Copyright Q 1992 by the Genetics Society of America
Selection for Early Meiotic Mutantsin Yeast Aaron P. Mitchell*lt>* and Katherine S. Bowdish* *Institute of Cancer Research, +Department ofMicrobiology, and *Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, New York 10032
Manuscript received November 14, 1991 Accepted for publication January 24, 1992 ABSTRACT In the yeast Saccharomyces cereoisiae, only a / a cells can enter meiosis; a and a cells cannot. Because a / a cells are typically diploid and a and a cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied byan increase in expression of the IMEl gene; the IMEl product (IME1) then activatesIME2 and other meiotic genes. We have found that IMEl expression is toxic to starved haploid cells, presumably because IMEl directs them into meiosis. IMEl toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IMEl toxicity include recessive mutations in two genes, RIMl 1 and RIM16 (Regulator of Inducer of Meiosis), that are required for IMEl to activate IME2 expression. RIMl 1 maps near CIN4 on chromosomeXIII.
N Saccharomycescerarisiae, meiosis and spore formation are restricted to one type of cell, the a/a cell, and are induced by starvation (see E~POSITOand KLAPHOLZ1981). The other types of cells, a and a cells, are unable to enter the meiotic S phase; they respond to starvation through arrest of growth and cell division. Because a/a cells are typically diploid and a and a cells are typically haploid, the cell type regulatory system ensures that only diploid cells enter meiosis. In effect, this restriction protects haploidcells fromattemptingareductional (meiosis I) division, which they could not successfully complete. Three lines of evidence indicate that theI M E l gene product (referred to as IMEl), a meiotic activator, transmits cell type signals. First, I M E l is essential for meiosis andfor meiotic gene expression (KASSIR, GRANOTand SIMCHEN 1988;Smith and MITCHELL 1989;, Mitchell, Driscoll and SMITH 1990; ENGEBRECHT and ROEDER1990). Second,I M E l RNA levels are higher in a/a cells than in non-a/a cells under starvation conditions (KASSIR, GRANOTand SIMCHEN 1988). Third, increased I M E l dosage permits cells of any type toenter meiosis (KASSIR, GRANOTand SIMCHEN 1988),andinappropriate expression of I M E l (from the GAL1 promoter) permits a anda cells to express meiotic genes (SMITHet al. 1990). Therefore, the natural low level of I M E l expression in a and a cells is insufficient for activation of meiotic genes. IMEl also transmits nutritional signals. I M E l expression is elevated in starved cells (KASSIR, GRANOTand SIMCHEN1988); constitutive IMEl expression permits elevated meiotic gene expression in unstarved cells (SMITHet al. 1990). However, even in cells that express I M E l constitutively, starvation is
I
Genetics 131: 65-72 (May, 1992)
necessary to induce meiosis and to further stimulate meiotic geneexpression. Inaddition,a multicopy I M E l plasmid permits only low levels of sporulation in mutants that fail to arrest growth in response to starvation (MATSUURA et al. 1990). Thus nutritional signals act both upstream and in parallel or downstream of IMEl to govern meiosis. In this paper, we report that expression of IMEl is toxic to haploid cells under starvation conditions. IMEl toxicity is exacerbated by a rad52 mutation, which results in inability torepairdouble-stranded chromosome breaks;such breaks occurduring meiosis (SUN et al. 1989; CAO,ALANIand KLECKNER1990). Suppressors of I M E l toxicity includemutations in IME2, the next known gene in the regulatory hierarchy that controls meiotic gene expression (SMITH and MITCHELL1989; MITCHELL,DRISCOLL and SMITH 1990; YOSHIDA et al. 1990, reviewed in MALONE 1990). Such suppressors also include recessive mutations in two newly identified genes,R I M l 1 and RIM16 (Regulator of Inducer of Meiosis). The IMEl product may act upstream or in parallel with the RIM11 and RIM16 gene products to stimulate IME2 expression and meiosis. MATERIALS AND METHODS Strains: All strains(Table 1) werederivatives of the SKI except for strain MAY 1140, rapidly sporulating diploid which was provided by ANDREWHOYT. Mutations previura3, leu2::hisG, trpl::hisG, lys2, ouslydescribedinclude ho::LYS2 (ALANI,CAO and KLECKNER1987), ga180::LEUZ (TORCHIA et al. 1984),imel-12::TRPl (SMITH and MITCHELL 1989), IME2-6::LEU2, his4,ade3,arg6 (MITCHELL, DRISCOLL and SMITH 1990), PCALI-IME1-14::TRP1,ime2-4lacZ::LEU2 (SMITHet al. 1990),his3, and met4 (NEIGEBORN and MITCHELL 1991). The PGALI-IME1-14::TRP1allele,
