1 7Ma”)

GTl53-63B (“17Ma”)

S181 S185

GT16045C GT161-31B

GT160-34B

GTl61-67D

Genotype

a/a/a; adeb/aded/ade6 a/a/a; adeb/adeb/ade& adel gal7 leu,? a trp1 ura3 his2 leu1 arg4 -l- adel jf leu2 01 trpl ura3 his2 leu1 argl his6 ilv3 f f lys7lysPade2 f met2 ilv3 met14 asp5 lys7 lys9 ade2 aro7 met2 adel gal7 leu2 a trpi ura3 his2 leu1 arg4 his6 ilv3 met14 asp5 lys7 lys9 ade2 aro7 met2 adel gal7 leu2 a trpl ura3 his2 leu1 arg4 his6 ilu3 met14 asp5 lys7 lys9 ade2 aro7 met2 a adeb leu1 lys7 hisX a adeh leu1 a adel leu2 his6 met14 lys9 N adel leu2 his6 met14 lys9 a ade2 leu1 his2 met2 lys7 asp5 a ade2 leu1 his2 met2 lys7 argl


TABLE 2

Sources of genetic markers

Chromosome Marker Source (strains)

I 2 3

4

5 6 7

8

9 10 I1 12 13

14 15 16 17

ade1 gd7 leu2

trpl

ura3 his2 leu1

adeb

his6 ilu3 met14 asp5 lys7

lys9 ade2 aro7 met2

Berkeley (X3 127-2A) Berkeley (uia A. HOPPER) (G7-a) Berkeley (X3104-8C, X3255-7C) This lab (5037-5A, 5037-5B, 5003-21A-1A) Berkeley (X1437-8C, X1049-2B) This lab (5003-21A-1A) This lab (5037-5A, 5037-5B) Berkeley (X3255-7C, X3104-8C) Berkeley (X3127-2A, G7-a) Thislab (381, S185) FOGEL (X1687-16C) This lab (S181, S185) Berkeley (X3127-2A) FOCEL (XI 687-1 6C) Berkeley (X1437-8C) Berkeley (X3255-7C) Berkeley (X3104-8C) Berkeley (X 1049-2B) Berkeley (X3 127-2A) This lab (S181) Berkeley (JB101) Berkeley (X3255-7C, G70.) Berkeley (X3255-7C, X3104-8C) This lab (5003-21A-1A)

Strains designated “Berkeley” are from the Yeast Genetic Stock Center, Berkeley, CA. Strains from our own collection have been described previously (CAMPBELL, FOGEL and LUSNAK 1975).

(e.g., translocations) between our laboratory strains and those of other laboratories contributing to the Stock Center collection; (2) poor spore coIony morphology and growth, including marked colony size differences independent of nutritional genotype, failure to grow on synthetic media and dry, “flaky” colonies that replica plated poorly; (3) poor sporulation and spore germination, including several instances in which germination was invariably 2 live : 2 dead in every tetrad; and (4) hidden suppressors that were revealed only in crosses to other marked strains. Since the desired 17M testers are haploid, our construction strategy was to create diploids at each stage with as many markers homozygous as possible, to reduce the number of ascus dissections needed to find the haploid segregants desired for the next series of crosses. We constructed 168 hybrid diploids and analyzed 3391 meiotic tetrads to derive the multiply marked strains listed in Table 2.

Whether 17M is disomic for one or more chromosomes does not interfere with its effective- ness as an identifier of disomy. If 17M were disomic, it would have to be homozygous for the recessive marker on that linkage group. (In the course of 17M construction, we detected no instances of disomy for any linkage group that would be indicated by abnormal meiotic segregations.) A diploid formed by a cross between a disomic segregant from the triploid and a disomic 17M would be tetrasomic (2N+2) and +/+m/m for the chromosome in question. But meiotic segregation will still yield frequent 4f:Om and 3 f : l m tetrads, so that disomy will still be efficiently detected. A diploid formed by a cross between a haploid segregant from the triploid and a disomic 17M would be trisomic (2N+1), and +Jm/m; meiotic segregation will nearly always be 2+:2m.

DISOME MITOTTC STABILITIES 243 Triploid construction: Triploids were constructed as outlined in Figure 1. A diploid (1)

was constructed from reference strains SI81 and SI85 and from haploid segregants derived from the later stages of GT153 construction. This diploid was sporulated, and asci were dissected. Tetrads were chosen as shown in (2). The centromere-linked leu1 marker identifies the meiotic sister pairs; mating of sisters produces an inbred diploid (3) in which all homologous centromeres are identical by descent. This diploid is homozygous wild type for all markers carried in 17M. The diploid was plated for single colonies on YEPD, and the plates were exposed t o sublethal doses of ultraviolet light (254 nm, 84 JJm2 maximum) to induce mitotic exchange in the diploid between the mating-type locus and its centromere, producing aJa and a/a homozygous, mating-competent diploids (4). The mating-competent diploids were detected by complementation, recovered from the original (premating) plates, purified by streaking for single colonies and then mated to the appropriate meiotic sister from which diploid (3) was originally constructed to produce am and m e a triploids ( 5 ) in which all homologous centromeres are identical by descent.

