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Current Genetics 9 Springer-Verlag 1992

Mitotic hyperploidy for chromosomes VIII and III in

Saccharomyces cerevisiae Lorraine M. Speetor and Seymour Fogel Department of Genetics and Plant Biology, University of California, Berkeley, CA 94720, USA Received November 1, 1991

Summary. The arg4-8 and cupl s markers comprise a copy-number-dependent signal device in the yeast Saccharomyces cerevisiae. These alleles permit reliable discrimination between euploid and disomic haploids as well as between euploid and trisomic diploids. To investigate and compare inherent inter-chromosomal differences as regards propensity for hyperploidy, we transplaced arg4-8 and cupl s by deleting them fi'om chromosome VIII and then re-introducing them at the leu2 locus on chromosome III. The rate of chromosome gain was significantly greater for the chromosome III construct compared to the native chromosome VIII, in both diploid and haploid strains. In addition, more coincident aneuploidy for other chromosomes was found among chromosome VIII hyperploids compared to chromosome III hyperploids. Key words: Yeast - Aneuploidy - Chromosomes

Introduction Eukaryotic organisms accurately apportion their chromosomes during mitotic and meiotic cell divisions and thereby produce cells with complete and balanced genome complements. Segregation failures are evidenced by aneuploidy. Genetic balance, first proposed by Bridges in 1922, refers to the "coordination and balance between coadapted genes and gene systems of a particular genotype" (Rieger et al. 1976); the primary effect of aneuploidy is to perturb the genetic balance of an organism. Chromosome segregation requires interaction between the chromosomes and the cellular machinery involved in the apportionment process. Among the chromosomal components, the centromere is of primary importance; acentric chromosomes fail to achieve orderly Offprint requests to: S. Fogel

segregation and are eventually excluded from the daughter nuclei, resulting in chromosome loss. Dicentric chromosomes may undergo breakage-fusion-bridge cycles (McClintock 1951). The yeast Saccharomyces eerevisiae provides a well-characterized, easily manipulated system for studying chromosome biology: yeast centromeres have been cloned and sequenced, and conserved regions and physical spacing requirements for those regions determined (reviewed in Fitzgerald-Hayes 1987; see also Gaudet and Fitzgerald-Hayes 1987, 1989). Sharon and Simchen (1990) found that the centromeric region plays a role in directing whether a pair of homologues will undergo reductional or equational division during singledivison meiosis in yeast strains homozygous for edc5 or cdcl4. Other specific elements are also essential for chromosomal maintenance and transmission. These include at least one functional origin of replication, or autonomously replicating sequence (ARS) (Wellinger and Zakian 1989; Huberman et al. 1988) of sufficient potency to support chromosome replication (Kearsey and Edwards 1987) (for multiple, weaker ARSs), and telomeres to prevent degradation of the chromosome (for review, see Walmsley 1987). Non-conserved chromosomal sequences affect the function of the conserved elements; e.g., Panzeri et al. (1985) found that transcription into a yeast centromere inhibits its segregational function. Also, Snyder et al. (1988) showed that ARS sequences may be transcriptionally inactivated, resulting in elevated mitotic loss of artificial yeast chromosomes. Physical characteristics also play a role in chromosome segregation. For instance, Murray and Szostak (1983) found a minimal size requirement of 20 kb necessary for proper segregation. Many trans-acting genes that affect chromosome transmission have been identified; several are mentioned here, although a lengthy discussion is beyond the scope of this paper. Most, such as CHL2, -3, -5 and -5 (Kouprina etal. 1988); DIS1 (Rockmill and Fogel 1988), RED1 (Rockmill and Roeder 1988), CL4 and CL8 (Karpova et al. 1987; Larionov et al. 1987), and NDC1 (Thomas and Botstein 1986), are not chromosome-specific. However, some mutations have been described which cause

310

non-random aneuploidy (Meeks-Wagner et al. 1986; Liras et al. 1978; Haber 1974; Culbertson and Henry 1975). Other chromosomal components or chromosomespecific factors may be involved in the chromosome apportionment process. Such elements, though not essential for segregation, as demonstrated by successful construction of artificial yeast chromosomes (Murray and Szostak 1983), might affect th e fidelity of the process. Hence, they could contribute to inherent differences between chromosomes in their propensity to become aneuploid. The work described in this paper was undertaken to investigate and characterize mitotic inter-chromosomal differences in the incidence of aneuploidy. Studies of the viable products of triploid meiosis have established that each yeast chromosome may be recovered as a primary disome. Typically, a given disomic chromosome is recovered in fewer than 50% of viable ascosporal colonies. This results from the poor viability conferred by multiple aneuploidy and from inter-chromosomal differences in mitotic stability (Campbell et al. 1981; Parry and Cox 1970; Pomper et al. 1954). Whittaker et al. (1988) reported a system wherein the copy number-dependent alleles arg4-8 and cupl s, normally resident on chromosome VIII in Saceharomyces cerevisiae, are used to determine the frequency of chromosome VIII trisomy. In the diploid yeast strain BR1669, euploid cells are prototrophic for arginine at 24 ~ but are auxotrophic at 30 ~ and 36 ~ and copper sensitive. Trisomic diploids may be distinguished from euploids by their arginine prototrophy at 30 ~ and increased copper resistance. To investigate whether other chromosomes are characterized by different levels of hyperploidy incidence, we transplaced the arg4-8 and cupP indicator genes by first deleting them from chromosome VIII, and subsequently reintroducing them via plasmid integration at the leu2 locus on chromosome III. Thus, different chromosomes are examined using the same selective phenotypes. The data demonstrate that chromosomes VIII and III have significant and characteristic differences relative to chromosome gain and stability. Hyperploidy rates, calculated from the median frequencies, showed the gain rate of chromosome III to be higher than that for chromosome VIII by 7.1-fold in haploids and 3.5-fold in diploids.

using an automatic plate-pouring apparatus that dispensed 20 ml per 100 x 15 mm plate. Potassium acetate plates, containing 2% potassium acetate, 0.23% yeast extract, 0.05% glucose, 1.8% Bacto agar, and a single addition of amino acids, purines and pyrimidines (at the same concentrations as in SC medium), were used both to induce sporulation and to determine respiratory competence. Uracil-requiring yeast colonies were selected on plates containing 5-fluoro-orotic acid (Bocke et al. 1984).