66
A. P. Mitchell and K. S. Bowdish TABLE 1 Yeast strains Originb
Strain
177 249 266 473 475 918 1003 1004 1005 1006 1007 1008 1142 1143 1144 1145
a a spol3 a his4 a IME2-6 his4
ade3
a IME2-6 his4 arg6 a
imel-12 ime2-4-lacZ met4
a P G A L I - I M E I - I ~ r a dhis3 52 a PGALI-IMEI-14 rad52 his3 aP
C ~ ~ ~ - I M Erad52 I - I ~arg6
(1) (2) (1) (2) (2) (3, strain AM1 345-3C) (1) (1) (1)
a PcALI-IMEI-14 rad52 arg6
(1)
a PcALI-IMEI-14
(1) (1) (1) (1) (1) 1151 X 918
his3 a PcALl-IMEI-14 arg6
a a rad52 aPGALI-IMEI-~~ a P G A L I - I M E Iime2-4-lacZ -~~
met4 his4
1146 1147
a rim1 1-4 p c ~ ~ l - I M E 1 - 1 4 a r g 6 1005r4 X 1008
a rimll-4 PcALl-IMEI-14ime24-lac2 his4 1148 a rimll-4 PcALl-IMEI-14 ime24-lac2 met4 1149 a rimll-4 PGALI-IMEI-14 met4 1150 a riml1-4 PcALI-IMEI-14his4 1151 a r i m l l - 6 p c ~ ~ l - I M E 1 -his4 14 1152 a rimll-6 PcALI-IMEI-14 his3 1153 a rimll-6 PcALl-IMEI-14 his3 1154 a rimll-6 PcALI-IMEI-14 ime24-lacZ his4 1155 a rimll-6 PCALI-IMEI-14 ime24-lac2 his4 1156 a rimll-6 PcALI-IME1-14 his4 1157 a rimll-6 PcALI-IMEI-14 his4 1158 a rim1 1-6 PCALI-IMEI-14 ime24-lac2 his4 1159 a r i m l l - 8 P c A L I - I M E I -his4 I~ 1160 a rimll-8 PcALI-IMEI-14 arg6 1161 a rimll-8 PcALI-IMEI-14 ime24-lacZ met4 his4 a rimll-8 PCALI-IMEI-14 ime21162 4-lacZ met4 his4 a rimll-8 PcALl-IMEI-14 his4 1163 1164 a r i m l l - 8 P c ~ ~ 1 - 1 h f E 1his4 -14 met4 a rimll-8 PcALl-IMEl-14 his4 1165 met4 1166 a rim16-12 PGALI-IMEI-I~ arg6 1167 a rim16-12 PcALl-IMEI-I4 ime2-4-lac2 met4 his4 a rim16-12 PGALI-IMEl-14 1168 ime2-4-lacZ met4 a rim16-12 PcALl-IMEI-14 met4 1169 a rim16-12 PGALI-IMEI-14 his4 1170 MAY 1 140 a his3 leu2 ura3 lys2 cin4::URA3
1146 X 1145 1146 X 1145 1146 X 1145 1146 X 1145 1004r6 X 266 1004r6 X 1007 1004r6 X 1007 1151 X 1145 1151 X 1145 1151 X 1145 1151 X 1145 1151 X 918 10061-8 X 266 1006r8 X 1007 1159 X 1145 1159 X 1145 1159 X 1145 1159 X 1145 1159 X 1145 10051-12X 1008 1166 X 1145 1166X 1145 1166 X 1145 1166 X 1145 A. HOYT
~~
All strains except MAY1 140 carry additional markers ura3 leu2::hisG trp1::hisG lys2 ho::LYS2 gal80::LEU2. Strains were (1) derived from crosses or transformations of DRISCOLL SK1-derived strains or were described in (2) MITCHELL, and SMITH(1990) or (3) SMITHet al. (1 990). a
which we refer to as PCALI-IMEIin this paper, is a replacement of the chromosomal IMEl promoter with the GAL1 promoter (directing expression of the IMEl coding region) and the selectable marker TRPl (SMITHet al. 1990). The spol3::hisG mutation was provided in an SK1 derivative by E. ALANIand N. KLECKNER.The rad52::LEUZ mutation RAD52 BglII site was an insertion of the LEU2 gene into the (D. SCHILD,personal communication; see SCHILDet al. 1983); it was introduced by transformation from plasmid pSM20 and was scored by sensitivity to ultraviolet light. l'he genotypes of all SK1derivatives includes markers uraj leu2::hisG trp1::hisG lys2 ho::LYS2 gal80::LEUZ. We designate the two genes identified in this study RIM1 I and RIM16 because mutations defining genes RIMI-RIM15 have been isolated through anotherapproach that identified one r i m l l mutation (S. SU, personal communication). In general descriptions of experiments carried out with all of the r i m l l and rim16 mutations, we refer to the mutations as Spo'-suppressors or, in genotypes, as s u p mutations. Media: Yeast and bacterial media, including YEPD, YEPAC,SD, SC and galactose indicator plates, followed standard recipes (ROSE,WINSTONand HIETER 1990;SMITHand MITCHELL1989). Liquid sporulation medium contained 2% potassium acetate and 20 mg/liter of necessary auxotrophic supplements. KAcXgal plates, for monitoring ime2-ZacZ expression, contained 20 g potassium acetate, 20 g Bactoagar, and 900 ml water; after autoclaving, 50 ml of 1 M sodium phosphate (pH 7.0), 3 ml of 20mg/mlXgalin dimethylformamide, and auxotrophic supplements (as in SC medium) were added. Spo plates, for inducing sporulation, contained 1.5% potassium acetate, 2% agar, 0.25% Difco Yeast Extract, 0.1% dextrose, 40 mg/liter adenine sulfate, 40 mg/liter uracil, 8 mg/liter tyrosine, and these supplements, each at 20 mg/liter: histidine HCI, leucine, lysine