The triploids were shown to carry no hidden aneuploidies (e.g., 3 N f l ) by crossing one of the parental spore colonies to 17M and demonstrating 2+:2m segregations for all 17 indicator markers. Since the triploid is constructed from sister spores, and since any hidden trisomy in diploid (3) wil always segregate N : N f l at the first meiotic division (CAMPBELL, FOGEL and LUSNAK 1975), determination that one sister spore colony is disome-free establishes that the other sister (and, hence, the triploid) is disome-free as well.

Disome recovery from triploid meiosis: The inbred triploids were sporulated and dissected. This procedure assures that every spore colony from the triploid will have arisen from a single spore, an assurance that cannot be guaranteed in random spore platings. The visible spore

1 meiosis

leu1 a leu1 a

a a segregation

a

a ar - (3N)

a

a FIGURE 1 .-Breeding scheme to produce inbred triploids in which homologous centromeres

are identical by descent. An inbred diploid (1) was sporulated and dissected. Meiotic sisters (2) were mated to produce a second diploid (3) that was induced by UV exposure t o undergo mitotic segregation, producing mating-competent aJa and diploids (4). These were then mated to the appropriate sister spore clones to produce the triploids ( 5 ) . (See MATERIALS AND

METHODS for further details.)

244 D. CAMPBELL et al. colonies were transferred to YEPD master plates, incubated 1 or 2 days at 30°, and then mated by cross-stamping with 17M(a) and 17M(a) haploid testers. After 1 day of incubation, the mating plates were replicated to adenineless medium. Diploids were thus selected by complementation between the ade6 marker carried by the meiotic segregants of the triploid and the adel and ade2 markers in 17M (Table 1). This procedure also allowed determination of the mating-type phenotype of each spore colony (Table 4). The complementation diploids were replica plated without further purification directly to sporulation medium. After 3-5 days incubation, the sporulation plates were stored in the cold and provided material for ascus dissection for several weeks with no detectable loss in spore viability.

Genetic structures of the trisomic diploids (and, hence, of the original spore isolates from the triploid) were based on tetrad analyses of dissected asci. In general, 11 asci from each diploid were dissected; when spore germination was poor, an additional 11 or 22 asci were dissected and characterized. Asci were dissected by micromanipulation on the surface of freshly poured (2 to 24 hr) GNA plates; the dissection plates (each accommodating 2.2 tetrads) served directly as the master plates for subsequent replica platings. In two experiments, 3260 asci from 279 trisomic diploids were dissected and analyzed. Six diploids were eliminated from consideration because of poor spore germination (<25%) and three for technical reasons.

Complementation tests: Meiotic segregations of the five marker pairs with common requirements (adel-ade2, leul- led, his2-his6, lys7-lys9, met-2-met14) were identified by complementation tests with the four multiply marked testers listed in Table 1. Inclusion of five phenotypically distinct markers in each tester pair simplifies the complementation tests such that four complementation crosses serve to identify all ten markers. Crosses were made on YEPD plates, which were incubated for 1 day, and then replica plated to the five appropriate dropout media. Met14 and lys7 are leaky markers and exhibit some ambiguity in scoring. We found, however, that incubation of the lysineless and methioneless complementation test plates at 35" noticeably improved the test resolution.

RESULTS

Experimental design: The experimental design to evaluate the spectrum of disomy among the meiotic segregants of triploids embodies several features in- tended to maximize recovery of information bearing on the genomic structure of the meiotic segregants themselves. First, inbred triploids were induced to sporulate, and asci were dissected. This assures that each spore colony arose from a single spore. Second, spore colonies were crossed without purification to 17- marker haploid testers, and diploids were selected by complementation. The diploids were immediately induced to sporulate without hr ther purification. Asci from these diploids were then dissected and tetrads analyzed in order to determine disomic configurations.

These procedures were based on the following scenario. A multiply disomic meiotic spore segregant of the triploid could be imagined to carry an aneuploid burden such that, upon germination, rapid proliferation of cells in the spore colony would be prevented or retarded. Under these conditions, a strong selective pressure may exist in favor of rapidly growing mitotic segregants that have lost one or more of the disamies present in the original spore. Repeated and non- identical chromosome-loss events of this kind may arise irdependently within the spore colony, with the result that the spore colony may contain numerous cell lineages with unique disomic configurations that only collectively reflect the disomic state of the original spore. Clonal purification of such a mixed colony at any stage in the disome recovery procedure could result in substantial loss of this col- lective information. Hence, by mating spore colony cells en masse to the 17M

DISOME MITOTIC STABILITIES 245

tester (s) , and by sporulating the resultant trisomic diploids for ascus dissection without purification, we optimize our chance of obtaining a reflection of the actual chromosome configuration of the meiotic segregant of the triploid. Finally, we suspect (without direct evidence) that the multiply trisomic diploids are mitotically more stable, per aneuploid chromosome, than the corresponding multiply disomic haploids; we have nonetheless stored these diploids in the ascal state, pending dissection and tetrad segregation analysis, in order to stop any further changes in aneuploid configuration (s) within each diploid mixture.

