meiotic recombination-deficient mutants of … · 2002-07-08 · 46 a. s. ponticelli and g. r....

Copyright 0 1989 by the Genetics Society of America

Meiotic Recombination-Deficient Mutants of Schizosaccharomyces pombe

Alfred S. Ponticelli*’+*’ and Gerald R. Smith* * Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, and ?Department $Pathology, University of Washington,

Seattle, Washington 98155 Manuscript received February 7, 1989 Accepted for publication May 20, 1989

ABSTRACT A mutant screen employing the ade64426 recombination hotspot was developed and used to isolate

Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to >300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome IIZ and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic us. intergenic) specificity of the corresponding ret+ gene products. None of the mutations specifically inactivated the ade64426 hotspot. Additional rec genes may be identified with these methods.

S TUDIES of the molecular mechanisms involved in homologous recombination have utilized both ge-

netic and biochemical approaches. An important genetic approach is the isolation of mutants deficient in recombination. The analysis of such mutants can identify genes whose products are required for recombination and can define the components of the recombination pathway(s). The subsequent cloning of mutant and wild-type rec genes and the determination of the biochemical activities of their encoded gene products are crucial steps in elucidating the molecular mechanisms of recombination.

Previous attempts to define mutations affecting meiotic recombination in the fission yeast Schizosac- charomyces pombe have been largely unsuccessful. Four recombination (rec) mutants of S. pombe were previously isolated. recl, isolated by GOLDMAN and GUTZ (1974), is a dominant mutation that blocks mitotic intragenic recombination but has no effect on meiotic intragenic recombination. rec3-8, rec2-5 and rec5-11, isolated by THURIAUX (1975), are mutations that in- crease or reduce the ectopic meiotic recombination between the unlinked tRNA suppressor genes sup3, sups, and supl2. These three mutations, however, have no effect on intragenic or intergenic recombination between homologous sequences at homologous chromosomal positions, suggesting that these mutations affect illegitimate pairing.

In addition to the four rec loci, a large number of radiation-sensitive mutations ( rad ) have been assigned to 22 unlinked genes (SCHUPBACH 1971; FABRE

’ Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02 11 5.

The publication costs of this article were partly defrayed by the payment ofpage charges. This article must therefore be hereby marked “aduerlisement” in accordance with 18 U.S.C. $1734 solely to indicate this fact.

Genetics 123: 45-54 (September, 1989)

1972a,b; NASIM and SMITH 1975). GROSSENBACHER- GRUNDER and THURIAUX (1 98 1) examined 18 of these rad mutations for their effect on intragenic recombination in crosses homozygous for the rad alleles. It was concluded that none of the rad mutations examined reduce spontaneous mitotic or meiotic recombination.

The interconversion of yeast mating-type involves recombination, and mutations in 10 identified switching (swi) genes reduce mating-type switching in homothallic strains of S. pombe (GUTZ and SCHMIDT 1985). Eight of these swi genes have been tested for their influence on cell viability and meiotic recombination (SCHMIDT, KAPITZA and GUTZ 1987). Of the 8 genes tested, mutations in only 3 (swi5, swi9 and milo) cause an increased sensitivity to UV-irradiation, while only one, swi5, reduces the frequency of meiotic intragenic recombination.

Taken together, 30 genetic loci of S. pombe involved in DNA metabolism have been tested for their effects on meiotic recombination. Only one, swi5, has a detectable effect. We therefore developed a screen to isolate S. pombe meiotic rec mutants.

A screen to isolate meiotic rec mutants should meet several requirements. First, two heteroallelic loci should be present in order to monitor recombination by the production of recombinant prototrophs. Sec- ond, a meiotic environment must be generated, and third, the meiosis should be the result of a selfing to allow the isolation of recessive rec mutations. In addition, a high frequency of recombinants in the ret+ parent is desired to allow the easy detection of mutants with a reduced frequency.

To meet the last requirement, we took advantage of the ade6-M26 recombination hotspot of S. pombe.

46 A. S. Ponticelli and G. R. Smith

T h e ade6-M26 mutation, a single G:C += T:A base- pair change, has no effect on mitotic recombination, yet gives an unusually high frequency of ade6 intragenic recombinants in two-factor meiotic crosses, up to fifteen times more than the closely linked ade6- M375 mutation (GUTZ 19'71; PONTICELLI, SENA and SMITH 1988; SZANKASI et al. 1988). Importantly for our screen, ade6-M26 can exhibit its hotspot activity when recombining during meiosis with the ade6-L469 gene carried on a high-copy plasmid (PONTICELLI, SENA and SMITH 1988).

We chose this plasmid X chromosome recombination event as the basis of the mutant screen. The screen monitored meiotic adeb intragenic recombination between a chromosomal ade6-M26 gene and the ade6-L469 gene carried on a high copy plasmid in a homothallic (mating-type switching) S. pornbe strain. After growing for 6 days on minimal agar + adenine, colonies of this strain contain many asci due to meiotic selfings. Such selfings, the result of mating-type switching within each colony during vegetative growth followed by mating and sporulation, allow the isolation of recessive rec mutations. Individual mutagenized colonies were then harvested into a solution to liberate spores from the asci and to kill vegetative cells. The spore suspensions were analyzed for recombinants by spotting them on a rich medium, on which the parental adeb mutant cells form a red lawn and the ade6+ recombinants form white papillae. We an- ticipated that even highly deficient recombination mutants would form sufficient viable spores for detection and analysis, since S. pombe contains only three chromosomes (GUTZ et al. 1974); random chromosome segregation in meiosis without recombination should produce at least 12.5% as many viable spores as or- dered segregation in a wild-type meiosis (see DISCUS-

SION). We describe here the details of this screen and the

subsequent characterization of thirteen meiotic recombination-deficient mutants.

MATERIALS AND METHODS

S. pombe media: Yeast-extract agar (YEA), yeast-extract liquid (YEL), Mitchison's minimal agar (MMA), and syn- thetic sporulation agar (SPA) were prepared as described by GUTZ et al. (1974). In some experiments, the minimal agar used was NBA (0.67% Difco yeast nitrogen base without amino acids, 1 % glucose, 2% agar). Modified EMM2 (minimal liquid) was prepared as described by NURSE (1975). EMM2, MMA, NBA and SPA were supplemented with required amino acids, purines, and pyrimidines (1 00 pg/ml).