HCI, arginine HCI, tryptophan, phenylalanine and methionine. Rich Spo broth had the same composition as Spo plates except that agar was omitted. Spo" and Spo' phenotypes: We routinely monitored the ability of strains to survive under sporulation conditions by a replica-plating test. Patches were grown on rich medium (YEPD) for 1 or 2 days at 30°, then replica-plated to a Spo plate. After 3 or 4 days at 30°, the Spo plate was replicaplated to asecond YEPD plate. Growth on the second YEPD plate was scored after 1 or 2 days at 30". Strains that are Spo' ("sporulation medium resistant") display strong, homogeneous growth onthe second YEPD plate (see examples in Figure 1, patches A, B and E). Strains that are Spos ("sporulation medium sensitive") display weak, heterogeneous growth on the second YEPD plate (Figure 1, patches C and D). We have quantitated survival after incubating strains in rich Spo broth at 30" for 3 days. Comparison with replicaplating tests indicates that Spo"strains have a plating density 10- 100-fold lower than Spo' strains under these conditions. We noteone discrepancy that may result from peculiar replica-plating properties: PGALI-IMEl sf1013 strains display a weak Spo' phenotype based on replica-plating but a strong Spo' phenotype in quantitative tests. Mutant isolation: We used a selection to isolate spontaneous mutants resistant to the toxicityof IMEl incells starved on Spo plates. Single colonies of haploid PCALI-IMEI rad52::LEU2 strains were grown as patches on YEPD plates, then replica-plated to Spo plates. After 4 days at 3 0 ° , SPO plates were replica-plated to YEPD. After 2 days at 30°, one papilla per patch was picked and retested. Because mitochondrial defects suppress PCALI-IMEItoxicity, larger papillae were chosen preferentially. Strains were then purified on YEPD and retested for survival on Spo plates. Gal-
Meiotic
Early
67 Mutants in Yeast
l M E l KAD.52 KIM11 mutations, which suppress P[;.,I./-IMEI toxicity by blocking A + + + its expression, were detected on Galactose indicator meR + + dium. Complementation tests were accomplished by examC /%A/./ + + ining sporulation or ime2-lacZ expression of diploids obD PCA/./ + tained from crosses to i m e l , ime2, rimI I and rim16 mutant E PGM / testers. Cosegregation of sporulation defects and Spo'-suppressors: In order to test whether each Spo'-suppressor caused a defect in sporulation, we first constructed a and cy testers carrying each Spa'-suppressor. The testers weremeiotic progeny from a/. P[;.~I.l-IMEI/Pc.~l.,-IMEI sup/+ ime2-lacZl + diploids;theywerechosenas segregants that failed to express ime2-lacZ activity (Lac- phenotype) from tetrads displaying 2 Spo'Lac-:2Spo'Lac+ segregation, including strains 1149, 1150, 1156,1157,1163,1165, 1169 and 1 170. To see whether each Spa'-suppressor cosegregated @-Galactosidaseassays: Quantitative P-galactosidase aswith a sporulation defect, the testers were mated to all says were performed on pernleabilized cells as described segregants of three tetrads from an a/a P[;,.,l.l-IMEI/P~;,~,.I(SMITHet al. 1990). Cells had been shifted from exponential I M E I sup/SUP diploid; sporulation of the resulting diploids phase in YEPAc to sporulation medium for 4 hr. Numbers was quantitated after 2 days at 30" on Spo plates. are the avcrage of determinations with three cultures. Suppression of r i m l l - 6 by IME2-6 r i m I l - 6 IMEP-6 segregants were isolated from crosses 1 157 x 473 and 1 153 x 47.5. rim1I-6 was followed in segregants by failure to RES U LTS complement the rim 11-6 testers 1 156 and 1157 for sporuToxicity of ZMEl expression to haploids: The lation. IME2-6,anallele from which expression of IME2 depends on the GAL regulatory system, was followed by I M E I product is a positive regulator of meiosis and fiilure to complement GAL80 ime2-2::LEUZ testers for spormeiotic gene expression. Expression of I M E I increases ulation (MITCHELL, DRISCOLI.and SMITH1990). Appropriin responsetonitrogenstarvation, which induces ately marked segregants were mated, diploids were selected, IiMEI during starvation is meiosis. Expression of and sporulation ability was determined after 3 days at 30" greater in a / a cells, which enter meiosis, than in a and o n Spo plates. Cloning of R I M I I : Haploid Pa.l/.I-IMEI rim1 1-6 ime2N cells, which arrest prior to meiotic DNA synthesis. lacZ ura3 strains were transformed with genomic libraries Prior studies suggested that the P c ; . . l ~ . ~ - I ~allele, ~lEl a carried in YCp50, a low copy-number UKA3-bearing plasfusion of the I M E I coding region to the CALI promid (ROSE et al. 1987). Transformants were replica-plated moter, would permit a and N cells to enter meiosis in from SC-Ura to KAcXgal plates (lackinguracil). Transformresponsetostarvation (SMITHet d l . 