Molecular techniques. All DNA manipulations were performed using standard techniques (Maniatis et al. 1982). Restriction enzymes, calf-intestine alkaline phosphatase, DNA polymerase large (Klenow) fragment, and DNA ligase were purchased from Boehringer-Mannheim and New England Biolabs, and used according to the manufacturers' directions. Amersham kit N5000 was used for nick translations. Yeast transformations were performed using a PEG/bicine procedure (Klebe et al. 1983). Yeast DNA for Southern analysis was prepared as per Denis and Young (1983). The single-copy yeast metallothionein gene, cupl s, was cloned in this laboratory as a 5.2 kb EcoRI restriction fragment (unpublished). The temperature-sensitive arg4-8 allele was cloned in this laboratory by gap-repair of plasmid pSZ524 (Ann Blechl, personal communication). LEU2 was obtained from plasmid CVI3, a gift from Michael Holland's laboratory. Yeast strah7 constructions. Chromosome gain detection in diploid strain BR1669 via the copy number-dependent phenotypes of arg4-8 and cup1~ was previously described (Whittaker et al. 1988). Euploid BR1669, a diploid strain, is arginine prototrophic at 24~ but auxotrophic at 30~ and 36 ~ and copper sensitive. Colonies trisomic for chromosome VIII may be distinguished from euploid colonies by their arginine prototrophy at 30 ~ and increased copper resistance, phenotypes that result from increased arg4-8 and cupP copy numbers, from one per haploid genome in the euploid, to 1.5 per haploid genome in trisomic diploids. The genotypes for haploid strains LS70-3B and LS70-6D are given in Fig. 1. They possess a BR1669-derived genetic background, and carry the arg4-8 and cupP alleles. Haploids LS70-3B and LS70-6D may be used to detect chromosome VIII disomy, or crossed together to form a diploid for studying chromosome VIII trisomy. In addition, they carry Ieu2-3,i12 and ura3-52 as cloning markers, his4-280 and his4290 are complementing heteroaUeles on chromosome III; cyhlO is a centromeric marker on chromosome II in LS70-3B. The ade2 allele on chromosome XII is utilized for determination of mating type: isolates to be tested are replicated to rich medium and sprayed separately with MATa, or MATs, adel tester strains. These plates are incubated overnight at 30 ~ and then replicated to SC,- -ade. Mating type was determined by adenine complementationresponse. All other strains used are listed in Table 1. The ARG4 and CUP1 loci were deleted as described in Fig. 2, and confirmed by Southern blot analysis, shown in Fig. 3. Construction of plasmid pSL15Leu2, and integration of pLS15Leu2 at the leu2 locus of strains LS70-3B and LS70-6D to generate strains 3BLeu and 6DLeu, is shown in Fig. 4.

Materials and methods Media. YEPD and YEPAD (YEPD supplemented with 160 mg/1 L-adenine HCl) are complex undefined media composed of 2% D-glucose, 2% Bacto peptone, 1% Bacto yeast extract, and 1.5% Bacto agar (all from Difco). Synthetic complete (SC) and omission media (SC, -x; lacking a single amino acid, purine or pyrimidine) were prepared as described by Whittaker et al. (1988, 1989), except that Difco Bacto agar (1.5%) replaced Gibco Phytagar. Hyperploids were selected on trace arginine (trace arg) medium, and copper resistance was scored on SC medium containing CuSO 4 (SCcup), both prepared as described and modified by Whittaker et al. (1988, 1989). Trace arginine medium consists of SC, -arg containing 1.5% Gibco Phytagar (instead of Bacto agar), supplemented with 0.1 mg/1 L-arginine HC1 after autoclaving. Copper resistance was scored on plates containing 40, 50 and 60 p.M CuSO4 added after autoclaving. Both trace arg and SCcup media were prepared

LS70-3B (II) cyhl0

(uI) his4-290 leu2-3r112 oMATa MAL2 ,.

(VII) met13 (VIII) larg4-8 THR1 cup1 s (IX) LYS1

(V) ura3-52 (XIV) ade2-1

LS70-6D

(1I) ,CYI-I+ (VII) met13

(IXI) his4-290 leu2-3r112 =MATs mal~ ,.

(V) ura3-52

(VIII),earg4-8thrl-4 cup1 s (IX) Iysl-1 (XIV) ade2-1

Fig. 1. Oenotypes of strains LS70-3B and LS70-6D

311 a)

Chromosomal deletion of cup1 s

HindlII SalI I

i

SspI SspI I HpaI ISaeIEcoRVSaeI X

BgIII SnaBI HindIII I , / Hpal

BCII D;aIII

HindIII

i~/ HindII! (Hpal/SHpaI naaI)

I

I

I

I

~g4A

b) Chromosomaldeletion of arg4 8

Ndel

Ndel

I?

EcoRI I

~--807bp--~ Ndel ~ Ndel /EcoRV._~ J

KpnI

q

5.2kb ~

EooRI

KpnI

EcoRI I

~/

\

~ /

Fig. 3A, B. Southern blot analysis of ARG4 and CUP1 deletions. Genomic DNA was digested with EeoR1 (CUPt) or HindIII (ARG4) and electrophoresed through 0.8% agarose. Lanes are LS70-3B (1) undeleted and (2) with ARG4 and CUP1 deletions and LS70-6D (3) undeleted and (4) with ARG4 and CUPI deletions. A the CUP1 deletion reduces the 5.2 kb EcoR1 fragment to approximately 4.3 kb. The radiolabeled probe was pBR322, into which the 5.2 kb CUP1 and a 1.5 kb TRP1-ARSI fragment had been cloned. The 1.5 kb band represents the TRP1-ARS1 fragment. B deletion of ARG4 reduces the approximately 3 kb HindIII fragment to approximately 1.4 kb. The radiolabeled probe was the ARG4 HindIII fragment, cloned into pBR322