Strains: S. pombe strains used in this work are listed in Table 1. Genetic nomenclature follows the recommenda- tions of KOHLI (1987). The homothallic mating-type (mating-type switching) is designated h"', and the heterothallic mating-types h+ and h-.

Nitrosoguanidine mutagenesis of S. pombe strain GP66: Strain GP66 was grown in EMM2 + adenine to a density of 1 X lo7 cells/ml. In EMM2 medium and MMA medium

used below, S. pombe grows vegetatively and undergoes mating-type switching, but mating and sporulation occur only upon starvation at high cell densities (GUTZ et al. 1974). One milliliter of culture was harvested by centrifugation, and the cells were washed with water and then with 0.1 M sodium citrate (pH 5.5). Cells were resuspended in 1 ml of the sodium citrate buffer, 110 pl of 2 mg/ml nitrosoguanidine (Sigma) was added, and the mixture was incubated for 30 min at 30". Three milliliters of citrate buffer was added, and the cells were harvested by centrifugation and washed once with buffer and twice with YEL. The mutagenized cells were resuspended in 5 ml EMM2 + adenine and incubated at 30" for 20 hr. Approximately 5% of the cells were viable after the mutagenesis and subsequently under- went 2-3 cell divisions during the recovery period. Glycerol was added to the cell suspension to a final concentration of 15%, and 2 ml aliquots were stored at -70".

Screening of mutagenized GP66 colonies: Mutagenized GP66 was plated on MMA + adenine to give approximately 100 colonies per plate and incubated at 32" for 6 days. Colonies of relatively uniform size (medium to large) were picked with a sterile flat toothpick and dispersed into a microtiter well (96-well cell culture clusters, Costar) containing 100 PI of 0.6% glusulase in water. The microtiter trays were covered and incubated 16 hr at 30". 100 pl of 60% ethanol was added to each well using a Transtar 96 96-well channel pipettor (Costar), and the plates were incubated at room temperature for 30 min to kill any remaining vegetative cells. Using the 96-channel pipettor, the spore suspension in each well was mixed by repeated pipetting and diluted 1:7 by transferring 25 PI into 150 111 of water in a fresh microtiter well. After mixing, 5 pl from each well was spotted onto YEA and onto MMA + uracil, using an 8- channel pipettor. 576 spore suspensions (from 6 96-well trays) were spotted on each 22.5 cm X 22.5 cm plate (Nunc) containing YEA or MMA + uracil. After 3 days incubation at 32"C, the plates were examined. Candidates yielding no or very few Ade+ recombinant (white) papillae (on YEA) or colonies (on MMA + uracil), but yielding many viable spores (on YEA), were analyzed further as described in the Results section.

Meiotic crosses: Samples of 1-5 X lo7 cells of each parent, freshly grown in YEL, were mixed in an Eppendorf tube, centrifuged 10 sec in an Eppendorf microcentrifuge, and washed with 0.5 ml saline (0.85% NaCI). Approximately 10 PI of saline was left in the tube when aspirating the wash supernatant, and the cells were resuspended in this saline and spotted on SPA. After 2 days at 25 O , asci and unmated cells were harvested from each spot into 1 ml of 0.5% glusulase (Dupont) in water. The suspension was incubated 16 hr at 30" to liberate spores from the asci and to kill vegetative cells. In some experiments, an equal volume of 60% ethanol was added and the suspension incubated at room temperature for 30 min. The spores were centrifuged 10 sec in an Eppendorf microcentrifuge and resuspended in 1 ml of water. Dilutions were plated on the appropriate media to select recombinants or on YEA to allow growth of all viable spores.

Construction of heterothallic rec mutant derivatives: Mutant candidates [hgoade6-M26 ura4-294 rec (pade6-L469)] were grown nonselectively in YEL + adenine + uracil and streaked on YEA. Individual colonies were tested on MMA + adenine uracil to identify Ura- segregants which had presumably lost the plasmid pade6-L469 (described below). 1-2 X IO7 cells of the Ura- derivatives were mated with 5 x 10' cells of GP13 (h- ade6-L52) on supplemented SPA as described above. The spore suspensions were plated on MMA + adenine and incubated for 5-6 days at 32". The

S. pombe Meiotic rec Mutants 47

TABLE 1

S. pombe strains used in this study

Strain Known genotype reference" Source or

GP6 GP13 GP24 GP27 GP66 GP67 GP2 17 GP2 19 GP220 GP22 1 GP222 GP225 GP229 GP230 GP23 1 GP235 GP236 GP237 GP243 GP245 GP247 GP248 GP249 GP250 GP253 GP257 GP258 GP259 GP263 GP264 GP265 GP271 GP273 GP274 GP275 GP277 GP278 GP279 GP288 GP289 GP290 GP291 GP292 GP294 GP3 1 1 GP3 12 GP3 13 GP314 GP3 15 GP3 16 GP317 GP3 18 GP3 19 GP320 GP323 GP324 GP349 GP350