Since decisions on the disomic content of meiotic segregants of triploids are based on marker segregation patterns among tetrads derived from probable diploid mixtures, one for each spore isolate, disome detection sensitivity is necessarily a function of the number of tetrads analyzed. But it is also limited by (1) the frequency of mitotic exchanges between the indicator markers and their centromeres, which can lead to homozygosis and 4+: Om segregations, and (2) by the frequency of meiotic gene conversion of indicator markers, which can yield 3 f : l m segregations. In the first case, misidentification of disomy due to pre- meiotic mitotic exchange can be reduced by using centroniere-linked markers. Of the 1 7 indicator markers, all but two (ade2 and m e t 2 ) are centromere-linked. Chromosome 15 (ade2) exhibits one of the lowest disome recovery frequencies (Table 6), suggesting that any loss in disome detection sensitivity from this source must be small.

In the second case, however, the upper limit of marker gene conversion frequency sets of the lower limit of sensitivity for the detection of disomy. The most frequently converting markers in 17M are his2 (3+: Im and 5+: 3m ~ 8 . 7 X and arg4 ( 3 f : l m = 3.6 x (FOGEL et al. 1978). Our testing method dis- tinguishes between 3+: Im gene conversions and 5+: 3m postmeiotic segregations at arg4; his2 is identified by complementation, and 3+: l m and 5+: 3m segregations are not distinguished. This 5 to 10% noise level due to conversion of the most frequently converting indicator markers suggests, then, that a detection level of 10% (i.e., assignment of disomy for a given chromosome to the original spore when 10% of the tetrads analyzed exhibit 3+: Im or 4+:0m segregations) represents the maximal practical sensitivity achievable in this system. Accord- ingly, we have ordinarily dissected and analyzed 11 tetrads from each isolate and have assigned detection criteria as “10%’’ (one or more aberrant segregations), “20%” (two or more), or “30%” (three or more). Data analyses in subsequent sections of this paper are referenced to these detection criteria.

Given this form of data evaluation, one prediction of our scenario is that the detected frequency of disomy for all chromosomes should increase toward the expected maximum frequency of 0.50 as the detection sensitivity is increased. Moreover. differences in disome recovery frequency among the several chromo- eomes ought to persist, independent of detection criterion, if these are the result of inherent differences in disome mitotic stability.

Triploid meiosis: When yeast triploids are induced to sporulate and asci are dissected, most of the spores fail to produce visible spore colonies. Table 3 presents spore viability data for meiotic tetrads of two inbred triploids that differ genotypically only at the mating-type locus. The data have been arranged according to

246 n. CAMPBELL et al.

the numbers of spores in each tetrad that produced spore colonies. The three data sets are internally consistent and have been pooled. The overall viability frequency is 0.1 79 [665/4( 927)].

That the great majority of spores failed to produce visible colonies does not mean that these spores failed to germinate. For example, among 3M spores (86 tetrads) from the a m triploid examined 7 days after ascus dissection, we saw 53 (15.4%) visible colonies (2 0.5 mm diameter), and microscopically (150X): 61 microcolonies consisting of 20 to several hundred cells, I28 groups of 1-10 cells, 52 spores with a single bud and 50 ungerminated spores. Hence, most of the spores that did not produce a visible colony (241/291 = 0.83) did in fact germinate. The overall spore germination frequency after triploid meiosis (294/344 = 0.85) is comparable to that found in our experience for typical normal diploids. The spore viability patterns presented in Table 3 are thus not attributable to poor spore germination, but rather to the failure of germinating spores to proliferate into visible spore colonies.

Segregation of MAT in triploid meiosis: The sole heterozygous marker in the triploids is the mating-type locus ( M A T ) . Segregation of the mating-type locus among the viable meiotic segregants can provide a test of the expectation that triploid homolog segregation at meiosis I is two-by-one, since it leads to the prediction that half the segregants should be disomic for the chromosome (chr. 3 ) bearing the mating-type locus. The same analysis also provides information on whether the subsample of testable segregants (i.e., those that produced a visible spore colony) is representative of all meiotic segregants of the triploid in terms of randomness of disome recovery.

The mating-type phenotypes of viable spore colonies recovered from triploid meiosis are shown in Table 4. The nonmating phenotype unambiguously signals the disomic genotype a/a. As expected, the a phenotype predominates among the segregants of triploid aacy, and the 01 phenotype predominates among the segregants of triploid ma. The a and cy phenotypes clearly consist of both haploid a and disomic ala, and haploid cy and disomic ./cy genotypes, respectively. An approxi- mate partition of these phenotypes into haploid and disomic configurations can

TABLE 3

Spore viability patterns among tetrads from lriploid meiosis

Colony forming:Non colony forming Total Total Percent 3N 4:O 3.1 2:2 1.3 0:4 spore colonies asci viability

aaa: 3 9 48 72 198 207 330 15.7

aaa 1 7 50 83 148 208 289 18.0

aail 0 19 55 83 151 250 308 20.3 Total 4 35 153 238 497 665 927 17.9

(Expt. 1)

(Expt. 2)

(a%.)