EcoRI

I

I

Ndel Fig. 2a, b. Deletions of the cup1 and arg4 loci. a an 807 base pair fragment carrying the entire cupl s gene was deleted from chromosome VIII. Strains LS70-6D and LS70-3B were transformed with a derivative of plasmid yRP17 carrying the EeoRI-NdeI (left) and NdeI-EcoRI (right) restriction fragments which flank the eupl s gene. Stable transformants (URA +) genetically verified to carry the insertion at the eupl locus, were then screened for spontaneous plasmid excision. This was signalled by loss of one of the eupl (eupls or cuplA) alleles and the URA3 phenotype. Cells which had lost the plasmid were tested for copper resistance phenotype, and the deletions confirmed by Southern blot analysis (see Fig. 3). b a 1.9 kb HpaI-SnaBI restriction fragment was deleted from the arg4 locus in cupA derivatives of LS70-3B and LS70-6D by integration of plasmid yIParg4A, a derivative of yIP5 digested with HindIII and BamHI, into which the HindIII-HpaI (left) and SnaBI-HindIII (right) restriction fragments which flank arg4-8 had been cloned. Transformants were selected by URA3 complementafion and the locus of the plasmid insertion confirmed genetically. Excision events removing the plasmid sequences and one of the arg4 alleles (either arg4-8 or arg4A) were selected by growth on plates containing 5-fluoro-orotic acid (Bocke et al. 1984). Arg4 deletions were confirmed by phenotypic, genetic and Southern blot analysis (see Fig. 3)

Phenotypie characterization of integrants. Discrimination between euploids and hyperploids is based on the copy-number-dependent phenotypes of the arg4-8 and cupl ~ alleles. Since position effects or multiple integration events would affect marker gene expression and confound hyperploid detection, the arginine and copper phenotypes of each strain carrying the integrated arg4-8 and cupl s mark-

ers were characterized. Integrants of pLSl5-Leu were crossed to two tester strains. One carries arg4A and euplA (226-5, 3BD10-30, or 315A-4C), the other is sing/e-copy for both these alleles (LS70-3B or LS70-6D). The resultant diploids were sporulated and subjected to tetrad analysis. Only those integrant strains that were established by genetic and Southern analysis to: (1) possess a single, stable insertion of the plasmid at the desired location, and (2) express the arginine and copper phenotypes at a level indistinguishable from the native, single-copy chromosome VIII marker alleles, were used for chromosome gain determinations.

Mitotic chromosome gain frequency determinations. In various experiments, from ten to 19 cultures were inoculated with approximately 200 cells each in 5 ml YEPAD in Klett tubes, and incubated on a roller drum at 30 ~ A 2 ml aliquot was taken in mid to late log phase after 40-48 h incubation (107-108 cells per ml), pelleted in a clinical centrifuge, and washed twice with sterile, gIass-distilled water at room temperature. Cells were diluted appropriately in 1.5% Tergitol NP-40 (Sigma), plated to synthetic complete (SC) and trace arginine selective media, and incubated at 30~ Trace arginine plates were sealed in plastic bags 1 day after plating to minimize evaporation. SC plates were counted after 3 days, trace arg plates after 7 days. About 100 of the colonies arising on trace arginine were patched to YEPAD medium for subsequent phenotypic characterization. A 32-prong multiple inoculation device, described by Whittaker et al. (1988), was used to transfer comparable inocula to each diagnostic plate. This protocol enhances the reliability of assessing the cupl" and arg4-8 phenotypes. The arginine phenotype was scored at 24, 30 and 36~ and the copper resistance level at 40, 50 and 60 micromolar Cu 2+. Each isolate was thus assigned to a single phenotypic category described in Table 2. Although the copy number per haploid genome of the arg4-8 and cupl ~ alleles is higher in a disomic haploid (2.0) than in a trisomic

312 Construction and integration of Leu2 plasmid a) Construction of pLS15-Leu2

LEU2 XhoI

KpnI

pTZ19 polylinker

I

Sail

KpnI

EcoRI 2.2kb

q

I EcoRI

II

Sal[ KpnI SmaI

~ Circularize, digest with EcoRI

Ligate to pLS15, digested with EcoRI

Ii I EcoRI

11 SalIlKpn[I SmaI EcoRI

diploid (1.5), disomes and trisomes were readily assignable to the same phenotypic categories. Petite colonies were not evaluated. For each independent culture tested, at least one presumptive hyperploid was subjected to tetrad analysis to confirm hyperploidy of the marked chromosome.

Reconstruction experiments. Reconstruction experiments were performed to evaluate the selectivity of the trace arginine medium for detecting hyperploids. The recovery efficiency for cells expressing an arg 30 ~+ cupR phenotype was assessed as follows: overnight YEPAD cultures of both a euploid strain (LS70-6D or LS73) and a hyperploid (disomic haploid or trisomic diploid) were grown, washed as described above, and diluted to four parallel series of tubes that were then mixed with similarly washed hyperploid cells diluted to yield 0, 25, 50, or 100 hyperploid colonies per plate. These

Integration of pLS15-1eu2 atleu2-3,112

b)

Table 2. Phenotypes of colonies arising on trace arg selective medium a

c u p1~s pLS15-1eu2

Growth on selective medium

arg4-8

CHROMOSOME II

SC, -arg

~ ~ / ~

~

lWmOoe|lJmm

iiiiImmll

KpnI

EcoRI

iilll

SalI

leu2-3,1 t2

Fig. 4 a, b. Integration of the arg4-8 and cupl s alleles at leu2-3,112. a The yeast LEU2 gene was obtained from plasmid CV13 as a SalI-XhoI restriction fragment, ligated into Sa/I-digested plasmid pTZ19. It was then excised as an approximately 1.4 kb KpnI fragment, circularized, digested with EcoRI, and ligated to EcoRI-digested pLS15 to generate plasmid pLSt5-Leu2, b Integration of pLSt 5-Leu2 was directed to the LEU2 locus by transformation with SmaI-digested plasmid. The drawing shows the synapsis configuration of the desired integration event