h+ ade6-M?i5 h- ade6-L52 h+ ade6-M26 h+ ade6-M210 sup9 h'" ade6-M26 ura4-294 (pade6-L469) hYo ade6-M?i5 ura4-294 (pade6-L469) hy" ade6-M26 ura4-294 rec6-10? (pade6-L469) h'" ade6-M26 ura4-294 rec l l -108 (pade6-L469) hyO ade6-M26 ura4-294 rec-I01 (pade6-L469) h'" ade6-M26 ura4-294 rec7-102 (pade6-L469) h" ade6-M26 ura4-294 rec9-104 (pade6-L469) hyo ade6-M26 ura4-294 rec-106 (pade6-L469) hyO ade6-M26 ura4-294 rec10-109 (pade6-L469) h 'I' ade6-M26 ura4-294 rec8-I10 (pade6-L469) hYo ade6-M26 ura4-294 rec l l - I I I (pade6-L469) h'" ade6-M26 ura4-294 rec-I 12 (pade6-L469) hy" ade6-M26 ura4-294 rec-1 I? (pade6-L469) hy" ade6-M26 ura4-294 reci-I14 (pade6-L469) hy" ade6-M26 ura4-294 rec8-1 I5 (pade6-L469) h'" ade6-M26 ura4-294 rec6-103 h'" ade6-M26 ura4-294 recII-I08 h'" ade6-M26 ura4-294 rec-I01 hSo ade6-M26 ura4-294 reci-102 hyo ade6-M26 ura4-294 rec9-104 h'" ade6-M26 ura4-294 rec-106 hYO ade6-M26 ura4-294 recI0-109 hy" ade6-M26 ura4-294 rec8-110 h'" ade6-M26 ura4-294 recll-I I 1 h'" ade6-M26 ura4-294 rec-I12 h'" ade6-M26 ura4-294 rec-113 h'" ade6-M26 ura4-294 rec7-I14 h'" ade6-M26 ura4-294 r e d - 1 15 h- ade6-L52 rec6-I03 h+ ade6-M375 rec6-103 h+ ade6-M26 rec6-103 h- ade6-L52 rec7-102 h+ ade6-M?75 reci-102 h+ ade6-M26 rec7-102 h- ade6-L52 rec9-104 h- ade6-L52 recl0-I09 h- ade6-L52 red-110 h- ade6-L52 recll-I I I h- ade6-L52 reci-I14 h- ade6-L52 rec8-I15 h+ ade6-M375 rec9-104 h+ ade6-M26 rec9-104 h+ ade6-M3i5 rec10-109 h+ ade6-M26 rec10-109 h+ ade6-M375 sec8-110 h+ ade6-M26 rec8-110 h+ ade6-M375 recll-I 1 I h+ ade6-M26 recl l - I I I h+ ade6-M375 reci-114 h+ ade6-M26 reci-114 h+ ade6-M375 rec8-1 15 h+ ade6-M26 rec8-I15 h- arg3-124 ura4-294 h+ bro2-1 ura4-595

S. GOLDMAN S. GOLDMAN PONTICELLI, SENA and SMITH (1 988) PONTICELLI, SENA and SMITH ( 1 988) This studyb This studyb NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 NG of GP66 s of GP217 s of GP219 S of GP220 s of GP22 1 s of GP222 S of GP225 S of GP229 S of GP230 S of GP23 1 S of GP235 S of GP236 S of GP237 S of GP243 GPl3 X GP245 GP6 X GP273 GP24 X GP273 GP 13 X GP249 GP6 X GP277 GP24 X GP277 GPI 3 X GP250 GPl3 X GP257 GP13 X GP258 GP13 X GP259 GPl3 X GP265 GPI 3 X GP27 1 GP6 X GP288 GP24 X GP288 GP6 X GP289 GP24 X GP289 GP6 X GP290 GP24 X GP290 GP6 X GP291 GP24 X GP291 GP6 X GP292 GP24 X GP292 GP6 X GP294 GP24 X GP294 This studyb This studyb

a NG, nitrosoguanidine mutagenesis; S, spontaneous derivative; X, mating and meiosis of the indicated strains. Conlplete lineages are available upon request.


TABLE 1 Continued

Strain Source or

Known genotype reference"

GP363 h+ ade6-M26 arg3-124 ura4-294 GP349 X GP24 GP369 h- ade6-L52 pro2-1 ura4-595 GP350 X GP13 GP421 h- ade6-L52 pro2-1 ura4-595 rec7-102 GP350 X GP277 GP422 h- ade6-L52 pro2-1 ura4-595 rec7-102 GP350 X GP277 GP423 h- ade6-L52 pro2-1 ura4-595 rec7-102 GP350 X GP277 GP424 h- ade6-L52 pro2-1 ura4-595 recIO-109 GP350 X GP289 GP425 h- ade6-L52 pro2-1 ura4-595 recl0-119 GP350 X GP289 GP428 h- ade6-L52 pro2-1 ura4-595 recll-1 I 1 GP350 X GP291 GP429 h- ade6-L52 pro2-I ura4-595 recll-111 GP350 X GP291 GP430 h- ade6-L52 pro2-1 ura4-595 recll-111 GP350 X GP291 GP43 1 h+ ade6-M26 arg3-124 ura4-294 rec7-102 GP349 X GP279 GP432 h+ ade6-M26 ag3-124 ura4-294 rec7-102 GP349 X GP279 GP433 h+ ade6-M26 arg3-124 ura4-294 rec7-102 GP349 X GP279 GP434 h+ ade6-M26 arg3-124 ura4-294 reclO-109 GP349 X GP3 14 GP435 h+ ade6-M26 arg3-124 ura4-294 recIO-109 GP349 X GP3 14

GP439 h+ ade6-M26 arg3-124 ura4-294 recll-111 GP349 X GP3 18

GP437 h+ ade6-M26 arg3-124 ura4-294 recll-1 I 1 GP349 X GP3 18 GP438 h+ ade6-M26 arg3-124 ura4-294 recll-111 GP349 X GP3 18

plates were treated with iodine vapors to stain sporulating hgO colonies dark brown (GUTZ et al . 1974). Nonstaining (yellow) h- colonies were purified by streaking on YEA, which distinguishes ade6-L52 (light-red) from ade6-M26 (dark-red) colonies. The h- ade6-L52 isolates were tested for rec by backcrosses to their respective homothallic ade6- M26 parents. 3 X lo7 cells of each hgo parent were mixed with approximately 1 X 10' cells of each ade6-L52 isolate being backcrossed, mated on SPA and harvested as described above. An aliquot of 15 pl (of l ml) of each spore suspension was spotted on NBA + adenine (2 pg/ml) and the plates were incubated at 32 " for 4 days. On this medium Ade- colonies are red and Ade+ colonies are white. Since only Ura+ cells grow, only spores from matings between the hgO and h- cells, and not from hgO selfings, could grow. Hence, the density of white papillae on the red spot lawn reflects the recombinant frequency. ade6-L52 isolates carrying rec were identified by spots with no or few Ade' papillae.

Heterothallic h+ ade6-M375 rec and h+ ade6-M26 rec strains were constructed by mating the h- ade6-L52 rec strains with h+ ade6-M375 (GP6) or h+ ade6-M26 (GP24). The spore suspensions were plated on YEA, and the plates were incubated at 32" for 4-5 days. Dark-red (ade6-M375 or ade6-M26) colonies were tested for rec by backcrossing to the respective ade6-L52 rec parent and analyzing the spores by the spot test described above.

