Chromosome Specificity of Polysomy Promotion by Disruptions of the Saccharomyces Cerevisiae Rnal Gene

Chromosome Specificity of Polysomy Promotion by Disruptions of the Saccharomyces cerevisiae RNAl Gene

Nigel S. Atkinson' and Anita K. Hopper* Department of Biological Chemistry, The M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 Manuscript received November 4, 1986 Accepted April 3, 1987

ABSTRACT Previously, we showed that a disruption of the yeast RNAI gene with LEU2 sequences promotes polysomy for chromosome XZZZ. Here we demonstrate that this phenotype is due to sequences specific to the RNAl gene and that the disruption allele does not affect nondisjunction of three other chromosomes or polysomy of a minichromosome. Hence polysomy appears to be restricted to chromosome XZZI.

HE mal-1 allele of Saccharomyces cerevisiae is a If the promotion of polysomy phenotype is specific T recessive temperature-sensitive lethal mutation to RNAl sequences and mutated RNAl alleles are affecting the production of mature rRNA, tRNA and capable of promoting polysomy for other chromo- mRNA (HUTCHISON,HARTWELL and MCLAUGHLIN somes, this could suggest that the RNAl gene product 1969; HOPPER,BANKS and EVANCELIDIS1978; ST. is involved in the replication or segregation of chro- JOHN and DAVIS1981). mal-1 strains grow at 23" mosomes. Alteration of a gene involved in the main- but not at temperatures exceeding 30". Generally, tenance of nuclear structure might be expected to 37 " is used as the restrictive temperature. affect nuclear processes as diverse as RNA accumula- Previously, we described the construction and phe- tion and chromosome segregation. Alternatively, if notype of a recessive lethal mutation of RNAl, disrupted RNAl alleles promote polysomy only for mal::LEU2[ATKINSON, DUNST and HOPPER1985 (in chromosome XZZZ, this could suggest that the trun- this reference this allele was referred to as RNAl:: cated RNA1 product establishes a selection for ampli- LEUZ)]. In the mal::LEU2 allele the RNAl coding fication of the mal-1 allele and that this amplification region is disrupted approximately 620 nucleotides results via polysomy for chromosome XZZZ. from the 5' end of the RNAl transcription unit by a 3-kb DNA fragment encoding the yeast LEU2 gene. MATERIALS AND METHODS This allele produces a truncated transcript and cir- cumstantial evidence suggested that it is translated Yeast strains: All yeast strains used in this study are into a truncated polypeptide (ATKINSON,DUNST and described in Table 1. 2bx3b, 13dx22c and 3ax9d are diploid strains that are derived from the following strains: HOPPER1985). rnal::LEU2/mal-l diploids become 4795303 (a gift from D. KOSHLAND),21R (JOHNSTONand polysomic for the chromosome XZZZ bearing the rnal- HOPPER 1985), a6131-20 and EElb (this laboratory). The l allele at a frequency of 2-5% (ATKINSON,DUNST strains 2bx3brnal::URA3 and 2bX3brnal::LEU2 are de- and HOPPER1985). Amplification of the mal-1 allele rived from 2bx3b by gene replacement (ROTHSTEIN1983). due to polysomy permits growth at 34", but not at 2bx3brnal ::LEU2A and 2bxSbrnal::LEU2B are independent isolates of a diploid heterozygous for the rnal::LEU2 37". This phenotype was referred to as partial tem- disruption allele (ATKINSON,DUNST and HOPPER 1985). perature resistance (PTR). Episomal copies of Construction of YEpma1::URAS: YEp24 was digested mal::LEU2 also promoted partial temperature re- with EcoRI and overhanging ends were converted to blunt sistance (ATKINSON,DUNST and HOPPER1985). ends. A BamHI linker was inserted at this site by blunt end In order to