Classification SCcup

24 ~

30 ~

36 ~

+ + + + +

+ + + -

+ -

+ +

Euploid (parental) Hyperploid Type I revertant Type II revertant Undetermined

Colonies arising after 7 days incubation on trace arginine selective medium are replicated to three SC, -arg plates, incubated at 24, 30 and 36 ~ and three concentrations of CuSO 4, 40, 50 and 60 gm, all incubated at 30 ~ G r o w t h is scored relative to known euploid and hyperploid controls carried on the same plate. Hyperploids exhibit enhanced copper resistance and arginine prototrophy at 30~ Type I and II revertants display the indicated levels of arginine prototrophy without associated copper resistance " Table adapted from Whittaker et al. (1988)

Table 1. Yeast strains employed

Strain

Ploidy

Description

3BLeu

n

Genotype as for strain LS70-3B (Fig. 1), with chromosomal cup1 and arg4 deletions, and pLS15Leu2 plasmid insertion at Ieu2 : cyhlO, his4-290, leu2-3,112: :arg4-8, cupP, MATa, MAL2,

6DLeu

n

Genotype as for strain LS70-6D (Fig. 1), with chromosomal cup! and arg4, deletions, and pLS15Leu2 plasmid insertion at leu2: his4-280, leu2-3, 112: : arg4-8, cupP, MAT~, ura3-52, met13, arg4d, thrl-4,

LS73 LS379 315A-4C, 3BD10-30

2n 2n n

LS70-3B x LS70-6D 3BLeu • 6DLeu Genotype as for strain LS70-3B (Fig. 1), with chromosomal cupl and arg4 deletions: cyhlO, his4-290,

226-5

n

Genotype as for strain LS70-6D (Fig. 1), with chromosomal cupl and arg4 deletions: his4-280,

n n 2n

Tester: MATa, his4-290, adel Tester: MAT~, his4-290, adel Tester:

ura3-52, met13, arg4A, cuplA, ade2-1.

cuplA, lysl-1, ade2-1

leu2-3,112, MATa, MAL2, ura3-52, met13, arg4A, cuplA, ade2-1 Ieu2-3,1t2, MAT~, ura3-52, met13, arg4A, thrl-4, cuplA, lysl-t, ade2-1 BR731a BR731c~ g721-2

leu2 MATa chnl-lohom3 his1-7 + ade2 + MATa + + his1-1 trp2 + Chromosomal deletions of arg4 and cupl are described in Fig. 2. Strains BR731 a and BR731 ~ originated in this laboratory, and served as mating type and his4-280 testers via complementation response for adenine and histidine. Strain g721-2 was obtained from John Game

313 mixed suspensions were plated, incubated, and the presumptive hyperploids phenotypically analyzed as for mitotic chromosome gain frequency determinations.

overlap between calculated 95% confidence limits of the rates for the two chromosomes in either haploids or diploids.

Results

Comparison of diploid and haploid chromosome gain rates

Chromosome gain determinations

I f we assume that each m e m b e r of an homologous chrom o s o m e pair acts as an independent unit relative to mitotic c h r o m o s o m e apportionment, we m a y expect that c h r o m o s o m e gain in diploids will occur at twice the comparable haploid rate. High spore viability and uniform spore colony size a m o n g dissected tetrads obtained f r o m presumptive c h r o m o s o m e I I I aneuploids indicated comparatively little effect on viability or colony growth due to c h r o m o s o m e I I I hyperploidy. In turn, this suggests that m o s t c h r o m o s o m e III grain events are probably detectable via the arg4-8, cup1 ~signal system. The observed ratio of diploid to haploid c h r o m o s o m e I I I gain rates was 3.81 x 10-s/1.32 x 10 -5, or 2.9. Thus, we conclude that the c h r o m o s o m e III gain rate was significantly higher in diploids than in haploids, and approximated the predicted two-fold difference. C h r o m o s o m e V I I I hyperploids were recovered at 5.8 times the rate in diploids as in haploids i.e., 1.08 x 1 0 - s vs. 1.85 x 10 -6. The difference between these values and the corresponding ratios for c h r o m o s o m e III m a y reflect the more severe biological consequences of hyperploidy events signalled by the same markers localized on chrom o s o m e VIII. It m a y be noted that c h r o m o s o m e V I I I has a D N A content 1.5 times that of c h r o m o s o m e III. The results f r o m ascus dissections of aneuploid isolates crossed to euploid tester strains and subsequent tetrad analysis demonstrated that putative c h r o m o s o m e V I I I disomes were often multiply aneuploid, and displayed associated low ascospore viability when crossed to euploid tester strains. In contrast, trisomic diploid isolates

Several independent cultures from each of four strains were assayed for c h r o m o s o m e gain as described. LS706D and 6DLeu are haploid strains which carry the arg4-8 and cupl s m a r k e r genes on chromosomes V I I I and III, respectively. As shown in Table 1, LS73 and LS379 are diploid strains homozygous for arg4-8 and cupP located on chromosomes V I I I and III, respectively. The collective data from all the independent cultures of a given strain were analyzed as follows: c h r o m o s o m e gain frequencies were calculated as events per viable cell and ranked in ascending order. F o r statistical purposes, cultures in which a presumed early event caused near-confluent growth on trace arginine were ranked as having the highest frequencies in the data set. The median chromosome gain frequency was determined and used for subsequent rate calculations. C h r o m o s o m e gain rates and 95% confidence limits (assuming a normal distribution) were calculated by the method of the median (Lea and Coulson 1949; Von Borstel 1978).