Plasmids and transformations: PAS-1 and pade6-L469 have been previously described (SZANKASI et al . 1988). They contain the wild-type ade6+ and mutant ade6-L469 genes, respectively, the S. cerevisiae URA3 gene (which comple- ments at high copy S. pombe ura4 mutations), and an S. pombe ARS element, and are maintained at approximately 80 copies per cell. For transformations, the Ura- rec derivatives were grown in YEL at 30" to a density of about 5 X lo7 cells/ml. An aliquot of 1.5 ml of culture was centrifuged 10 sec in an Eppendorf microcentrifuge, and the cells were washed with 10 mM Tris-HCI (pH 7.5), 1 mM EDTA (TE 7.5). Cells were resuspended in 150 pI of solution I (0.1 M lithium acetate in TE 7.5) and incubated with occasional agitation at 30" for 60 min. For each transformation, 50 pl was transferred to a new Eppendorf tube, 1 gg of plasmid was added, and the mixture incubated at 30" for 30 min. 175 pl of solution I1 (40% polyethylene glycol 3350, 0.1 M lithium acetate in TE

7.5) was added and the mixture incubated at 30" for 30 min. The cells were heat-shocked at 42 for 15 min, and 20 pl was spotted and streaked for isolated colonies on the appropriate medium (NBA for PAS-1 transformations, NBA + adenine for pade6-L469 transformations).

Crosses to test for M26 suppressibility: The Ura- ade6- M26 rec strains were mated with GP27 (h+ ade6-M210 sups). Aliquots of 30 pI of the spore suspensions were spotted and streaked on MMA, and the plates were incubated at 32" for 4-5 days. Ade+ (recombinant) colonies on MMA are white; Ade+ colonies growing due to suppression of ade6-M26 are pink.

Determination of meiotic ade6 recombinant frequencies: Plasmid X chromosome: The homothallic rec strains carrying pade6-L469 [hgO ade6-M26 ura4-294 rec (pade6- L469)] were plated on MMA + adenine and incubated at 32" for 6 days. Spores from individual colonies were harvested as for meiotic crosses as described previously. The spore suspensions were plated on YEA to determine the frequency of ade6+ (white) recombinant colonies among total (red + white) colonies.

Chromosome X chromosome: Strains were crossed on supplemented SPA, and the spore suspensions were plated on YEA to determine the total viable spores and on NBA + guanine (50 pg/ml) to determine ade+ recombinants. Gua- nine (kept as a 1 mg/ml stock in 50 mM KOH) prevents adenine uptake in S. pombe (CUMMINS and MITCHISON 1967) and was added to prevent residual growth of Ade- cells.

RESULTS

Isolation of S. pombe mutants deficient in meiotic plasmid x chromosome recombination: When un- mutagenized strain GP66 (ade6-M26) was sent through the screening procedure (Figure l), individual colonies reproducibly yielded approximately 50 Ade+ (white) papillae on the spot lawns of the ade6 mutant parent. In comparison, strain GP67 (ade6- M 3 7 5 ) yielded 2-5 Ade+ papillae. This difference was similar to the difference in recombinant frequencies with ade6-M26 and ade6-M375 when analyzed by standard procedures (GUTZ 197 1). We therefore mu-


s. cereuwae uRA3 S. pombe c strain GP66 PlasmId

Homothallfc. ade6-MZ6.

pade6-LA69. ad&.M69 um4-294 I + P b d d

chr. nr I

ade6-MZ6

1 I 1 1 1

Mutagenize with nitrosoguanidine.

Grow colonies on minimal agar ( " A I + adenine. After 6 days. colonies contain many asci due to meiotic se f igs .

Plck individual colonies to microtiter wells + glusulase [to liberate spores).

+ ethanol [to W any remaining vegetative cells).

Dllute.

Spot on yeast extract agar IyEAl. Spot on "A + uacfl. Red lawn of parental ade6 mutant cells Colonies of

+

plus white papillae of ade6 + recombinants. recombinants.

I Analyze candidates yielding

no or very few ade6 recombinant papillae or colonies.

FIGURE 1 .--Screen used to isolate S. pombe mutants deficient in meiotic plasmid X chromosome recombination.

tagenized strain GP66 with nitrosoguanidine, plated the cells on MMA + adenine, and screened the mutagenized colonies (see MATERIALS AND METHODS).

From approximately 4900 mutagenized colonies examined, we found three classes of mutants: (1) Approximately 500 colonies produced no or few viable spores; these were not analyzed further. (2) Ap- proximately 50 colonies produced no visible papillae on YEA but did produce small Ade+ colonies on MMA + uracil. When retested (as for class 3), these candidates were not recombination-deficient. (3) 242 colonies produced few or no white papillae on YEA or colonies on MMA + uracil. From the YEA plates, these were streaked for single colonies on MMA + adenine, and four colonies were retested as in the initial screen. About 40 of these 242 candidates gave the same phenotype as they had in the initial screen and were analyzed further.

These 40 candidates were analyzed to eliminate those that yielded a low frequency of Ade+ recombinants because they had acquired (through the mutagenesis procedure) additional mutations in either the chromosomal ade6-M26 gene or the plasmid-borne ade6-L469 gene. Candidates were grown non-selec- tively in rich medium to obtain Ura- segregants which

TABLE 2

Effect of rec mutations on meiotic plasmid X chromosome recombination

ade6-MZ6 X pade6-L469 rec Ade+ recornbinants/lO'

Strain genotype viable spores

GP66 rec+ 25, 63 (109/4,000; 63/1,000) GP217 rec6-103 <0.5 (0/5,000) GP219 r e c l l - I 0 8 C0.5 (0/5,000) GP220 rec-IO1 <1.5 (0/2,000) GP221 rec7-102 <0.5 (0/4,000) GP222 rec9-104 5 (51/10,000) GP225 rec-106 0.5 (1/2,000) GP229 rec10-109 0.8 (8/10,000) GP230 rec8-110 0.4 (4/10,000) GP231 r e c l l - I l l 0.6 (3/5,000) GP235 rec-I12 6.0 (9/1,500) GP236 rec-I13 0.5 (1/2,000) GP237 rec7-114 0.1 (1/10,000) GP243 rec8-115 0.5 (1/2,000) GP67 Tee+, ade6-M375 2.5, 2.0 (5/2,000; 3/1,800)