1990). Weexants that turned blue on KAcXgal were purified, retested, checked for co-curing of Ura+ and ime2-lacZ expression pected haploids that entered meiosis to become inviphenotypes. able because of errors i n chromosome segregation as Production of anti-IMEl antibodies: Antibodies were theyattempt meiosis I division. T h u s Pc;,.,l.l-I~VIEl produced in rabbits against an 1ME1 fusion protein purified should be toxic to haploidcells starved on sporulation from E. coli. The IMEI open reading frame and 12 addiplates. tional 5' codons were excised as a RamHl fragment from plasmidYCpPC;,,I,/-IMEI (SMITHet al. 1990) and inserted ~lEl conferred We observed that P ~ ; , . l ~ . ~ - I iexpression into the BamHI site of plasmid pAR3039 (STUDIER and sensitivity to sporulation medium (Figure 1). A wild MOFFAT 1986). The resulting plasmid expresses IMEI and type haploid strain remained viable after incubation a total of 24 5' codons from the phage T7 gene 10 proon Spo plates for 4 days [Spo" phenotype (patch A)]. and MOFFAT 1986) moter. E. coli strain B12 1(DE3) (STUDIER However, a haploid strain that expressed Pc;,.,/.I-I~bfEl carrying this plasmid was induced with IPTG and lysed with lysozyme-EDTA, freeze-thaw cycles, and sonication. The survived the incubation poorly[Spo' phenotype (patch fusion protein was pelleted with cell debris, washed, solubiC ) ] .A wild-tvpe GAL80 allele, jvhich blocks functional lized in 7 M urea, passed through CM Sepharose, fractionP~;.,,/.~-IMEI expression in sporulationmedium,conated on DEAE Sepharose and dialyzed to remove urea. New fers a Spo' phenotype to Pc;,.,~-l-IMElhaploids(not Zealand White rabbits received subcutaneous primary in-jecshown). We note that Pc;,.,~,l-IMElis not toxic to a / a tions of 250 pg of fusion protein emulsified with Freund's complete adjuvant. Subsequent injections (boosts)of 250 pg diploids: a / a P~;~,/./-Ii~lEl/P~;;,/,,-IMEl diploids sporuof fusion protein in Freund's incomplete adjuvant were late efficiently and yield >9.5% spore viability. 'These accomplished at 30-day intervals. Immune serum used for results suggest that 'the I M E I gene product, I M E I , is the experiment in Figure 2 was collected 7 days after the toxic to starved haploid cells. fourth boost. We considered two modelsfor I M E l toxicity: IMEI Immunoblots: Cell extracts were prepared fromlog phase YEPAc cultures by vortexing with glass beads in 50 may directhaploidstoenter meiosis andthusdie; m~ Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride. 50 alternatively, I M E l may interfere with some vital pg of soluble protein was fractionated on a 10% polyacrylprocess in which it normallypl;~ys norole. IMEI amide SDS gel, transferred to nitrocellulose, and incubated normally activates meiosis through activation of I M E 2 sequentially w i t h rabbit serum and peroxidase-conjugated expression. T h e first model predicts thata P
Selection for Early Meiotic Mutantsin Yeast Aaron P. Mitchell*lt>* and Katherine S. Bowdish* *Institute of Cancer Research, +Department ofMicrobiology, and *Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, New York 10032
Manuscript received November 14, 1991 Accepted for publication January 24, 1992 ABSTRACT In the yeast Saccharomyces cereoisiae, only a / a cells can enter meiosis; a and a cells cannot. Because a / a cells are typically diploid and a and a cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied byan increase in expression of the IMEl gene; the IMEl product (IME1) then activatesIME2 and other meiotic genes. We have found that IMEl expression is toxic to starved haploid cells, presumably because IMEl directs them into meiosis. IMEl toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IMEl toxicity include recessive mutations in two genes, RIMl 1 and RIM16 (Regulator of Inducer of Meiosis), that are required for IMEl to activate IME2 expression. RIMl 1 maps near CIN4 on chromosomeXIII.