Asci from the two inbred triploids were dissected on the surface of GNA plates. Visible spore colonies ( 2 0.5 mm diameter) were scored after 16 days incubation at 30".


TABLE 4

Mating-type phenotypes of tesled uiable spore colonies from triploid meiosis

3N Phenotype

a a Nonmating Total

a m (Expt. 1) am (Expt. 2) m a

105 58 27 190 102 37 69 208 55 132 51 238

PhenDtypes “a” and “a” include both haploid a and disomic a/a, and haploid a and disomic Q/OI genotypes, respectively. The nonmating phenotype is disomic genotype a /a .

be had by reference to those mating-competent segregants from triploid am that were tested for disomy by tetrad analysis. (No segregants from aaa were tested by tetrad analysis.) From triploid a m (Expt. I), 20 of 58 phenotypic a’s were disomic a/a, and 12 of 41 phenotypic a’s were disomic a/a; from triploid aaa! (Expt. e), 22 of 93 phenotypic a’s were disomic a/a, and 7 of 33 phenotypic a’s were disomic a/a. Application of these proportions to the data in Table 4 results in the geno- typic assignments shown in Table 5. The estimated proportion of disomy for chr. 3 among the meiotic segregants is 0.45 (181/398); this value does not differ significantly ( P > 0.05) from the expected proportion of 0.50.

Comparison is also made in Table 5 between meiotic segregation of the mating- type locus inferred for the triploid, and the meiotic segregation pattern observed in a group of analogous diploids trisomic (2NS-1) for chr. 3. In trisomic diploids, meiotic segregation has been shown unambiguously to be two-by-one at MI (CAMPBELL, FOGEL and LUSNAK 1975). Tetrad data €or the trisomic diploids are given in the legend to Table 5 and are entered in the Table partitioned by spore genotype. The fractional proportions of the two data sets are in good agreement except for the 3-fold excess, among segregants of the triploid, of the minority a/a class. Thus, the two data sets, taken in entirety, differ significantly ( P < 0.001), but this difference disappears when the discrepant a/a class is omitted ( P > 0.05). The ,./a! disomic class arises from meiotic segregations in which exchange between the centromere and the mating-type ~QCLIS is accompanied by co-migration of the two recombining homologs to the same pole (tetrad class E2 in the legend to Table 5). The differences observed between triploid and trisomic diploid meiotic segregation may therefore reflect differences only in the relative frequencies of this comparatively rare meiotic event.

The main conclusions from this analysis are that (1 ) triploid homolog segregation at meiosis I is clearly two-by-one, and (2) the subset of colony-forming (and so testable) meiotic segregants of the triploid is an unbiased sample of all segregants (for at least one chromosome) with respect to the incidence of disomy. Disome recovery from triploid meiosis: We determined by tetrad analysis the

complete disomic content of 270 spore colony meiotic segregants of the aaa triploid in two independent experiments. In experiment 1,102 (58 a’s, 41 a’s, 3 a/a’s) of 190 isolates (54%) were characterized; in experiment 2, 168 (93 a’s, 33 a’s, 42 a/a’s) of 208 isolates (81 %) were characterized.

248 D. CAMPBELL et al. TABLE 5

Distribution of mating-type genotypes among uilrble meiotic segregants of the triploid and of the corresponding trisomic diploid

Expt. Spore colony genotype

a a/a 01 a/a a /a Total

3N (aaol) 69 36 41 17 27 190 (Expt. 1) 3N (am) 78 24 29 8 69 208 (Expt. 2) Total 147 60 70 25 96 398 Fraction 0.369 0.151 0.176 0.063 0.241 2N + 1 (am) 1200 654 586 68 1064 3572 Fraction 0.336 0.183 0.164 0.019 0.298

The mating-type phenotypes of recovered meiotic segregants of the aaa triploid were partitioned genotypically according to the method described in the text. The comparison data derive from dissection and analysis of 893 tetrads from a group of interrelated diploids trisomic (2N4-I) for chr. 3. Assignment of mating-type genotypes was based on: (1) mating-type phenotypes, (2) knowledge that, in these trisomic diploids, segregation is invariably two-by-one (N+1 : N ) at meiosis I (CAMPBELL, FOGEL and LUSNAK 1975): and (3) correlations with segregation at heteroallelic loci on the opposite chromosome arm, through which disomy can be ascertained directly. The data:

Tetrad type PI P2 El E2

N-l-1 a/= a / a a/a a/a a h a/a a /a a h N i - 1

Number found 127 366 332 68

N a a a a N a a ff a

In experiment 1 , we found that all of the isolates exhibited segregation patterns indicative of disomy for chromosome I at the 30% detection level (see Experi- mental Design section). The triploid was shown not to be aneuploid (3N-l-1) for chr. I (see MATERIALS AND METHODS). ' f i e 17M testers, however, were found to carry a recessive suppressor of adel, an indicator marker for chr. I (Table 2). The presence of the ade2 (chr. I S ) marker in the 17M haploids prevented detection of the suppressor in the complementation tests used to identify the two ade markers. We therefore isolated new, suppressor-free 17M testers from diploid strain GT153 (Table 1 ) for the second experiment.