Isolated colonies of homothallic plasmid-bearing strains on MMA + adenine were picked after 6 days incubation at 32" and assayed for Ade+ (white) recombinants on YEA as described in MATERIALS AND METHODS. The denominator in parentheses is an estimate of the total (red + white) colonies on YEA; the numerator is the number of white Ade+ recombinants scored. Two independent determinations were made for the ret+ controls GP66 and GP67.

had presumably lost pade6-L469. About 20 candidates gave no Ura- segregants and were not analyzed further. T o test for an additional mutation on the plasmid, the Ura- derivatives were retransformed with pade6-L469, and the Ura+ transformants retested by the original screen. Nearly all of the transformants had the mutant phenotype (low Ade+ frequency) upon retesting and were tested for additional mutations in the chromosomal ade6-M26 gene. This was achieved by mating the Ura- derivative to a strain containing sup9 (GP27) to confirm that the ade6 gene in the candidates was still suppressible for the adenine requirement caused by the M26 mutation. About 10 candidates were not suppressible and were deemed to have an additional ade6 mutation. Transformation of the Ura- derivative of each candidate with a plasmid containing the ade6+ gene (PAS-1) verified that the adenine requirement for growth was solely due to the ade6-M26 mutation.

Thirteen candidates passed these tests and were considered to contain mutations that reduce meiotic plasmid X chromosome recombination. The frequency of Ade+ recombinants obtained for each mutant is listed in Table 2. With respect to the rec+ (ade6- M26-containing) control, 1 1 of the 13 mutants yielded at least 50-fold reduced Ade+ recombinant frequencies. The Ade+ frequency of two mutants, designated rec9-104 and rec-112, was reduced approximately 10- fold. (We assigned allele numbers to these rec mutations arbitrarily, beginning with 101, and gene numbers beginning with rec6 based upon the complemen-


tation data described later. For clarity we use the complete designations throughout this paper.)

Rec mutations reduce interchromosomal meiotic recombination: T o determine whether the rec mutant candidates were deficient for standard interchromosomal recombination, we constructed for each rec mutant the heterothallic derivatives h- ade6-L52 rec, h+ ade6-M375 rec, and h+ ade6-M26 rec. Crosses between these strains would test for their interchromosomal recombination proficiency at ade6, when each rec mutation was homozygous, and for a specific effect on ade6-M26-stimulated recombination.

T o construct heterothallic h- ade6-L52 rec strains, the Ura- derivative of each mutant (hgo ade6-M26 ura4-294 rec) was mated to GP13 (h- ade6-L52). h- ade6-L52 ura4+ progeny were backcrossed to their hgo ade6-M26 ura4-294 rec parents and the spores were analyzed with the spot test to estimate the Ade+ recombinant frequencies. For 8/13 candidates, approximately 50% of the progeny gave a rec phenotype in these backcrosses, suggesting that (1) the mutations are recessive, (2) they are not tightly linked to either matl, ade6, or ura4, and (3) they are likely to be due to lesions in single genes. These 8 were rec7-102, rec6- 103, rec9-104, reclO-109, rec8-110, recll-I I I, rec7- 1 14 and rec8-115. Tetrad analysis has been performed for rec7-102, rec6-103, recIO-109 and recll-Il l , and 2+:2- segregation of rec was observed in all of 8, 5 , 8 and 8 tetrads from rec heterozygous dipoids, respectively (N. HOLLINGSWORTH, personal communication). Additionally, it was noted that the ade6-L52 rec isolates usually yielded fewer total viable spores, as judged by the density of growth of the red spot lawn (see MATERIALS AND METHODS). Since meiotic recombination plays a critical role in the proper segregation of chromosomes (BAKER et al. 1976), the reduced viable spore yield in the homozygous rec matings was expected. (The effect of the rec mutations on spore viability is discussed later.) We assumed that the rec candidates had reduced yields of viable spores due to their recombination deficiencies and continued to use the spot test as a simple guide in the construction of additional derivatives (see below).

Of the remaining five mutants, one (rec-101) appar- ently carried a dominant mutation, i.e., it yielded a 3- fold reduced meiotic adeb recombinant frequency in the hgO ade6-M26 ura4-294 rec-101; X h- ade6-L52 cross (data not shown). We recognized that an additional mutation in the ade6-M26 gene could explain this phenotype. However, the ade6-M26 gene in this strain was opal-suppressible for the adenine requirement, demonstrating that, if there is an additional mutation in the ade6 gene, it must also create an opal nonsense codon. More importantly, ade6-L52 rec-IO1 derivatives were obtained (6/32 from the above cross), demonstrating that this rec mutation is separable from

ade6. This mutant has not been further analyzed. For the remaining four candidates (recll-108, rec-

106, rec-112 and rec-113), no h- ade6-L52 rec derivatives were identified among eight ade6-L52 segregants tested for each. For rec-106, rec-112 and rec-113, this may be due to the limited number of segregants tested or to their mutations being genetically linked to mating-type (mat l ) , ura4 or ade6, as only h- ade6-L52 ura4+ progeny were analyzed by backcrosses. Alter- natively, these mutations may affect only plasmid X chromosome recombination. For recll-108, we have not further attempted to make an ade6-L52 derivative, since recll-I08 fails to complement recll-11 I (see below).

From the h- ade6-L52 rec derivatives for eight of the mutants, h+ ade6-M375 rec and h+ ade6-M26 rec derivatives were constructed by mating the h- ade6- L52 rec strains with h+ ade6-M375 (GP6) or h+ ade6- M26 (GP24), backcrossing the dark-red (ade6-M26 or ade6-M375) progeny to the respective ade6-L52 parent, and analyzing the spores for rec by a spot test (see

of the progeny tested for each mutant carried rec. From the results of crosses between strains carrying

ade6-L52 rec and ade6-M26 rec or ade6-M375 rec (Table 3) we grouped the mutants into two classes. Class I mutants (rec7-102, rec6-103, rec8-110, rec7-114 and rec8-115) yielded only a few Ade+ recombinants per lo6 viable spores in both ade6-M375 and ade6- M26 crosses. Compared to the ade6-M375 X ade6-L52 rec+ control crosses, the class I mutants exhibited approximately a 50-fold to 300-fold reduction in their adeb intragenic recombinant frequencies, confirming that these mutants are severely impaired in meiotic interchromosomal intragenic recombination.