N Saccharomycescerarisiae, meiosis and spore formation are restricted to one type of cell, the a/a cell, and are induced by starvation (see E~POSITOand KLAPHOLZ1981). The other types of cells, a and a cells, are unable to enter the meiotic S phase; they respond to starvation through arrest of growth and cell division. Because a/a cells are typically diploid and a and a cells are typically haploid, the cell type regulatory system ensures that only diploid cells enter meiosis. In effect, this restriction protects haploidcells fromattemptingareductional (meiosis I) division, which they could not successfully complete. Three lines of evidence indicate that theI M E l gene product (referred to as IMEl), a meiotic activator, transmits cell type signals. First, I M E l is essential for meiosis andfor meiotic gene expression (KASSIR, GRANOTand SIMCHEN 1988;Smith and MITCHELL 1989;, Mitchell, Driscoll and SMITH 1990; ENGEBRECHT and ROEDER1990). Second,I M E l RNA levels are higher in a/a cells than in non-a/a cells under starvation conditions (KASSIR, GRANOTand SIMCHEN 1988). Third, increased I M E l dosage permits cells of any type toenter meiosis (KASSIR, GRANOTand SIMCHEN 1988),andinappropriate expression of I M E l (from the GAL1 promoter) permits a anda cells to express meiotic genes (SMITHet al. 1990). Therefore, the natural low level of I M E l expression in a and a cells is insufficient for activation of meiotic genes. IMEl also transmits nutritional signals. I M E l expression is elevated in starved cells (KASSIR, GRANOTand SIMCHEN1988); constitutive IMEl expression permits elevated meiotic gene expression in unstarved cells (SMITHet al. 1990). However, even in cells that express I M E l constitutively, starvation is
I
Genetics 131: 65-72 (May, 1992)
necessary to induce meiosis and to further stimulate meiotic geneexpression. Inaddition,a multicopy I M E l plasmid permits only low levels of sporulation in mutants that fail to arrest growth in response to starvation (MATSUURA et al. 1990). Thus nutritional signals act both upstream and in parallel or downstream of IMEl to govern meiosis. In this paper, we report that expression of IMEl is toxic to haploid cells under starvation conditions. IMEl toxicity is exacerbated by a rad52 mutation, which results in inability torepairdouble-stranded chromosome breaks;such breaks occurduring meiosis (SUN et al. 1989; CAO,ALANIand KLECKNER1990). Suppressors of I M E l toxicity includemutations in IME2, the next known gene in the regulatory hierarchy that controls meiotic gene expression (SMITH and MITCHELL1989; MITCHELL,DRISCOLL and SMITH 1990; YOSHIDA et al. 1990, reviewed in MALONE 1990). Such suppressors also include recessive mutations in two newly identified genes,R I M l 1 and RIM16 (Regulator of Inducer of Meiosis). The IMEl product may act upstream or in parallel with the RIM11 and RIM16 gene products to stimulate IME2 expression and meiosis. MATERIALS AND METHODS Strains: All strains(Table 1) werederivatives of the SKI except for strain MAY 1140, rapidly sporulating diploid which was provided by ANDREWHOYT. Mutations previura3, leu2::hisG, trpl::hisG, lys2, ouslydescribedinclude ho::LYS2 (ALANI,CAO and KLECKNER1987), ga180::LEUZ (TORCHIA et al. 1984),imel-12::TRPl (SMITH and MITCHELL 1989), IME2-6::LEU2, his4,ade3,arg6 (MITCHELL, DRISCOLL and SMITH 1990), PCALI-IME1-14::TRP1,ime2-4lacZ::LEU2 (SMITHet al. 1990),his3, and met4 (NEIGEBORN and MITCHELL 1991). The PGALI-IME1-14::TRP1allele,
66
A. P. Mitchell and K. S. Bowdish TABLE 1 Yeast strains Originb
Strain
177 249 266 473 475 918 1003 1004 1005 1006 1007 1008 1142 1143 1144 1145
a a spol3 a his4 a IME2-6 his4
ade3
a IME2-6 his4 arg6 a
imel-12 ime2-4-lacZ met4
a P G A L I - I M E I - I ~ r a dhis3 52 a PGALI-IMEI-14 rad52 his3 aP
C ~ ~ ~ - I M Erad52 I - I ~arg6