The cumulative incidence of disomy for all 17 yeast chromosomes is presented in Table 6. Data for chr. I (ade l ) are from experiment 2 only. Also included in the data are the results of tests of 45 isolates that were nonmating and disomic a/. (chr. 3 ) . These were characterized by mixing them with the 17M haploid tester (s) and selecting by complementation for diploids resulting from rare matings between 17M and mitotic segregants of the disome that had lost one of the two chromosomes 3 , thereby becoming haploid a or (Y and mating competent (36 of 45), or that had undergone mitotic exchange to produce mating-competent a/a or disomes (9 of 45) (CAMPBELL, FOGEL and LUSNAK 1975). This data subset was analyzed separately for disome recovery frequencies of the 16 remaining chromosomes; the results were identical to those in the data taken as a whole. These data have therefore been included in Table 6, but with the chr. 3 entries omitted.


TABLE 6

Disome recovery among meioiic segregants from the triploid

Chromosome ,(marker)

1 ( d e l ) 2 (gal7) 3 (leu2) 4 (trp1) 5 (ura3) 6 (his.!?) 7 ( l e d ) 8 (ark741 9 (his6)

10 (ilu3) 13 (met l4) 12 (asp51 13 (lys7) 14 (lys9) 15 (ade2) 16 (aro7) 17 (met.!?)

Cumulative number of disomes found Tested 30% 20% 10% isolates No. Freq. No. Freq. No. Frep

168 11 0.065 32 0.190 57 0.339 270 225 270 270 270 270 270 270 270 270 270 270 270 270 270 270

57 28 60 37 10 6

29 38 4>3 59 65 68 41 10 35 33

0.21 1 0.124 0.222 0.137 0.037 0.022 0.107 0.141 0.159 0.219 0.241 0.252 0.152

0.130 0.122

0.037

81 39 88 49 25 15 49 58 59 79 88

107 57 23 51 58

0.300 0.173 0.326 0.181 0.093 0.055 0.181 0.215 0.219 0.293 0.326 0.396 0.211 0.085 0.189 0.215

1M 61

1 22 70 54 39 85 82 95

120 133 148 92 60 91 86

0.385 0.271 0.452 0.259 0.200 0.144 0.315 0.304 0.352 0.444 0.493 0.548 0.341 0.222 0.337 0.319

~ _____

The cumulative number and frequency of detected disomy for each chromosome are listed as a function of the three detection criteria (see text). The 30% level is the least sensitive and most rigorous criterion; the 10% level is the most sensitive and least rigorous criterion. Data for chr. 1 are from Expt. 2 only. Data for chr. 3 include only those isolates that were originally mating competent.

As shown in Table 6, disome recovery frequencies among the several chromosomes are not the same. The frequency range is 11-fold at the least sensitive (and most rigorous) detection criterion (30%) , and Cfold at the most sensitive criterion (10%). In four cases (chromosomes 4 , I I , 12 and I 3 ) , the observed disome recovery frequencies at the 10% level do not differ significantly ( P > 0.05) from expected maximum disome frequency of 0.50. We have already shown that chr. 3 belongs in this group as well; as noted earlier, the data in Table 6 for chr. 3 include only those isolates that were initially mating competent and omit those that were disomic and nonmating.

Chromosomes I , 6, 7 and I 5 are least frequently recovered as disomes. The contrast between these four and the remaining thirteen chromosomes (or all seventeen chromosomes) is most apparent at the 30% level ( P < O.OOl), but persists even at the less discriminating 10% level ( P < 0.001),

The frequency of detected disomy for all chromosomes increases as the detection criterion becomes less stringent. This is precisely the result expected if the differences in disome recovery frequency are due to intrinsic differences in post- germination disome stability. This result contradicts that expected if all the disome recovery differences were the consequence of nonrandom segregational errors during triploid meiosis o r to selective spore lethality of certain diu comes or disome combinations.

Distributions of recovered disomy: Nearly all the meiotic segregants of the triploid characterized here are disomic for more than a single chromosome. The


distributions of the numbers of chromosomes identified as disomic in each isolate are shown in Figure 2. The three distributions have means of 2.6 (30% level), 3.8 (20%) and 5.8 (10%) disomic chromosomes per isolate. If all meiotic segregants of the triploid were viable and recoverable, we would expect a binomial distribution of numbers of disomic chromosomes per isolate, with a mean of 8.5 (N/2, N = 17). The observed distributions show that cellular tolerance of multiple disomy among the colony-forming meiotic segregants of the triploid is restricted. The maximum number of chromosomes we detected as disomic (at the 10% level) in a spore colony was 11. That lesser maxima were found at the more rigorous detection criteria is wholly consistent with our previously drawn inference that the failure of meiotic segregants of the triploid to proliferate into visible spore colonies is due to their burden of multiple disomy. These results are also consistent with our proposal of progressive mitotic loss of disomy among the cells in spore colonies derived from triploid meiosis.