Class I1 mutants showed reduced intragenic recombination with either ade6-M375 or ade6-M26 but still displayed an ade6-M26 stimulation. Three mutants exhibited this phenotype. recIO-109 and recll-I I 1 yielded only a few Adef recombinants per lo6 viable spores in the crosses containing ade6-M375, similar to the values observed for the class I mutants. However, unlike the class I mutants, ade6-M26 had an approximately 10-fold stimulation in rec10-109 crosses and a %fold effect in recll-11 I crosses. The third mutant in class 11, rec9-104, exhibited greater recombination proficiency than the other two mutants in this class; rec9-104 displayed a 3-fold reduction in recombinant frequencies for crosses involving either ade6-M26 or ade6-M375 and maintained the 10-fold ade6-M26 stimulation.

We next tested the effects of three of the recessive rec mutations (rec7-102, recIO-109 and recll-I I I ) on ura4 intragenic and pro2-arg3 intergenic meiotic recombination. Appropriate ade6 ura4 pro2 (or arg?) rec strains were constructed and crossed as listed in

MATERIALS AND METHODS). Again, approximately 50%


TABLE 3

Effect of rec mutations on meiotic ade6 interchromosomal recombination

TABLE 4

Effect of rec7-102, rec10-109 and recl l -I l l mutations on ura4 intragenic and pro2-arg3 intergenic meiotic recombination

Ade+ recombinants/

Ade+ recombinants/

spores GP M375 X L52 CP

spores M26 X L52

l o 6 viable 1 O6 viable

strains Mutation crossed Expt. 1 Expt. 2 crossed Expt. 1 Expt. 2

rec+ 6, 13 190 260 24, 13 2500 4100 (320) (290) (247) (344)

rec6-103 273, 274 3 <6 273, 275 4 4

rec7-102 277, 278 3 <9 277, 279 4 <6

rec8-110 290, 315 2 7 290,316 6 2

rec7-114 292, 319 1 <6 292, 320 1 1

strains

Class I

(5) (0) (4) (2)

(3) (0) (5) (0)

( 1 ) (3) (3) ( 1 )

(1) (0) ( 1 ) ( 1 )

(3) (4) ( 1 ) (2) rec8-115 294, 323 5 8 294, 324 2 4

Class I1 rec9-104 288, 311 53 100 288, 312 900 1100

recl0-109 289, 313 4 7 289, 314 26 35 (23) (51) (208) (591)

(3) (9) (25) (52)

(5) (3) (12) ( 1 1 ) r e c l l - l l l 291,317 5 3 291, 318 13 11

~ ~ ~~

Heterothallic strains were crossed on supplemented SPA and assayed for total viable spores on YEA and for ade6+ recombinant spores on NBA + guanine as described in MATERIALS AND METHODS. The data in the table are the Ade' recombinant frequencies (per IO6 viable spores) calculated from the number of Ade+ colonies counted and listed in parentheses. At least 200 colonies were counted to determine the number of viable spores. For entries with no Ade+ colonies, the upper limit of the recombinant frequency was calculated by assuming 3 Ade+ colonies in the sample plated.

Table 4. The results demonstrated that these rec mutations reduced ura4 intragenic and pro2-arg? intergenic in addition to ade6 intragenic recombination. The class I mutant rec7-102 showed a greater reduction than the class I1 mutants recIO-109 and rec l l - 111. These results suggest that the products of the corresponding wild-type rec genes are required for general meiotic recombination.

In summary, the 13 mutants isolated on the basis of reduced meiotic chromosome X plasmid recombination were examined for their effects on meiotic interchromosomal recombination. One mutant displayed a dominant phenotype for reducing ade6 recombination. Three mutants have not yielded any segregants which affect interchromosomal recombination, suggesting either that their mutations are linked to the genetic markers used in selecting the progeny to be tested (see above) or that they affect only plasmid X chromosome recombination. The remaining nine mutations were recessive and, when homozygous, reduced intragenic recombination at ade6 3-fold to 300-fold. Three of these nine allowed an ade6-M26 stimulation to be detected, while none

Recombinants/l O6 viable spores GP strains

rec crossed Ade+ Ura+ Pro+ Arg+

+ 363, 369 2900 360 120,000 (429) (272) (181)

rec7-102 421, 431 4 5 4,600

rec7-102 422, 432 2 <3 3,200 (3) (4) (169)

(2) (0) (1 56)

(0) (0) (98)

(18) (36) (493)

(19) (33) (386)

(5) (40) (1 70)

(13) (81) (217)

(6 ) (27) (220)

rec7-102 423, 433 <IO <lo 3,600

rec10-109 424, 434 18 36 49,000

rec10-I09 425, 435 34 59 69,000

recl l - I 1 I 428, 437 6 49 21,000

recl l -1 I I 429, 438 7 40 22,000

r e c l l - I l l 430, 439 14 64 26,000

Heterothallic strains were crossed on supplemented SPA and assayed for total viable spores on YEA, for ade6+ recombinants on NBA + guanine + uracil + proline + arginine, for ura4+ recombinants on NBA + adenine + proline + arginine, and for pro2+ arg3+ recombinants on NBA f adenine + uracil. The data in the table are the Ade+, Ura+, or Pro' Arg' recombinant frequencies (per lofi viable spores) calculated from the number of Ade+, Ura', or Pro+ Arg' colonies counted and listed in parentheses. At least 200 colonies were counted to determine the number of viable spores. For entries with no recombinant colonies, the upper limit of the recombinant frequency was calculated by assuming three recombinant colonies in the sample plated.

appeared to affect the action of the ade6-M26 hotspot without also affecting recombination in its absence.