characterize the promotion of polysomy ligation. This produced plasmid pBR:URA, which is lacking the 2 p s6quences and carries a URA3 gene flanked by the phenotype associated with mal::LEU2 it is necessary newly inserted BamHI linker on one side and the BamHI to determine: (1) whether this phenotype is the result site within the Escherichia coli tetracycline gene on the other of RNAl-specific sequences or the LEU2 sequences side. The 1.54-kb BamHI DNA fragment from pBR:URA present in rna1::LEUZ; and (2) whether polysomy is containing the URA3 gene was ligated into the BamHI site promoted for chromosomes other than chromosome of the plasmid YEpRNAl(7.7) to yield the plasmid YEprna1::URAJ (Figure 1). xzzz. Measurement of the frequency of loss of chromosomal markers: Cells were inoculated into rich medium (YEPD) at ' Current address: Laboratory of Genetics, University of Wisconsin, Mad- ison, Wisconsin 53706. a density of 1~10~cells per ml with a freshly grown inocu- * To whom correspondence should be addressed. lum and were grown for 48 hr at 23" to a density of 1-

Genetics 116: 371-375 (July, 1987) 372 N. S. Atkinson and A. K. Hopper TABLE 1 Genotype of yeast strains

Strain Genotype 2bX3b MATalMATa, ura3-52/ura3-52, leu2-3,112/leu2-3,112, mal-l/rnal-1,ade2/ade2, tyrl fTYR1, his7/HIS7 2bXSbrnal::URA3 MATaIMATa, ura3-52/ura3-52, leu2-3, I12/leu2-3,112, rna l::URA3/rna 1-1, ade2lade2, tyr IITYR 1, his7/HIS7 2bX3brnal::LEU2A or B MATaIMATa, ura3-52/ura3-52, leu2-3,112/1eu2-3,112, rnal::LEU2frnal-l,ade2/ade2, tyrl fTYR1, his71HIS7 13dx22c MATaIMATa, ura3/ura3,leu2/leuZ, rna 1-1f rnal-1, ade2/ade2, ade3fADE3, tyrllTYR1, his7/HIS7, can1 fCAN1 3ax9d MATafMATa, ura3/ura3,leu2/leu2, mal-llrnal-1, ade2/ade2, ade31ade3, tyrl/TYRI, his71his7 a thr MATa, thr

(Y thr MATa, thr

1.7~lo8. One hundred to 400 colony-forming units of these strain 2bx3b (Table 1) to Ura+. Tetrad analysis was cultures were spread onto YEPD medium and were incu- performed on a stable Ura+ diploid, 2bX3b mal:: bated at 23" for 72 hr. Resulting colonies were replica URA3. Twenty-eight tetrads segregated 2:2 and 3 plated to medium lacking tyrosine or histidine (HOPPER, BANKSand EVANGELIDIS1978) to monitor the loss of TYRl showed 1 :3 segregation for viability to lethality. None and HIS7, respectively (chromosome II), or to canavanine- of the spores was Ura+. These data are consistent with containing medium (medium lacking arginine that contains the interpretation that the strain 2bX3brnal:: URAS 80 mg of canavanine sulfate per liter) to monitor chromo- is heterozygous for a URA3 disruption at the RNAl some V. Loss of a MAT allele (chromosome ZII) was scored locus and that this allele behaves as a recessive lethal. by replica plating the colonies to freshly prepared lawns of the MATa and MATa mating type tester strains, a thr and a We have previously determined that mal::LEU21 thr. The replicas were incubated for 24 hr to permit mating mal-1 diploids give rise to PTR isolates and, using and then replica plated to minimal medium. Growth of a genetic and physical techniques, demonstrated that colony on minimal medium indicates that cells within the PTR isolates are polysomic for chromosome XZZZ (AT- colony have mated with the tester strain. All incubations were performed at 23". Two controls were used in all KINSON,DUNST and HOPPER1985). We have used this experiments: (1) an untransformed isolate of the parent convenient phenotype as an assay for polysomy of strain and (2) a mock transformed isolate of the parent chromosome XZZZ. Cells were inoculated in rich me- strain. The loss of genetic markers