Patterns of multiple disomy: Among the 270 characterized isolates, all 136 pairwise disomic combinations were found. This fact alone means that disome formation in triploid meiosis is unbiased with respect to any pairwise combination, and also that disomy for any given chromosome is not necessary for the co- existence of any other disomy, nor that disomy for any given chromosome neces-

6 a .Y

h v)

Number of disomic ch"s FIGURE 2.-Distributions of the number of different chromosomes found to be disomic among

the meiotic segregants of the aaa triploid. The three distributions were derived by considering the same data set at the three different detection levels (IO%, 20% and 30%), as described in the text.

DISOME MITOTIC STABILITIES 25 1

sarily excludes any other disomy. This latter point must be tempered by the understanding that our detection method defines a minimal disomic content of the germinating ascospore from the triploid, and not the disomic content of individual cells within the spore colony. Nonetheless, examinations of the tetrad data confirms the occurrence in individual trisomic diploid cells of all 136 pairwise combinations.

We have tested the incidence of recovered double disomy against the null hypothesis that disomy for any chromosome is produced in triploid meiosis inde- pendently of that of any other chromosome. The data are shown in Table 7 for chromosomes 2 and 4 through 17 (for which complete data are available). The analysis reveals a number of significant positive and negative associations. These associations suggest the possibility that disomy for certain chromosomes may serve in mitosis to stabilize or destabilize disomy for other chromosomes.

We have also examined the data for the occurrence of 3-disome and 4-disome combinations. An isolate disomic for, say, 6 different chromosomes carries 20 unique 3-disome and 15 unique 4-disome combinations. Among the 270 isolates, 679 of 680 (> 99%) possible 3-disome combinations, and 2209 of 2380 (93%) possible 4-disome combinations were found. The presence of nearly all 3-disome 4-disome combinations in only 270 isolates means again that there i s no inherent bias o r selection in the formation, during triploid meiosis, of any disome or disome combination.

These findings strengthen our contention that the observed differences in individual disome recovery (Table 6) are due to differences in post-germination disome mitotic stability. The results also imply that the truncation of the distributions of numbers of disomes per isolate (Figure 2) at means below that expected if recovery were complete is due principally, if not exclusively, to the poor viability conferred by high multiple disomy, and not to certain lethal combinations of multiple disomy.

DISCUSSION

We have described measurements o€ the incidence of chromosomal disomy among the colony-forming segregants of yeast triploids. The experiments were designed so that disomy for all seventeen yeast chromosomes could be assessed simultaneously and in a homogeneous genetic background. The principal results include these points. ( 1 ) Ascospores irom triploid meiosis germinate at frequencies comparable to those of normal diploids, but the majority fail t~ proliferate into visible colonies. (2) Detailed analyses of disome recovery for one chromosome (chr. 3 ) confirm that meiotic (MI) homolog segregation in the triploid for this chromosome is two-by-one and imply that the subset of testable (colony-forming) meiotic segregants of the triploid is an unbiased sample of all segregants with respect to disome recovery for this chromosome. (3) The frequencies of recovered disomy for the several chromosomes fall into three classes. Chromosomes 1,6,7 and 15 are least frequently recovered, chromosomes 3 , # , 11, 12 and 13 are found at frequencies that closely approach the expected maximal

252

2 *$ %

s * - . - 0 2

2 M

!w 2 $ g s g

%

0 h Q

I-.

g

4 g

% 2 U Qi

s

D. CAMPBELL cut al. i r w . 5

i $j ;B 23 g!g-&gzag$&& * * * E2i; g g h 3

2 0x3 &'IC) g $ FI%Q .-, * * * b$;>

g y 9 . E

25 2.z ,-. * * G .5 .I! 0"

3 - ; 84: $ * E 2

m u D m * E U3

0 ) - 2 $2 g 0 s.3

* W g k @ B 03.5 4-2 as s a m ; a - a m a E " m u a , g

I w a g s

- , X h & d c s C T ) h * * 9 gAg 2 g m w m a i m m * h k m 2 E P).u $ m w q

*p&3-sj 5iZ.F 2 E B " 2,z.z:g %s -C.lYJ-ahl t.ot-az g; E a u4-"E,

* a$ g %

g v) $2 &$ a"$ .Z .GmF

a&?%-; .Z g m . t ; g ,Z,mo -*a- E;-. go x= $ 5v

3 s " && m 2 o m $2 p=:-

g&%- .e u s 0 - +.E r g o

-2 U $ a $3 gv b ,B-& SE$%.. E z as: E Z % h\n" a,853z9

$ E E m O * g agv .z 3 3 .sa, -7i d.F .. g$ 8 8 O d g g 3 g g g g gj 8 g $ g E

v v w m u $32 2g Q ~ l n w h c Q m Q - . Q m Y ! . l n w ;qJ;

ggea,: 22322

- S " X % r= %ad -a c M +-' h-d

* * %**2

2s 2 g g 2 :: g 3 g 3 E 5 z G R * ,

M O 10 m i i h 10 h h a a a m -E m a - c 4 + a + - a m r o c a

d. a, 2% w - d m a w w o d m e m a - c 4 - a a m * a m

c a 2% % i T 3 % % % ? E 3 2 .2 * m

r o c s c n a h . L F 1 0 1 0 e f i r;; 2::: 1 0 w m a 4 * + a m

* h h i # " C O O

*

Q E

'E. hm M v

- 5 a U * *

h - m e 0 h* - m m

.-. a% ,-. cI\ ---,-. n -- - n- - n

;& cz w v v v v v i - -vu- m *

o e

hlhhlhhlhh OF: U


frequency of 50%, and the remaining chromosomes are recovered as disomic at intermediate levels. (4) Among 270 tested isolates, all possible 2-disome, and nearly all possible 3-disome and 4-disome combinations were found.