(1) (2) (1) (2) (2) (3, strain AM1 345-3C) (1) (1) (1)
a PcALI-IMEI-14 rad52 arg6
(1)
a PcALI-IMEI-14
(1) (1) (1) (1) (1) 1151 X 918
his3 a PcALl-IMEI-14 arg6
a a rad52 aPGALI-IMEI-~~ a P G A L I - I M E Iime2-4-lacZ -~~
met4 his4
1146 1147
a rim1 1-4 p c ~ ~ l - I M E 1 - 1 4 a r g 6 1005r4 X 1008
a rimll-4 PcALl-IMEI-14ime24-lac2 his4 1148 a rimll-4 PcALl-IMEI-14 ime24-lac2 met4 1149 a rimll-4 PGALI-IMEI-14 met4 1150 a riml1-4 PcALI-IMEI-14his4 1151 a r i m l l - 6 p c ~ ~ l - I M E 1 -his4 14 1152 a rimll-6 PcALI-IMEI-14 his3 1153 a rimll-6 PcALl-IMEI-14 his3 1154 a rimll-6 PcALI-IMEI-14 ime24-lacZ his4 1155 a rimll-6 PCALI-IMEI-14 ime24-lac2 his4 1156 a rimll-6 PcALI-IME1-14 his4 1157 a rimll-6 PcALI-IMEI-14 his4 1158 a rim1 1-6 PCALI-IMEI-14 ime24-lac2 his4 1159 a r i m l l - 8 P c A L I - I M E I -his4 I~ 1160 a rimll-8 PcALI-IMEI-14 arg6 1161 a rimll-8 PcALI-IMEI-14 ime24-lacZ met4 his4 a rimll-8 PCALI-IMEI-14 ime21162 4-lacZ met4 his4 a rimll-8 PcALl-IMEI-14 his4 1163 1164 a r i m l l - 8 P c ~ ~ 1 - 1 h f E 1his4 -14 met4 a rimll-8 PcALl-IMEl-14 his4 1165 met4 1166 a rim16-12 PGALI-IMEI-I~ arg6 1167 a rim16-12 PcALl-IMEI-I4 ime2-4-lac2 met4 his4 a rim16-12 PGALI-IMEl-14 1168 ime2-4-lacZ met4 a rim16-12 PcALl-IMEI-14 met4 1169 a rim16-12 PGALI-IMEI-14 his4 1170 MAY 1 140 a his3 leu2 ura3 lys2 cin4::URA3
1146 X 1145 1146 X 1145 1146 X 1145 1146 X 1145 1004r6 X 266 1004r6 X 1007 1004r6 X 1007 1151 X 1145 1151 X 1145 1151 X 1145 1151 X 1145 1151 X 918 10061-8 X 266 1006r8 X 1007 1159 X 1145 1159 X 1145 1159 X 1145 1159 X 1145 1159 X 1145 10051-12X 1008 1166 X 1145 1166X 1145 1166 X 1145 1166 X 1145 A. HOYT
~~
All strains except MAY1 140 carry additional markers ura3 leu2::hisG trp1::hisG lys2 ho::LYS2 gal80::LEU2. Strains were (1) derived from crosses or transformations of DRISCOLL SK1-derived strains or were described in (2) MITCHELL, and SMITH(1990) or (3) SMITHet al. (1 990). a
which we refer to as PCALI-IMEIin this paper, is a replacement of the chromosomal IMEl promoter with the GAL1 promoter (directing expression of the IMEl coding region) and the selectable marker TRPl (SMITHet al. 1990). The spol3::hisG mutation was provided in an SK1 derivative by E. ALANIand N. KLECKNER.The rad52::LEUZ mutation RAD52 BglII site was an insertion of the LEU2 gene into the (D. SCHILD,personal communication; see SCHILDet al. 1983); it was introduced by transformation from plasmid pSM20 and was scored by sensitivity to ultraviolet light. l'he genotypes of all SK1derivatives includes markers uraj leu2::hisG trp1::hisG lys2 ho::LYS2 gal80::LEUZ. We designate the two genes identified in this study RIM1 I and RIM16 because mutations defining genes RIMI-RIM15 have been isolated through anotherapproach that identified one r i m l l mutation (S. SU, personal communication). In general descriptions of experiments carried out with all of the r i m l l and rim16 mutations, we refer to the mutations as Spo'-suppressors or, in genotypes, as s u p mutations. Media: Yeast and bacterial media, including YEPD, YEPAC,SD, SC and galactose indicator plates, followed standard recipes (ROSE,WINSTONand HIETER 1990;SMITHand MITCHELL1989). Liquid sporulation medium contained 2% potassium acetate and 20 mg/liter of necessary auxotrophic supplements. KAcXgal plates, for monitoring ime2-ZacZ expression, contained 20 g potassium acetate, 20 g Bactoagar, and 900 ml water; after autoclaving, 50 ml of 1 M sodium phosphate (pH 7.0), 3 ml of 20mg/mlXgalin dimethylformamide, and auxotrophic supplements (as in SC medium) were added. Spo plates, for inducing sporulation, contained 1.5% potassium acetate, 2% agar, 0.25% Difco Yeast Extract, 0.1% dextrose, 40 mg/liter adenine sulfate, 40 mg/liter uracil, 8 mg/liter tyrosine, and these supplements, each at 20 mg/liter: histidine HCI, leucine, lysine HCI, arginine HCI, tryptophan, phenylalanine