was used to estimate the dium (YEPD) at a density of 1X 1O4 cells per ml with loss of the respective chromosome. Transformation was a freshly grown inoculum and were grown for 48 hr performed as described by DUNNet al. (1985). Measurement of the mitotic segregation of pDK243: at 23 " to a density of 1 to 1.7X 108. One hundred to An isolate of 3ax9d that carried the minichromosome 400 colony-forming units of these cultures were pDK243 was transformed with YEp24, YEprnal(7.7) spread onto YEPD medium and were incubated at or YEprna1::URAJ. Cells were maintained on medium that 23" for 72 hr. Resulting colonies were replica plated selected for the retention of the plasmids. Three individual clones from each transformant were picked and suspended to three YEPD plates. Each replica was incubated for into YEPD broth and plated onto YEPD medium lacking 48 hr at a different temperature, 23", 34" or 37", supplemental adenine. The plates were incubated at 23" and the frequency of PTR colonies was recorded. for 72-96 hr and then the colonies were scored for colora- The frequency of PTR isolates was compared be- tion. tween 2bx3brnal::URAJ and two control strains: (1) RESULTS the untransformed 2bX3b parental strain and (2) To determine whether the promotion of polysomy mt2bX3b, a mock transformed parental strain. Nei- is due to sequences specific to the RNAl gene or to ther of the control strains produced PTR isolates (827 the disrupting LEU2 sequences, we constructed an- and 1404 colonies were tested, respectively). The other RNAl disruption allele, mal::URA3(see MATE- strain 2bX3brnal::URAS produced 32 PTR isolates RIALS AND METHODS). mal::URA3is disrupted at the out of 434 colonies examined. Experiments of this same position as in mal::LEU2 (Fig. 1). Neither type were repeated a number of times using two YEprna 1(7.7)::LEU 2 nor YEprna 1:: URAS comple- independently derived disruptions and the frequency ments the mal-1 mutation. of PTR colonies was shown to vary between 1.9 and Gene replacement (ROTHSTEIN1983) was used to 13.3%. These values are comparable to the frequen- introduce the mal::URA3allele at the RNAl locus. cies previously reported for the ma1::LEUZ allele [2- A 5.5-kb SstI fragment derived from the plasmid 5% (ATKINSON,DUNST and HOPPER 1985)l. YEprna1 ::URA3, was used to transform the diploid Two PTR isolates from 2bX3brnal::URA3 were Promotion of Polysomy 373 grown at 34” and then transferred to 23” for sporu- TABLE 2 lation, dissection and growth of spore clones. Of 46 Frequency of loss of chromomme markers in the derivatives of tetrads from one isolate, the following segregation the strain 2bxSb classes were obtained: 1:3, 2:2, and 3:l for viability to lethality in the ratio of 17:28:1. Two spores, one Percent of cob Percent of cob No.. of col- nies exhibiting nies exhibiting from the-3:1 and one from a 2:2 ascus, were Ura+. onies ex- loss of TYRl and loss of MAT. or Thirty-nine tetrads from the other PTR clone segre- Strain amined HIS7 MATO! gated 1:3, 2:2 and 3:l in the ratio of 11:25:2. Eleven 2bX3b 827 0 0.36 (2 from the 3:1, 6 from the 2:2, and 3 from the 1:3 mt2bX3b 1404 0.07 0 2bX3bma 1::URAS 434 1.15 0.92 ascus types) of these spores were Ura+. The increased 2bX3bma 1::LEU2A 1394 0 0.07 frequency of 1:3 segregation in the PTR isolates com- 2bX3bma 1 ::LEU2B 5 1 5 0 0.39 pared to the ts isolates is not understood. However, YEpma1::URAS 985 0.2 0 previously we showed that 3:l and 4:O segregation in strain 2bX3b ~ classes resulting from the presence of rnal::LEU2and suppression of the haplo-lethal phenotype of the dis- the same arm of chromosome ZZ and these dominant ruption were due to chromosome XZZZ polysomy. The alleles are in coupling. Loss of the TYRl and HIS7 3:l segregation of viability to lethality and the pro- bearing homologue would “uncover” the recessive duction of Ura+ meiotic products that carry a gene tyrl and his7 alleles. Loss of either MAT allele on that is a recessive lethal (mal::URA?) provides evi- chromosome ZZZ would enable the strain to mate with dence that the mal::URA3-induced PTR isolates are haploid tester strains. 