Comparison of chromosome 111 and VIII gain rates Diploid and haploid c h r o m o s o m e gain rates and corresponding 95% confidence intervals are given in Table 3. C h r o m o s o m e III grain rates were significantly higher than the corresponding c h r o m o s o m e V I I I gain rates, by 3.5-fold in diploids and 7-fold in haploids. There was no

Table 3. Median frequencies and rates of chromosome gain Strain Haploids LS70-6D (Chr. VIII) 6DLeu (CHr. III) Diploids LS73 (Chr. VIII) LS379

Number Titer of cultures (cells/ml)

Number of cells plated

Number of colonies

Proportion aneuploid

Median frequency

Rate

95% confidence intervals

11

9.64 x 107

2.17 • 106

175

17/112

1.22 • 10- 5

1.85 • 10 -6

1.6-2.1 x 10 -6

10

5.82 X 10 7 6.91 x 107

1.31 x 1 0 6 1.56 x 106

237 224

86/111 90/112

1.28 x 10 -4

1.32 x 10 -5

1.07-1.59 x 10 -5

19

6.07 x 107

2.73 • 106

353

99/112

1.14 • 10 -4

1.08 • 10- 5

0.97-1.18 • 10 -5

13

3.51 X 10 7

1.27 • 10s

55

34/55

2.69 x 10 -4

3.81 • 10 -5

3.35-4.28 x I0-5

Comparisons: Haploids: 6DLeu/LS70-6D Diploids: LS379/LS73 Chromosome VIII: diploid/haploid Chromosome III: diploid/haploid

7.1 3.5 5.8 2.9

Chromosome gain rates are expressed as events per cell per mitotic division. Number of colonies: colonies counted on trace arg plates. Proportion aneuploid: number of isolates classified as aneuploid according to the criteria presented in Table 2, as a fraction of the total number tested

314

for chromosome III were seldom multiply aneuploid, and spore viability was comparable to the euploid strain.

Genetic analysis of presumptive chromosome VIII hyperploids At least one random, verified arg30 ~+, cup R colony from each independent culture was subjected to tetrad dissection and genetic analysis. Presumptive disomes from haploid strain LS70-6D, which carries the signal markers on chromosome VIII, were crossed to haploid LS70-3B (see Table 1), and the resultant hybrids were sporulated and subjected to ascus dissection and tetrad analysis. Ordinarily, such euploid hybrids would constitute diploid LS73, a strain homozygous for the signal markers located on chromosome VIII, which is not expected to exhibit detectable segregations for either arg4-8 or cupl ~. Of the isolates from nine cultures, two yielded viable spore colonies and displayed a normal 2 + : 2- segregation pattern for all heterozygous markers as well as for arg4-8 and cupP. This agreed with expectations for simple chromosome VIII disome segregation wherein the disomic spore colonies were prototrophic for arginine at 30 ~ and copper resistant, and the remaining spore colonies were arginine requiring at 30 ~ and copper sensitive. Isolates from the remaining seven cultures either failed to sporulate or sporulated poorly, yielding mostly 2- and 3-spored asci that failed to produce viable spore colonies. A second isolate from each of these cultures was analyzed in the same fashion, and similar results were obtained. To test the notion that poor sporulation and low spore viability exhibited by the 14/16 haploid chromosome VIII isolates represented an effect of multiple aneuploidy, we crossed several isolates to a diploid tester (g721-2, see Table 1). Hybrids involving multiply aneuploid isolates would be expected to yield near-tetraploid hybrids and thus produce viable spores. Three verified disomic isolates from the two independent cultures which yielded the simple disomic segregations discussed above, and 19 other arg+cup R isolates taken randomly from five other independent cultures, were crossed to the a/a diploid g7212. The hybrids were selected by a positive histidine complementation response. All hybrids produced abundant four-spored asci. One of the isolates that produced viable spores and was confirmed as being disomic when crossed to haploid LS70-3B (culture 5, Table 4) yielded only five viable spores among 30 tested spores. Two similar isolates from culture 6 produced zero and five viable spores of 40 spores examined. These results are consistent with triploid meioses. Of the 19 isolates from the other five cultures, seven produced significant numbers of viable spores (Table 4) with colony size variation and segregation patterns indicating multiple disomy (3:1 segregations for marker genes other than arg4-8 and cupl ~, as well as non-mating spore colonies indicative of chromosome III aneuploidy). But, the spore colonies were patently unstable; e.g., many of the non-maters displayed both a and ~ mating-type sectors. The remaining 11 isolates could not be diagnosed since they produced few

Table 4. Ascospore viability of hybrids from crosses between presumptive hyperploid LS70-6D isolates and diploid G-721 Isolate source

#Viable spores # Tested

% Viability

Culture 5 (verified disome) Culture 6 (two isolates, both verified disomes) Culture 6 (two additional isolates) Culture 1 Culture 2 Culture 3 Culture 9 (three isolates) Eleven other isolates

5/30 5/40, 0/40

17 13, 0

11/12, 0/28 92, 0 33/40 83 62/80 78 6/12 50 19/30, 20/28, 31/40 63, 71,78 2-4/40 avg. 8%

viable spores, e.g., about 3 out of 40 spores tested. These same isolates were simultaneously retested and confirmed as retaining the arg+cup R phenotype. When crossed to haploid strain 315A-4C and sporulated, histidine complementation was positive, though sporulation was infrequent. To evaluate the possibility that the observed spore inviability cited above resulted not from aneuploidy but rather some other effect of selection on trace arg medium, 44 isolates which were not phenotypically hyperploid (as defined in Table 2) were crossed to haploid 315A-4C (Table 1). The resulting hybrids were sporulated and ten or more tetrads from each hybrid were analyzed. The results are summarized in Table 5. Of the 44 LS70-6D isolates examined only two had spore viability insufficient for analysis. Of the remaining 42, four were classified as chromosome VIII hyperploids on the basis of 2 + :2- co-segregation of the arg4-8 and cupl ~ phenotypes. Thus, only two among 44 phenotypically non-hyperploid isolates that arose on trace arg medium at 30 ~ had poor spore viability when crossed to a standard haploid strain. This contrasts with poor sporulation and spore viability among 14 of 17 phenotypically hyperploid isolates subjected to ascus dissection, and poor sporulation among an additional 18 that were not subsequently dissected. These results support the notion that among phenotypically hyperploid isolates, spore inviability reflects multiple aneuploidy, and is not attributable to selection on trace arg medium. In summary, all examined arg30 ~+ cup R isolated from haploid LS70-6D displayed various genetic abnormalities ranging from simple chromosome VIII disomy to approximate diploidy. Aneuploidy involving multiple chromosomes provides a plausible explanation for the failure of several isolates to produce viable spores when crossed to both haploid and diploid tester strains. Thirty-four selected arg30 ~+ cup R isolates from 19 independent cultures of diploid LS73 bearing the homozygous signal markers in their native positions on chromosome VIII were sporulated and dissected. The 82% mean spore viability among the presumptive trisomes compared favorably with the 84% euploid level. This stands in contrast to the tetrad data obtained for haploid LS70-6D isolates after they were crossed to appropriate testers. All 34 were diagnosed as chromosome