The results of this work support the following inferences. (1) The failure of most ascospores from triploid meiosis to proliferate into colonies is due to their burden of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all yeast chromosomes. (3) There are no inherent chromosomal restrictions, other than absolute number, on the cellular tolerance of multiple disomy. That is, disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (4) During vegetative growth of multiply disomic cells in ascosporal colonies, progressive and nonrandom mitotic chromosome losses (N+l+N) occur that account ultimately for the observed differences in individual disome recovery. These considerations imply that disomes for the several yeast chromosomes are not all equally stable in mitosis.

Mitotic chromosome loss from disomy ( N f l ) to haploidy (N) represents an error in chromosome (chromatid) segregation. The mechanism of this process is unknown, though it has been suggested that mitotic recombinational events in or near the disomic centromere may act to potentiate disomic chromosome loss, since there is a strong positive correlation between these two events and since centromere-adjacent mitotic conversions associated with chromosome loss are markedly polarized toward the centromere (CAMPBELL, FOGEL and LUSNAK 1975; CAMPBELL and FOGEL 1977; CAMPBELL 1980). An understanding of mitotic chromosome loss could nonetheless provide clues to, or delimit explanations of, the more central problem of the control of mitotic chromosome segregation itself. The different mitotic stabilities of yeast disomes found here might be taken to suggest that segregational fidelity is assured by more than a single mechanism. That the mitotic chromosome-loss mutant, chl, effects the loss (2N+2N-I or N+l+N) of some chromosomes, but apparently not others, may also be a reflection of the same possibility (LIRAS et al. 1978; J. MCCUSKER, personal communication).

Because our estimates of individual disome stabilities are based on their recovery in a multiply disomic background, it should not be inferred that the relative mitotic stabilities of single disomes in isolation will necessarily be the same. The measured absolute loss (N+l+N) frequency of chr. 3 is of the order

per cell (CAMPBELL, FOGEL and LUSNAK 1975) ; this value might also apply to the disome stabilities of those other chromosomes ( 4 , I I , 12 ,13) found in the same stability grouping. But the relative nature of the present data disallows assignment of absolute loss frequencies to the remaining, apparently less stable, chromosomes. The measurement in singly disomic strains of chromosome loss for one or more of the chromosomes ( I , 6,7,15) judged to be least mitotically stable might serve to set boundaries for the range of disome mitotic stabilities for the entire yeast genome.

One source of ambiguity in our results stems from the phenomenon of partial disomy, in which disomic loss occurs for a single chromosome arm only (CAMP- BELL and FOGEL 1977). Though we doubt (without evidence) that partial disomes are generated during triploid meiosis, the progressive disomic chromosome loss


adduced to arise in our system could include instances of this sort. Our experimental design, depending as it does on a single indicator marker per chromosome, would prevent partial disome detection, and would lead us to assign cases of marked-arm partial disomy to the disomic recovery class and cases of unmarked- arm partial disomy to the haploid class. Of 83 conversion-associated disomic chromosome (chr. 3 ) losses that we identified in previous work, 11 (13.3%) were single-arm losses (CAMPBELL and FOGEL 1977; CAMPBELL 1980). This is a highly selected sample, to be sure, but it suggests that some fraction of our euploid- aneuploid assigments could be misplaced. This uncertainty could be assayed for selected chromosomes by means of haploid tester(s) constructed to carry two indicator markers per chromosome, one on each arm, We have not yet ventured to construct a 34-marker haploid tester, however. Partial disomy might ako explain certain internal contradictions in attempts to map genes by trisomic analysis, in which new linkage groups have been proposed on the basis of exclusively diploid (2f : 2m) segregations when single indicator markers for all seventeen linkage groups segregated trisomically (COHN, TABOR and TABOR 1978; HILGER and MORTIMER 1980; WICKNER 1979).

HILGER and MORTIMER (1980) reported the isolation 01 haploid strains apparently stably disomic for as many as five chromosomes. Our results suggest that this value may be close to the upper limit of stable aneuploid tolerance (PARRY and Cox 1970). A possible mechanistic explanation of this upper limit comes from electron microscope observations of the yeast mitotic spindle (PETERSON and RIS 1976), in which the number of hali-spindle microtubules closely approxi- mates the expected number of segregating units-about 20 in haploids, about 40 in diploids, Similarly, in studies of in uitro microtubule assembly initiated on isolated spindle pole bodies (SPBs), BYERS, SHRIVER and GOETSCH (1978) dcmon- strated equilibria centering on about 20 microtubules per SPB. These findings suggest that the number of spindle microtubules is determined by the SPBs themselves, and lead us to speculate that any substantial excess of segregating chromosomes may simply saturate the segregation apparatus, such that chromosomes are lost in consequence of competition for a limited and fixed number of mobilization sites.