13dx22c is also heterozygous also polysomic for chromosome XZZZ. Since the disrup- for the canl/CANl marker that is located on chro- tion of RNAl with either of two unrelated sequences mosome V. Uncovering of the can1 allele (loss of causes the production of PTR colonies and polysomy CANl)results in colonies that grow on media contain- for chromosome XZZZ, we conclude that the promotion ing canavanine. of polysomy is not a consequence of the sequences Table 2 summarizes the data from derivatives of used to disrupt the gene, but, rather, related to the 2bX3b. Loss of HIS7 and TYRl was concomitant. truncated RNAl coding region. Since these are unlinked markers on chromosome ZZ, Amplification (polysomy) of minichromosomes oc- we conclude that concomitant loss reflects chromo- curs primarily as a result of nondisjunction (KOSH- some loss. In comparison with the controls, the strains LAND,KENT and HARTWELL1985) and therefore, it 2bX3brnal::LEUSA and 2bX3brnal::LEUSB (two is possible that chromosome polysomy results from a independent isolates that are heterozygous for nondisjunction event. In order to determine whether rnal::LEU2 and mal-1) did not exhibit an increased the disruption alleles influence the frequency of mi- frequency of loss of either chromosome ZZ or IZZ. The totic nondisjunction, we determined whether the 2bX3brnal::URAS strain showed an increase in loss rnal::LEU2and mal::URA? alleles enhanced the fre- of chromosome ZZ, and to a lesser extent loss of quency of nondisjunction and the resultant chromo- chromosome ZZZ. The presence of mal::URA3 on a some loss for three other chromosomes. multicopy plasmid [5-30 copies per cell (STRUHLet The effect of chromosomal disruptions on chro- al. 1979)] appeared to stimulate loss of chromosome mosome loss was analyzed in the strains 2bx3brnal- ZZ slightly; however, the degree of stimulation was less ::URA3 and 2bX3bmal::LEU2, which were derived than that observed with a single copy of mal::URA? from 2bX3b by gene replacement (Table 1). We have and, therefore, was not dosage dependent. The pres- demonstrated that increasing the copy number of the ence of multiple copies of the rnal disruption alleles rnal::LEU2 disruption allele causes an increased fre- did not cause an increased frequency of loss of the quency of PTR isolates; that is, the frequency of PTR TYRl, HZS7, MAT or CANl loci in strain 13dX22c isolates is dependent upon the dosage of rnal::LEU2 (Table 3). (ATKINSON,DUNST and HOPPER1985). If the rnal To summarize, a slight increase in the frequency of disruption alleles promote chromosome polysomy in chromosome loss with respect to the controls was general, then the frequency of polysomy should in- observed only for chromosome ZZ in the strain crease with increased dosage of the allele. There- 2bx3brnal ::URA3. Since neither a chromosomal fore, we also determined whether a disruption allele copy of mal::LEU2 nor an episomal copy of carried on a multicopy plasmid [YEprnal::URA3, rnal::URA3 stimulated the frequency of loss of chro- (Figure 1)] stimulated the frequency of chromosome mosomes ZZ, ZZZ or V, we conclude that the rnal loss in the strains 2bx3b and 13dX22c. disruption alleles do not enhance the frequency of 2bX3b and 13dX22c are heterozygous for three nondisjunction of these chromosomes. genetic markers: tyrllTYR1, his71HZS7 and MATaI The disruption alleles did not increase the fre- MATa. The TYRl and HIS7 loci are 73 cM apart on quency of polysomy as assayed by chromosome loss. 374 N. S. Atkinson and A. K. Hopper