315 Table 5. Genetic analysis of phenotypically non-aneuploid haploid isolates Strain

Primary phenotype

Number analyzed

Classification Euploid

LS70-6D

6DLeu

Disome

Revertants Type I

Type II

Arg +, Cup-

20

9

-

8

-

Arg-, Cup + Arg-, Cup-

2 18

1 6

1 3

7

-

36~ revertant Arg +, CupArg-, Cup + Arg-, Cup36~ revertant

4 12 2 10 2

9

1 2a 1 1 -

1 -

2 2

8 -

Other

2 insufficient viability 1 chr. VIII indeterminate 1 type II revertant or euploid 1 unstable disome or euploid 1 2:2 size segregation 1 variable arginine phenotype 1 2:2 size segregation -

Haploid isolates which did not display hyperploid (arg30~ CUpR) phenotypes were crossed either to strain LS70-3B (LS70-6D chr. VIII isolates) or 315A-4C (6DLeu chr. III isolates). The resultant hybrids were sporulated and at least ten asci per isolate dissected. Hybrids were then classified on the basis of segregation patterns for copper and arginine among the spore colonies. Type I and II revertants are as defined in Table 2, and demonstrate arginine prototrophy at 30~ and 36~ respectively without co-segregation of copper resistance. Primary phenotype: classification upon original phenotype verification utilizing the multiple inoculation device One unstable

V I I I trisomes. Two isolates were additionally trisomic for c h r o m o s o m e III, as indicated by a/a/~ and a/e/~ mating type segregations and of these one displayed mating type segregations of 0 a: 3 0t: 1 non-mater, and 1 a: 2~: 1 non-mater. Two other dissected isolates were diagnosed as being doubly trisomic, for chromosomes V I I I and II, as indicated by an excess of cycloheximide-sensitive ascosporal colonies. There was no bias regarding which c h r o m o s o m e V I I I homologue was involved in the trisomies; equal numbers of isolates demonstrated threonine segregations of + / + / - and + / - / - .

Genetic analysis of presumptive chromosome III hyperploids Segregation ratios for M A T and the his4 heteroalleles allow unequivocal confirmation of c h r o m o s o m e I I I hyperploidy. One phenotypically aneuploid isolate from each of nine independent haploid 6DLeu cultures was crossed to arg4A, c u p l A strain 315A-4C. Hybrids were sporulated and dissected. M e a n spore viability a m o n g spores obtained f r o m the hybridized disomes was 82%, a value comparable the euploid LS379 (80%). Eight a m o n g the nine isolates were c h r o m o s o m e III disomes, the ninth was an apparent euploid. An additional isolate f r o m the ninth culture was analyzed and shown to be a chromosome I I I disome. O f the disomes, one was a meiotically unstable c h r o m o s o m e I I I disome, and one was a chromosome I I I disome that displayed low spore viability and marked spore colony size variation suggestive of multiple aneuploidy. The m e a n spore viability a m o n g putative trisomes obtained f r o m the m a r k e d c h r o m o s o m e I I I diploid LS379 was 88%, a value comparable to euploid LS379 (80%). O f 13 randomly selected isolates examined (one from each culture), ten displayed trisomy for c h r o m o s o m e III,

and three were tetrasomic. A m o n g the trisomes, one was meiotically unstable as indicated by mating-type segregations, three had indeterminate copper phenotypes, and two were either tetrasomes or trisomes with coincident conversion of one arg4A allele to arg4-8. There was no bias regarding which chromosome III homologue was involved in the trisomies; equal numbers of isolates demonstrated mating-type segregations of a/a/~ and a/~/~. The previous sections concerned genetic analysis of putative hyperploid isolates which carried the dosage-dependent signal markers on chromosomes III or V I I I in haploids and diploids. F r o m these same strains, we also analyzed isolates which arose on trace arg medium and were assigned to each of the four non-hyperploid categories specific in Table 2. Table 5 summarizes the genetic analysis of haploid isolates that arose on trace arginine medium within 7 days incubation at 30~ Subsequently, however, these failed to display diagnostic arg+(30~ cup s hyperploid phenotypes. Verifiable disomes were found a m o n g LS706D c h r o m o s o m e V I I I isolates f r o m all four non-aneuploid phenotypic categories and a m o n g 6DLeu isolates from three of the four classes, indicating that hyperploidy frequency determinations involving these haploid strains represent underestimates of the actual events. Euploids comprised 65% of 6DLeu non-aneuploid isolates, compared to 36% of the LS70-6D sample. Euploids represent a lower proportion of the phenotypically non-aneuploid isolates from the diploid strains (summarized in Table 6) c o m p a r e d to the haploid strains discussed above. Bona fide LS379 trisomes were identified in all four " n o n - a n e u p l o i d " phenotypic categories, LS70-6D disomes were probably represented in all four categories ( A R G § cup s was equivocal), and demonstrable LS73/358 trisomes and 6DLeu disomes in three of the four (not a m o n g type II revertants). Thus, hyperploidy

316 Table 6. Genetic analysis of phenotypically non-aneuploid diploid isolates

Strain

Primary phenotype

Number analyzed

Classification Euploid

LS73

LS379

Trisome

Revertants

Other

Type I

Type II

Arg +, CupArg-, Cup + Arg-, Cup-

11 10 13

4

4a 9 3

5 Ib 1

1 _ 1

1 tetrasome _ 2 2:2 lethal segregation 1 trisome II (excess cyhs) 1 homozygous arg + at 30~ cup heterozygous 1 undetermined (3 viable spores)

36~ revertant Arg +, CupArg-, Cup + Arg-, Cup36~ revertant

1 8 0 5 5

. 2 -

6~ . 1e

-

1

-

2

2d 1

2

.