We are grateful to CHRISTOPHER D. BURNS for conscientious technical assistance, to R. K. MORTIMER, S. FOGEL, M. S. ESPOSITO, J. HABER and J. MCCUSKER for helpful discussions, ideas and encouragemen6 to W. DOCTOR for help with computer analyses and to R. CONTOPOULOS of the Yeast Genetic Stock Center, Berkeley, for generously responding to our almost endless stream of requests for yeast strains. This work was supported by research grants from Research Corporation, from the Public Health Service (GM25749) and from the E. C. Campbell Foundation.

LITERATURE CITED

BYERS, B., K. SHRIVER and L. GOETSCH, 1978 The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiue. J. Cell Sci. 30: 331-352.

CAMPBELL, D., 1980 Association of disomic chromosome loss with EMS-induced conversion in yeast. Genetics 96: 613-625.


Association of chromosome loss with centromere- adjacent mitotic recombination in a yeast disomic haploid. Genetics 85: 573-585.

Mitotic chromosome loss in a disomic haploid of Saccharomyces cerevisiae. Genetics 79: 383-396.

Isolation and characterization of Sacchwomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and sper- mine. J. Bacteriol. 134: 208-213.

Genetic analysis of hybrid strains trisomic for the chromosome containing a fatty acid synthetase gene complex (fasi) in yeast. Genetics

Meiotic gene conversion: A signal of the basic recombination event in yeast. Cold Spring Harbor Symp. Quant. Biol. 43:

FOGEL, S. and R. ROTH, 1974 Mutations affecting meiotic gene conversion in yeast. Molec. Gen. Genet. 130: 189-201.

HILGER, F. and R. K. MORTIMER, 1980 Genetic mapping of argl and arg8 in Saccharomyces cerevisiae by trisomic analysis combined with interallelic complementation. J. Bacteriol. 141 : 270-274.

Characterization of a mutation in yeast causing nonrandom chromosome loss during mitosis. Genetics 88: 651-671.

Mitotic recombination in yeast: isolation and characterization of mutants with enhanced spontaneous mitotic gene conversion rates. Genetics 94: 825-839.

Genetic mapping in Saccharomyces. IV. Map- ping of temperature-sensitive genes and the use of disomic strains in localizing genes. Genetics 74: 33-54.

The tolerance of aneuploidy in yeast. Genet. Research 16: 333-340.

Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J. Cell Sci. 22: 219-242.

Genetic and bio- chemical studies of genes controlling the synthesis of threonine and methionine in Sac- charomyces. Genetics 53: 609-619.

Radiation-induced recombination in Saccharomyces: The genetic control of recombination in mitosis and meiosis. Rad. Research 49: 148-154.

Radiation-induced recombination in Sac- charomyces: Isolation and genetic study of recombination-deficient mutants. Rad. Research

A system selective for yeast mutants deficient in meiotic recom-

A mutational analysis of gene conversion and reciprocal recombination in the yeast, Saccharomyces cerevisiae. Ph.D. Dissertation, University of California, Berkeley.

A stable aneuploid of Sac- charomyces cerevisiae. Genetics 67 : 483-405.

Mapping chromosomal genes of Saccharomyces cerevisiae using an

Corresponding editor: F. SHERMAN

CAMPBELL, D. A. and S. FOGEL, 1977

CAMPBELL, D. A., S. FOGEL and K. LUSNAK, 1975

COHN, M. S., C. W. TABOR and H. TABOR, 1978

CULBERTSON, M. R. and S. A. HENRY, 1973

75: 444-458.

FOGEL, S., R. MORTIMER, K. LUSNAK and F. TAVARES, 1978

1325-1341.

LIRAS, P., J. MCCUSKER, S. MASCIOLI and J. E. HABER, 1978

MALONEY, D. H. and S. FOGEL, 1980

MORTIMER, R. K. and D. C. HAWTHORNE, 1973

PARRY, E. M. and B. S. Cox, 1970

PETERSON, J. B. and H. RIS, 1976

DE ROBICHON-SZULMAJSTER, H. Y., Y. SURDIN and R. K. MORTIMER, 1966

RODARTE-RAMON, U. S., 1972

RODARTE-RAMON, U. S. and R. K. MORTIMER, 1972

49: 133-147.

ROTH, R. and S. FOGEL, 1971

SANFILIPPO, D., 1976

bination. Molec. Gen. Genet. 112: 295-305.

SHAFFER, B., I. BRFARLEY, R. LITTLEWOOD and G. R. FINK, 1971

WICKNER, R. B., 1979 improved genetic mapping method. Genetics 92: 803-821.

differential mitotic stability yeast disomes derived … · 2003-07-30 · differential mitotic...

Documents