TABLE 3 YEpRNAI(7.71 2.b URA t- Loss of chromosome markers in the strain 13dx22c mI t A I CAN1 TYRl orHIS7 MLzr E EHH SH SH BH S E

C oI on i e s Colonies Colonies YEprnal(7.71::LEU2 2u- URA t Plasmid Percent Total Percent Total Percent Total I Mock transformed 0 832 0.12 832 0.24 832 I\ I YEp24 0 657 0 657 0.62 657 E EHH SH S H BH S E YEpRNAl(7.7) 0 3259 0.12 3259 0 3259 A YEprna 1 (7.7)::LEUZ 0 493 0 493 0.61 493 LZU2 Y Eprna 1 ::U RA3 0 1759 0.34 1759 0.46 1759 YEp rnal::URA3 YCpRNAI(7.7) 0 1510 0 1510 0.2 1510 2ur URA c- YCornal[7.71::LEU2 0 347 0 347 0 347 .I The loss of the CAN1 and MAT markers were measured as an I\ I estimate of the loss of chromosomes V and Ill, respectively. The E EHH SH S H BH S E 7YRl and HIS7 markers were measured as an estimate of the loss of chromosome 11. BBB However, the loss of these chromosomes may not be FIGURE1 .-Restriction maps of recombinant plasmids. The well tolerated in these strains or chromosome ampli- darkened regions are pBR322 sequences. The open regions are fication may occur in the absence of nondisjunction. sequences derived from yeast. The arrow represents the RNA1 To assess these possibilities, we investigated the effect transcript and the direction of transcription. Restriction sites and of the RNAl disruptions upon the segregation of a relevant labels: B, BamHI; S, SstI; E, EcoRI; H, HindlII; 2 p, 2 minichromosome. The minichromosome, pDK243, micron origin of replication; URA, URA3 gene; LEU2, LEU2 gene. YEp24 contains yeast 2 F replicator and the WR.43 gene. YEp carries a centromere sequence, LEU2 and a leaky ade3 RNAl(7.7) was derived from YEp24 by insertion of yeast genomic allele. In an ade2 ade3 genetic background it is possible sequences into the BamHI site of YEp24. Plasmids not shown: to visually quantitate the minichromosome copy num- YCpRNAI(7.7) and YCprna1(7.7)::LEU2. These plasmids are iden- ber within a colony (KOSHLAND,KENT and HARTWELL tical to YEpRNAl(7.7) and YEpma1(7.7)::LEU2, respectively ex- 1985). Cells that carry 0, 1 and 2 copies of the mini- cept that the 2 sequences have been replaced with CEN3 and chromosome produce colonies that are white, pink ARSl. and red, respectively. The frequency of white colonies TABLE 4 reflects the summation of the frequency of simple loss and nondisjunction of the minichromosome. The fre- Effect of mal disruptions on mitotic segregation of pDK243 quency of the red colonies reflects the summation of Second plasmid Average colonies Mean percent Mean percent the frequency of nondisjunction and oversynthesis of present examined/clone white colonies red colonies the minichromosomes (KOSHLAND,KENT and HART- None 610 6.16 (k0.94)” 3.67 (f1.75) WELL 1985). KOSHLAND, KENT and HARTWELLdid YEp24 469 9.24 (k2.84) 4.40 (k5.29) not observe amplification of the minichromosome in YEpRNAl(7.7) 472 6.27 (f0.55) 4.09 (k2.59) the absence of nondisjunction. Y Eprna 1:: URAS 577 6.18 (f0.73) 5.85 (k2.52) Strain 3ax9d was transformed with pDK243 alone or with pDK243 and one of the following plasmids: a The numbers in parentheses are standard deviations. YEp24, YEpRNA1(7.7), YEprna1::URAS (Figure 1). The cells were grown in