. 1 trisome II (excess cyhs) 1 undetermined

Diploid isolates which did not display hyperploid (arg30~+, cup R) phenotypes were sporulated and at least ten asci per,isolate dissected. They were then classified on the basis of copper and arginine segregation among the spore colonies. Type I and II revertants are as defined in Table 2, and demonstrate of arginine prototrophy at 30~ and 36~ respectively without co-segregation of copper resistance. Primary phenotype: classification upon original phenotype verification utilizing the multiple inoculation device One unstable b Homozygous One unstable d One homozygous Also a type II revertant

determinations involving diploid strains also underestimate the events that actually occur.

Reconstruction experiments Reconstruction experiments were performed to determine the efficiency with which previously characterized hyperploids would be recovered under the given selective conditions (trace arginine plates at 30 ~ as a function of different euploid cell background densities. Spontaneous c h r o m o s o m e V I I I hyperploids, verified by tetrad analysis, were used to inoculate various dilutions of euploid cells. In two separate experiments, haploid strain LS706D was mixed wifla two independently isolated LS70-6D disomes. An erythromycin-resistant (ery R) derivative of LS70-6D was used in one of these experiments, permitting spontaneous (cry R) disomes to be distinguished from the added (ery ~) disomes. In a third experiment, diploid LS73 was mixed with a trisomic LS73 isolate. In the absence of a euploid cell background, recovery of the two independent disomic isolates plated to trace arginine medium was essentially 100%. Trisome recovery was 61% (95% confidence limits were 5 0 - 7 4 % ) when plated to trace arginine medium without a euploid cell background. W h e n mixed with euploid cells, recovery of seeded hyperploids was essentially 100% at the 3.2 x 10 -4 plating dilution for all three seeded culture experiments. In our experience, both euploid and aneuploid cell densities clearly influence recovery efficiency with respect to the added hyperploids. The interaction a m o n g these

variables, as well as the characteristics of the particular added hyperploid makes prediction of hyperploid recovery complex. However, conditions were defined for each of the reconstruction experiments that resulted in complete recovery of the seeded hyperploids. U n d e r the conditions applicable to spontaneous hyperploidy determinations, a range of cell dilutions m a y be identified in which colony count on trace arg medium is linearly related to euploid cell dilution. C h r o m o s o m e gain frequency calculations, for each culture, are based on trace arg colony counts obtained from plates within that dilution range.

Discussion

Whittaker et al. (1988), using the diploid yeast strain BR1669, homozygous for arg4-8 and cupl ~, reported a median spontaneous mitotic c h r o m o s o m e V I I I gain rate of 1.2 x 10 .6 hyperploids per cell division with 95% confidence limits of 0.47-1.95 x 10 -6. Strain construction for the present study entailed multiple backcrosses to BR1669-derived haploids and the resultant strains have an average of 15/16 homology to the BR1669 genetic background. Nonetheless, the rate of native c h r o m o s o m e V I I I trisomy in diploid LS73 significantly exceeds the comparable BR1669 levels, by 1.6-fold. The results obtained in this study were generated using a single haploid and a single diploid strain, altered via molecular techniques. Thus, in effect, chromosomes III and V I I I were examined in isogenic strains. Characteristic and significant qualitative and quantitative variations

317 between the two chromosomes with respect to aneuploidy recovery are reported. The utility of the arg4-8, cupl ~gene dosage selection system for detection of hyperploidy, previously demonstrated in diploids, is now extended to haploids. If each chromosome exhibits a constant propensity for segregational error, we can predict that, for a given chromosome, gain would occur at twice the rate in diploids compared to haploid cells. The present results obtained for chromosome III support this notion. However, the observed diploid chromosome VIII gain rate reported here is 5.8-fold greater than the calculated haploid chromosome VIII gain rate. This may indicate reduced viability among chromosome VIII hyperploids and is consistent with the genetic profiles of the various hyperploids, discussed below. The distribution of events among recovered disomes is markedly dependent on the particular chromosome carrying the selectable signal markers. With the signal markers transplaced to chromosome III, the trisomy rate was twice the corresponding value for comparable disomic haploids. Among ten disomes subjected to tetrad analysis, only one showed detectable multiple aneuploidy. Spore viability was typically high and the size of spore colonies from euploids and aneuploids was generally uniform, indications of infrequent multiple hyperploidy occurrence. These observations of indicate moderate tolerance of chromosome III copy number changes relative to the genome as a whole. Interestingly, although there seems to be little or no pressure to maintain the genomic balance of chromosome III copy number relative to other chromosomes, the hyperploid chromosome III tended to be unstable. Thus, of ten phenotypically aneuploid 6DLeu isolates dissected, two or three displayed instability for the hyperploid chromosome III indicating selection against its maintenance. In contrast to chromosome III, the rate of chromosome VIII trisomy was significantly higher than the comparable disomy values, suggesting that chromosome VIII disomes exhibited reduced viability. Surviving chromosome VIII disomes were frequently multiply aneuploid, resulting in poor spore viability and often displayed irregular spore colony growth when crossed to appropriate tester strains. Chromosome VIII hyperploids were clearly more stable than was the case for chromosome III however, indicating that the genetic load due to chromosome VIII aneuploidy was relatively low despite the fact that chromosome VIII contains 1.5 times as much D N A as chromosome III. Thirty-five presumptive hyperploid isolates of LS70-6D were analyzed genetically and diagnosed as stable chromosome VIII hyperploids. Most were additionally hyperploid for other chromosomes. Of the phenotypically non-hyperploid isolates analyzed, 36% (16/44) of the LS70-6D and 65% (17/26) of the 6DLeu isolates were euploid for the chromosome carrying the arg4-8 and cupi ~ markers, a further indication of greater hyperploid instability of chromosome III than chromosome VIII. Campbell's analysis of the products of triploid meiosis (which excluded chromosomes I and III) reported a single positive disome pair correlation involving chromo-