and methionine. Rich Spo broth had the same composition as Spo plates except that agar was omitted. Spo" and Spo' phenotypes: We routinely monitored the ability of strains to survive under sporulation conditions by a replica-plating test. Patches were grown on rich medium (YEPD) for 1 or 2 days at 30°, then replica-plated to a Spo plate. After 3 or 4 days at 30°, the Spo plate was replicaplated to asecond YEPD plate. Growth on the second YEPD plate was scored after 1 or 2 days at 30". Strains that are Spo' ("sporulation medium resistant") display strong, homogeneous growth onthe second YEPD plate (see examples in Figure 1, patches A, B and E). Strains that are Spos ("sporulation medium sensitive") display weak, heterogeneous growth on the second YEPD plate (Figure 1, patches C and D). We have quantitated survival after incubating strains in rich Spo broth at 30" for 3 days. Comparison with replicaplating tests indicates that Spo"strains have a plating density 10- 100-fold lower than Spo' strains under these conditions. We noteone discrepancy that may result from peculiar replica-plating properties: PGALI-IMEl sf1013 strains display a weak Spo' phenotype based on replica-plating but a strong Spo' phenotype in quantitative tests. Mutant isolation: We used a selection to isolate spontaneous mutants resistant to the toxicityof IMEl incells starved on Spo plates. Single colonies of haploid PCALI-IMEI rad52::LEU2 strains were grown as patches on YEPD plates, then replica-plated to Spo plates. After 4 days at 3 0 ° , SPO plates were replica-plated to YEPD. After 2 days at 30°, one papilla per patch was picked and retested. Because mitochondrial defects suppress PCALI-IMEItoxicity, larger papillae were chosen preferentially. Strains were then purified on YEPD and retested for survival on Spo plates. Gal-
Meiotic
Early
67 Mutants in Yeast
l M E l KAD.52 KIM11 mutations, which suppress P[;.,I./-IMEI toxicity by blocking A + + + its expression, were detected on Galactose indicator meR + + dium. Complementation tests were accomplished by examC /%A/./ + + ining sporulation or ime2-lacZ expression of diploids obD PCA/./ + tained from crosses to i m e l , ime2, rimI I and rim16 mutant E PGM / testers. Cosegregation of sporulation defects and Spo'-suppressors: In order to test whether each Spo'-suppressor caused a defect in sporulation, we first constructed a and cy testers carrying each Spa'-suppressor. The testers weremeiotic progeny from a/. P[;.~I.l-IMEI/Pc.~l.,-IMEI sup/+ ime2-lacZl + diploids;theywerechosenas segregants that failed to express ime2-lacZ activity (Lac- phenotype) from tetrads displaying 2 Spo'Lac-:2Spo'Lac+ segregation, including strains 1149, 1150, 1156,1157,1163,1165, 1169 and 1 170. To see whether each Spa'-suppressor cosegregated @-Galactosidaseassays: Quantitative P-galactosidase aswith a sporulation defect, the testers were mated to all says were performed on pernleabilized cells as described segregants of three tetrads from an a/a P[;,.,l.l-IMEI/P~;,~,.I(SMITHet al. 1990). Cells had been shifted from exponential I M E I sup/SUP diploid; sporulation of the resulting diploids phase in YEPAc to sporulation medium for 4 hr. Numbers was quantitated after 2 days at 30" on Spo plates. are the avcrage of determinations with three cultures. Suppression of r i m l l - 6 by IME2-6 r i m I l - 6 IMEP-6 segregants were isolated from crosses 1 157 x 473 and 1 153 x 47.5. rim1I-6 was followed in segregants by failure to RES U LTS complement the rim 11-6 testers 1 156 and 1157 for sporuToxicity of ZMEl expression to haploids: The lation. IME2-6,anallele from which expression of IME2 depends on the GAL regulatory system, was followed by I M E I product is a positive regulator of meiosis and fiilure to complement GAL80 ime2-2::LEUZ testers for spormeiotic gene