a medium that selected for alleles that promote polysomy. This phenotype is due retention of the plasmids. Three independent clones to sequences specific to the RNAl gene and has been from each transformant were assayed for the copy shown not to affect three other chromosomes or a number of pDK243 (Table 4). We did not observe a minichromosome. Our hypothesis is that disruption significant increase in the frequency of white or red of the RNAl coding region at the BamHI site results colonies associated with the presence of the plasmid in a selection for cells that are amplified for the YEprna 1:: URAS. After colony color was scored chromosome XIIZ that carries the mal-1 allele. We the colonies were replica plated to media lacking uracil propose that a truncated RNAl product encoded by or leucine to detect the presence of the plasmids. No the disruption alleles inhibits an RNA 1-mediated proc- increase in loss of either plasmid was found to be ess and that this inhibition is deleterious to the cell. correlated with the presence or absence of a particular This inhibition establishes a selection for an increase RNAl allele (data not shown). in the concentration of the intact mal-1 gene product in order to compete out the truncated product. This DISCUSSION selection is satisfied by mal-1 gene amplification me- Our results show that interruption of the RNAl diated via polysomy for chromosome XZZZ. Precedence coding region at the BamHI site generates RNAl for the selection of sequence-specific amplification in Promotion of Polysomy 375 eukaryotes has been established for DHFR and FOGEL, S. and J. W. WELCH,1982 Tandem gene amplification PALA-encoding regions (SCHIMKE1984). In yeast mediates copper resistance in yeast. Prof. Natl. Sci. USA 79: similar iteration of DNA segments surrounding CUP1 5342-5346. HOPPER,A. K., F. BANKSand V. EVANGELIDIS,1978 A yeast results in resistance to copper (FOGELand WELCH mutant which accumulates precursor tRNAs. Cell 14: 211- 1982). Although there are few examples of amplifi- 219. cation of entire chromosomes, SCHATZ,SOLOMON and HUTCHISON,H., L. H. HARTWEUand C. MCLAUCHLIN,1969 BOTSTEIN(1 986) recently reported that a disruption Temperature-sensitive yeast mutant defective in ribonucleic acid production. J. Bacteriol. 99: 807-814. of the yeast TUB1 gene, which codes for (Y tubulin, JOHNSTON,S. A. and J. E. HOPPER, 1982 Isolation of the yeast, could be suppressed by the gain of an additional copy regulatory gene GAL4 and analysis of its dosage effects on the of the chromosome (XIZZ) that encodes this gene. Since galactose/melibiose regulon. Proc. Natl. Acad. Sci. USA 79: 6971-6975. chromosome XZZZ also codes for a second (Y tubulin gene (TUB3), this suppression is probably a result of KOSHLAND,D., J. C. KENT and L. H. HARTWELL,1985 Genetic analysis of the mitotic transmission of minichromosomes. Cell increased expression of TUB3. 40 393-403. We thank RALPHKEIL for critical reading of this manuscript and ROTHSTEIN,R. J., 1983 One-step gene disruption in yeast. Meth- useful suggestions. This research was supported by a U.S. Public ods Enzymol. 101: 202-2 1 1. Health Service grant from the National Institutes of Health to SCHATZ,P. J., F. SOLOMONand D. BOTSTEIN,1986 Genetically A.K.H. essential and nonessential a-tubulin genes specify functionally interchangeable proteins. Mol. Cell. Biol. 6: 3722-3733. SCHIMKE,R. T., 1984 Gene amplification in cultured animal cells. LITERATURE CITED Cell 37: 705-7 13. ATKINSON,N. S., R. W. DUNST and A. K. HOPPER, 1985 ST. JOHN, T. P. and R. W. 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