some VIII, namely that with chromosome X (Campbell et al. 1981). There was also a single negative association involving chromosome VIII, with chromosome XI, so there are at most four chromosomes with which chromosome VIII has detectable genomic balance requirements. The degree of multiple aneuploidy among selected chromosome VIII hyperploid haploids indicates that the underlying genetic events are of a more extreme nature than the hyperploidy III-generating events, and that most likely the differences between the two chromosomes do not merely reflect differing genomic balance requirements. The observed multiple aneuploidy associated with both chromosomes VIII and III indicates that the aneuploidy-generating events being observed are not chromosome specific. However, the rate difference between chromosomes VIII and III demonstrates that not all chromosomes are affected equally. This apparent paradox could be explained by a temporal sequence of chromosomal involvement in any of a number of mitosis-related events. The postulation of a temporal sequence predicts a correlation between aneuploidy rate and the degree of coincident aneuploidy, and is testable by utilizing this system to examine other chromosomes. Interchromosomal differences among trisomic diploid isolates were less pronounced than comparable differences among disomic haploids, although similar trends are evident. In meiosis and post-meiotically, the trisomic chromosome VIII was more stable than trisomic chromosome III. Effects on colony growth were reduced in comparison to haploids, and spore viability was unaffected in comparison to the isogenic euploid strains. Coincident aneuploidy occurred among chromosome VIII trisomes to a lesser degree than was the case with disomes. None of the 13 chromosome III trisomes examined displayed evidence of multiple aneuploidy. It is not clear whether the observed lower incidence of multiple aneuploidy mirrors a difference in the hyperploidy generating mechanism, or a greater ability of diploids to eliminate hyperploid chromosomes and return to pseudo-euploidy. The present results demonstrate that interchromosomal aneuploidy comparisons using a single selection criterion are both possible and significant in S. cerevisiae. The utility of the arg4-8, cupI S gene dosage selection system for detecting hyperploidy in haploids is also demonstrated. The data show that chromosomes VIII and III have significant and characteristic differences relative to chromosome gain and stability of the aneuploid chromosomes. Hyperploidy rates calculated from the median frequencies showed chromosome III to have a significantly higher gain rate than chromosome VIII, in both diploids and haploids. The data presented here suggest that the examined chromosomes have different propensities for involvement in aneuploidy-generating events, and it is postulated that this may reflect the timing of chromosomal involvement in mitotic events. The use of this system to examine the behavior of other chromosomes in mitosis may yield information about the normal pattern of chromosomal progression through the mitotic cycle. Hyperploid selection via the cloned arg4-8 and cupP alleles might also be useful in the search for chromosomespecific and other segregation functions, and in charac-

318 terizing cloned genes affecting c h r o m o s o m a l a p p o r t i o n m e n t in mitotic and meiotic cell division.

Acknowledgements. We thank Steven Whittaker and Daniel Maloney for manuscript review, critical suggestions, and excellent statistical and theoretical discussions. Thanks also to Michael Esposito for reviewing the manuscript and help in data interpretation, and to Michael Holland for manuscript review and for making this collaboration possible. Finally, we thank Karin Lusnak, Julie Welch, Charlotte Paquin, Marsha Williamson, Ann Blechl, and Barbara and Wilfreid Kramer for helpful discussions, and Kathe Hesterman and Jennifer Johnson for technical assistance.

References Bocke J, LaCroute F, Fink G (1984) Mol Gen Genet 197:345-346 Campbell D, Doctor J, Feuersanger J, Doolittle M (1981) Genetics 98:239-255 Culbertson MR, Henry SA (1975) Genetics 80:23-40 Denis C, Young E (1983) Mol Cell Biol 3:360-370 Fitzgerald-Hayes M (1987) Yeast 3:187-200 Gaudet A, Fitzgerald-Hayes M (1987) Mol Cell Biol 7:68-75 Gaudet A, Fitzgerald-Hayes M (1989) Genetics 121:477-489 Haber J (1974) Genetics 78:843-858 Humberman J, Zhu J, Davis L, Newlon C (1988) Nucleic Acids Res 16:6373-6384 Karpova T, Zhuravleva T, Pashina O, Nikolaishvili N, Larionov V (1987) Genetika 23:2148-2156 Kearsey S, Edwards J (1987) Mol Gen Genet 210:509-517 Klebe R, Harris J, Sharp Z, Douglas M (1983) Gene 25:333-341 Kouprina N, Pachina O, Nikolaishwili N, Tsouladze A, Larionov V (1988) Yeast 4:257-269

Larionov V, Karpova T, Zhouravleva G, Pashina O, Nikolaishvili N, Kouprina N (1987) Curr Genet 11:435-443 Lea D, Coulson C (1949) J Genet 49:264-285 Liras P, McCusker J, Mascioli S, Haber J (1978) Genetics 88:651671 Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York McClintock B (1951) Cold Spring Harbor Symp Quant Biol 16:1347 Meeks-Wagner D, Wood J, Garvik B, Hartwell L (1986) Cell 44:53 -63 Murray A, Szostak J (1983) Nature 305:189-193 Panzeri L, Landonio L, Stoltz A, Phillippsen P (1985) EMBO J 4:1867-1874 Parry E, Cox B (1970) Genet Res Cambridge 16:333-340 Pomper S, Daniels K, McKee D (1954) Genetics 39:351-355 Rieger R, Michaelis A, Green M (1976) Glossary of genetics and cytogenetics, 4th edn. Springer-Verlag, New York Rockmill B, Fogel S (1988) Genetics 119:261-272 Rockmill B, Roeder G (1988) Proc Natl Acad Sci USA 85:60576061 Sharon G, Simchen G (1990) Genetics 125:487-494 Snyder M, Sapolsky R, Davis R (1988) Mol Cell Biol 8:2184-2194 Thomas J, Botstein D (1986) Cell 44:65-76 Von Borstel R (1978) Methods Cell Biol 20:1-24 Walmsley R (1987) Yeast 3:139-148 Wellinger R, Zakian V (198w Proc Natl Acad Sci USA 86:973-977 Whittaker S, Rockmill B, Blechl A, Maloney D, Resnick M, Fogel S (1988) Molec Gen Genet 215:10-18 Whittaker S, Moser S, Maloney D, Piegorsch W, Resniek M, Fogel S (1989) Mutat Res 242:231-258 C o m m u n i c a t e d by C.W. Birky Jr.

Mitotic hyperploidy for chromosomes VIII and III in Saccharomyces cerevisiae.

The arg4-8 and cup1s markers comprise a copy-number-dependent signal device in the yeast Saccharomyces cerevisiae. These alleles permit reliable discr...
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