Complementation analysis: T o determine the number of complementation groups represented by the nine recessive mutations, h+ ade6-M26 rec and h- ade6-L52 rec strains were crossed (Table 5). The Ade+ recombinant frequencies obtained for all the rec com- binations demonstrated that the recessive mutations represented six complementation groups. Three of the complementation groups (rec6, rec9 and rec l0) contained one mutation each, while three (rec7, rec8 and r e c l l ) contained two mutations each. rec7-102 and rec7-114 did not complement, nor did rec8-110 and rec8-115. In addition, crosses with the homothallic ade6-M26 Ura- derivative of rec l l - IO8 (for which a heterothallic derivative was not obtained) and the various ade6-L52 rec strains demonstrated that recl I- 108 and r e c l l - I l l did not complement (data not shown).

From these results, we assigned rec gene numbers to the recessive mutants. Since four rec genes (1,2,?,5) have been previously reported (see Introduction), we began our numbering with rec6 and assigned gene numbers as indicated in Tables 1, 5 and 6.


TABLE 5

Complementation analysis of the recessive rec mutations

Ade+ recornbinants/1O6 viable spores

+ 6-103 7-102 7-114 8-1 10 8-115 9-104 10-109 11.111 rec GP # (24) (275) (279) (3 16) (324) (3 12) (314) (3 18) (320)

+ (13) 4100 2000 3800 3100 2200 2400 3300 2100 2000 6-103 (273) 2700 4 2500 2800 1900 1900 3400 1000 1500 7-102 (277) 3500 2600 <6 c3 1600 1800 3000 2000 1800 7-1 14 (292) 4300 2500 C4 1 2200 2300 3200 2300 2000 8-110 (290) 3100 2100 3400 3000 2 2 2500 2400 1900 8-115 (294) 3000 2500 3000 3000 2 4 3600 2100 3100 9-104 (288) 3000 2900 3700 3700 2100 2000 1100 2100 1900

10-109 (289) 2800 1900 2800 3100 1700 1800 2900 35 1300 1 1 - 1 1 1 (291) 2700 2100 3200 3000 1900 1900 2500 2000 11

Strains along the top row were of the genotype h' ade64426 rec; strains on the left column were of the genotype h- ade6-L52 rec. For the crosses that showed complementation, between 2.0 X 1 O4 to 1.4 X 1 O5 viable spores were plated and between 100 and 600 Ade' recombinants were scored for each recombinant frequency determination. For the crosses that were homozygous for rec, the data are those of Table 3, Experiment 2. For GP277 X GP320, 0 Ade+ recombinants were scored among 1 X lo6 viable spores plated; GP292 X GP279, 0 among 8.7 X 10'; GP290 X GP324, 1 among 5.8 X 10'; GP294 X GP316, 2 among 1.1 X lo6. In addition, crosses with the homothallic ade6426 derivative of recll-108 (strain GP247) and the ade6-L52 strains listed above demonstrated that recll-108 did not complement rec11-1 1 1 but did complement the other rec mutations listed (data not shown).

TABLE 6

Effect of rec mutations on spore viability ~~ ~~

Percent spore viability

Mutation Expt. 1 Expt. 2

rec+ 125 70 Class I

rec6-103 14 19 rec7-102 30 22 rec7-114 45 17 rec8- 1 10 12 19 r e d - 1 15 11 14

rec9-104 97 24 rec10-109 60 46 rec l l - I l l 75 50 recll-108 72 Not tested

Class I1

Others rec- 10 I 105 Not tested rec-106 18 Not tested rec-112 102 Not tested rec-I13 17 Not tested

Expt. 1: Selfings of the homothallic mutant isolates (GP217 to GP243) were conducted on SPA. The concentration of total spores from each harvested suspension was determined by microscopy, and the spore suspensions were plated on YEA to determine the concentration of viable spores. Expt. 2: The spore suspensions analyzed were from the rec homozygous crosses, ade6-M26 X ade6- L52, experiment 2, Table 3. At least 250 spores and at least 300 colonies were counted in all cases.

Reduced spore viabilities of some rec mutants: T o test the effects of the rec mutations on spore viability, two random spore experiments were performed. In the first test, selfings of the original homothallic mu-

determined by microscopy, and the spore suspensions were plated on YEA to determine the concentration of viable spores. The results presented in Table 6 show that the mutants exhibited reduced spore viabilities, with the percent viability correlating with recombination proficiency. The class I mutants, with severely reduced ade6 recombination and no detectable ade6-M26 effect, displayed the lowest spore viabilities. The more recombination proficient class I1 mutants exhibited higher spore viabilities.

Sensitivity of rec9-104 to DNA damage during mitotic growth The rec mutants were also tested for their mitotic sensitivity to UV-irradiation, to the DNA damaging agent methyl methanesulfonate (MMS), and to caffeine, which inhibits DNA repair (FABRE 1972a; GENTNER 1977; GENTNER, WERNER and HANNAN 1978). The rec9-104 mutant failed to form visible colonies after 4 days incubation at 32" in the presence of MMS (0.005% in YEA) while the wild-type did; in addition rec9-104 was slightly more sensitive to UV than the wild-type (data not shown). These sensitivities were observed in all cases to cosegregate with the recombination deficiency by random spore analysis. None of the remaining mutants were detectably more sensitive to these agents than the wild-type. These results suggest that the rec9+ gene product is involved in both meiotic recombination and the mitotic repair of DNA damage. Mitotic recombination in rec9-104 remains to be tested.

DISCUSSION

tant isolates were conducted. In the second experi- Using a screen for reduced meiotic plasmid X chro- ment, standard heterothallic crosses homozygous for mosome recombination, we have isolated thirteen mu- each recessive rec mutation were performed. The tants with reduced meiotic recombination. These mu- concentration of total spores from each cross was tants are likely to be independent, since the mutagen-


ized culture grew only 2-3 generations before assay and only 1 0-2 of the mutagenized cells were screened. Nine of the mutations, designated rec, were recessive and defined six rec genes unlinked to ade6. When homozygous, these mutations reduced meiotic interchromosomal ade6 recombination 3- to 2300-fold. Three of these rec mutations were tested for their effects at loci other than ade6. rec7-102, recIO-109 and recl l-I I I reduced meiotic intragenic recombination at ura4 (1 1 00-fold, 1 O-fold, and 1 O-fold, respectively) as well as intergenic exchange between pro2 and arg3 on chromosome Z (about 30-fold, 2-fold, and 6-fold, respectively). Hence, the meiotic recombination deficiencies due to these mutations are not adeb locus- specific. These results suggest that the products of the corresponding wild-type rec genes are required for general meiotic recombination.