expression. Expression of I M E I increases ulation (MITCHELL, DRISCOLI.and SMITH1990). Appropriin responsetonitrogenstarvation, which induces ately marked segregants were mated, diploids were selected, IiMEI during starvation is meiosis. Expression of and sporulation ability was determined after 3 days at 30" greater in a / a cells, which enter meiosis, than in a and o n Spo plates. Cloning of R I M I I : Haploid Pa.l/.I-IMEI rim1 1-6 ime2N cells, which arrest prior to meiotic DNA synthesis. lacZ ura3 strains were transformed with genomic libraries Prior studies suggested that the P c ; . . l ~ . ~ - I ~allele, ~lEl a carried in YCp50, a low copy-number UKA3-bearing plasfusion of the I M E I coding region to the CALI promid (ROSE et al. 1987). Transformants were replica-plated moter, would permit a and N cells to enter meiosis in from SC-Ura to KAcXgal plates (lackinguracil). Transformresponsetostarvation (SMITHet d l . 1990). Weexants that turned blue on KAcXgal were purified, retested, checked for co-curing of Ura+ and ime2-lacZ expression pected haploids that entered meiosis to become inviphenotypes. able because of errors i n chromosome segregation as Production of anti-IMEl antibodies: Antibodies were theyattempt meiosis I division. T h u s Pc;,.,l.l-I~VIEl produced in rabbits against an 1ME1 fusion protein purified should be toxic to haploidcells starved on sporulation from E. coli. The IMEI open reading frame and 12 addiplates. tional 5' codons were excised as a RamHl fragment from plasmidYCpPC;,,I,/-IMEI (SMITHet al. 1990) and inserted ~lEl conferred We observed that P ~ ; , . l ~ . ~ - I iexpression into the BamHI site of plasmid pAR3039 (STUDIER and sensitivity to sporulation medium (Figure 1). A wild MOFFAT 1986). The resulting plasmid expresses IMEI and type haploid strain remained viable after incubation a total of 24 5' codons from the phage T7 gene 10 proon Spo plates for 4 days [Spo" phenotype (patch A)]. and MOFFAT 1986) moter. E. coli strain B12 1(DE3) (STUDIER However, a haploid strain that expressed Pc;,.,/.I-I~bfEl carrying this plasmid was induced with IPTG and lysed with lysozyme-EDTA, freeze-thaw cycles, and sonication. The survived the incubation poorly[Spo' phenotype (patch fusion protein was pelleted with cell debris, washed, solubiC ) ] .A wild-tvpe GAL80 allele, jvhich blocks functional lized in 7 M urea, passed through CM Sepharose, fractionP~;.,,/.~-IMEI expression in sporulationmedium,conated on DEAE Sepharose and dialyzed to remove urea. New fers a Spo' phenotype to Pc;,.,~-l-IMElhaploids(not Zealand White rabbits received subcutaneous primary in-jecshown). We note that Pc;,.,~,l-IMElis not toxic to a / a tions of 250 pg of fusion protein emulsified with Freund's complete adjuvant. Subsequent injections (boosts)of 250 pg diploids: a / a P~;~,/./-Ii~lEl/P~;;,/,,-IMEl diploids sporuof fusion protein in Freund's incomplete adjuvant were late efficiently and yield >9.5% spore viability. 'These accomplished at 30-day intervals. Immune serum used for results suggest that 'the I M E I gene product, I M E I , is the experiment in Figure 2 was collected 7 days after the toxic to starved haploid cells. fourth boost. We considered two modelsfor I M E l toxicity: IMEI Immunoblots: Cell extracts were prepared fromlog phase YEPAc cultures by vortexing with glass beads in 50 may directhaploidstoenter meiosis andthusdie; m~ Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride. 50 alternatively, I M E l may interfere with some vital pg of soluble protein was fractionated on a 10% polyacrylprocess in which it normallypl;~ys norole. IMEI amide SDS gel, transferred to nitrocellulose, and incubated normally activates meiosis through activation of I M E 2 sequentially w i t h rabbit serum and peroxidase-conjugated expression. T h e first model predicts thata P