We believe that several features of the screen used here made it successful. By assaying plasmid X chromosome recombination, we were able to utilize homothallic strains to isolate recessive rec mutations. The use of the ade6-M26 hotspot gave a high frequency of recombination events, allowing a simple spot assay to be used to detect mutant candidates with reduced recombination. Such candidates would potentially in- clude both general rec mutants as well as mutants that specifically inactivate the ade6-M26 hotspot. In addition, by working with S. pombrwavoided the ex- tremely low viability of meiotic products associated with meiotic rec mutants in other organisms. Such inviability is due to the critical role that interhomo- logue recombination plays in the proper reductional segregation of chromosomes at meiosis I (BAKER et al. 1976). For organisms with a high chromosome number, such as the yeast Saccharomyces cerevisiae, these viability problems have hampered the isolation and analysis of meiotic rec mutants. Certain procedures to avoid these problems, such as the “return-to-growth” protocol and the s t013 bypass system have been used, but with only moderate success to date (see review by ESPOSITO and KLAPHOLZ 1981). However, since S. pombe has only three chromosomes [and produces only one class of viable aneuploids, disomic for the shortest chromosome ZZZ, which are highly unstable and be- come normal haploids (NIWA and YANAGIDA 1985)], random segregation of the chromosomes in the absence of recombination should still result in about 12.5% (0.53 ) of the spores being viable true haploids, allowing the direct isolation of severely impaired recombination mutants. As expected from these consid- erations, the mutants with the most severely reduced recombination frequencies had about 15% spore viability. Mutants with lesser reductions had higher spore viabilities. Finally, our procedure employed a simple treatment to kill unsporulated cells; by assaying only viable spores we eliminated from the analysis any

apparent rec mutants that were simply defective in mating-type switching, mating, meiosis, sporulation, or germination.

The screen used here yielded a wide spectrum of mutant types. Both recessive and dominant mutations were isolated. Mutants with recombination frequencies reduced %fold to 1000-fold were obtained. One mutation has a detectable phenotype during vegetative (mitotic) growth-increased sensitivity to the ra- diomimetic agent MMS-while the others do not. Three mutations may reduce plasmid X chromosome recombination but not chromosome X chromosome recombination (see RESULTS for other possible expla- nations of these three mutations). Although no mutations inactivating only the M26 recombination hotspot were obtained, we believe this screen could reveal them if they exist. Analysis of nine recessive mutations showed they fell into six complementation groups, three with two mutations each and three with one mutation each. These results suggest that many more meiotic rec genes can be identified with this screen. In fact, five more recessive rec mutations, defining three additional complementation groups, have been obtained (N. MCKITTRICK, personal communication).

Two mutations, rec7-102 and rec10-109, reduced ade6 and ura4 intragenic recombination more severely than they reduced pro2-arg3 intergenic recombination. This result may reflect locus specificity of the rec7+ and reclO+ gene products (see next para- graph). Alternatively, these mutations may reduce gene conversion (as measured by intragenic recombination) more severely than they reduce crossing over (as measured by intergenic recombination). This differential reduction could reflect two separate path- ways for gene conversion and crossing over. This interpretation is in contrast to one widely held view that gene conversion and crossing over reflect equally frequent outcomes of a common initiating event (reviewed by FOGEL, MORTIMER and LUSNAK 1981) but is in accord with another recently discussed view that they are separate events (reviewed by HASTINGS 1988). Another interpretation is that these mutations alter either the frequency of “cross-over” us. “non- crossover’’ resolution of a common precursor or the length of hybrid DNA in this precursor, or both. Further analysis of rec7 and reclO mutants is required to resolve this issue.

The differential reductions of recombination by rec7-102, recIO-I09 and r e c l l - I l l in the three intervals tested (adeb, ura4 and pro2-arg3) could reflect locus specificity of the corresponding rec+ gene products. Locus specificity of rec gene products has been described in Neurospora crassa (reviewed by CATCHE- SIDE 1977). Analysis of natural isolates of N . crassa revealed differences at several rec genes, the low frequency alleles of which are dominant and reduced

54 A. S. Ponticelli and G . R. Smith

recombination at some but not other test intervals. Typically the rec gene is unlinked to the interval in which recombination is depressed. Although the low frequency alleles of the S. pombe rec7, reclO and r e c l l genes are recessive, further analysis may reveal re- gional controls of recombination in S. pombe.

One mutation, re&-104, conferred sensitivity during mitotic growth to the DNA damaging agent MMS and reduced meiotic recombination approximately 3- fold. These properties are similar to those of sui5 (SCHMIDT, KAPITZA and GUTZ 1987). However, recY- 104 has no apparent deficiency in mating-type switching and therefore does not appear to be allelic with swi5 (P. SZANKASI, personal communication). The effect of re&-104 (as well as that of the other rec mutations) on mitotic recombination remains to be determined.

The wild-type rec genes might be isolated by screening an S. pombe genomic library for clones that complement the recombination defects. Physical, enzy- matic and genetic analysis of S. pombe rec mutants and rec genes should provide significant information about the molecular mechaniarns of meiotic recombination.

We thank PHILIPPE SZANKASI and JURG KOHLI for strains, plasmids and information about them before publication; NANCY HOL-

ing us to cite their unpublished data; and SUE HOLBECK, NANCY HOLLINGSWORTH, KAREN LARSON, MICHAEL LICHTEN, NIKI Mc- KITTRICK, PHILIPPE SZANKASI, and ANDREW TAYLOR for comments on the manuscript. This work was supported by the National Institutes of Health grants GM31693 and GM32194 to G.R.S. A.S.P. is the recipient of a Ginsberg predoctoral fellowship from the Fred Hutchinson Cancer Research Center.

LINGSWORTH, NIKl MCKITTRICK and PHILIPPE SZANKASI for allow-

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Communicating editor: R. L. METZENBERG

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