DEVELOPMENTAL GENETICS 13:392402 (1992)

Characterization of REClQ4, a Gene Required for Early Meiotic Recombination in the Yeast Saccharomyces cerevzszae ANNE M. GALBRAITH AND ROBERT E. MALONE University of Iowa, Iowa City The REC104 gene was initially ABSTRACT defined by mutations that rescued the inviability of a rod52 spo 13 haploid strain in meiosis. We have observed that reclO4 mutant strains undergo essentially no induction ofmeiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC704 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome Vlll near FUR1. Expression of the RECIO4 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed. 0 1993 Wiley-Liss, Inc

Key words: Meiotic recombination in yeast, spo 13, prototrophy, mitotic cells

INTRODUCTION Meiosis is a eukaryotic developmental process in which a diploid cell divides twice to produce four haploid daughter cells. In the budding yeast Saccharomyces cerevisiae, these daughter cells or spores are contained within a n ascal sac. Prior to the first meiotic division, a n extensive Prophase I occurs during which the homologous chromosomes pair with each other; associated with chromosome pairing is the formation of the synaptonemal complex (SC). The SC is a proteinaceous network that assembles early in Prophase I and is aligned along the length of each of the chromosomes, apparently holding homologs together [reviewed in Von Wettstein et al., 19841. In meiotic Prophase I, high levels of recombination occur; the frequency of meiotic recombination is 100-1,000 times higher than the frequency of recombination in mitosis. It has been proposed that the high levels of meiotic recombination are due to formation of the SC [Von Wettstein et al., 19841. Both pairing and recombination ensure that homologous chromosomes segregate properly a t the first meiotic division, a n essential step, since random segrega-

0 1993 WILEY-LISS. INC

tion leads to aneuploidy and death of the cell [see Baker el al., 1976 for review]. A number of mutants have been isolated in yeast that are defective in meiotic recombination. One type of mutation results in defective chromosome pairing, abnormal SC formation, and inviable spores, but only decreases levels of meiotic recombination to 10-20% of the normal frequency; the wild-type products of these genes have been proposed to be involved primarily in the pairing of the homologous chromosomes (e.g., R E D l , Rockmill & Roeder, 1988; H OPl, Hollingsworth and Byers, 1989; MEKl, Rockmill and Roeder, 19911. In the case of HOPl, it has been shown that antibodies to the gene product react with proteins associated with the chromosomes [Hollingsworth et al., 19901. Another type of meiotic Rec-mutant completely eliminates all meiotic recombination (e.g., rad50, Malone and Esposito, 1981; spoll, Klapholz et al., 1985; mei4, Menees and Roeder, 1989; reclO4 and recll4, Malone et al., 1991; reclO2 and mer2, Cool and Malone, 19921. The genes defined by these mutations have been proposed to be involved primarily in meiotic exchange (see below), although in several cases, mutations in genes of this class have been demonstrated to also affect SC formation (e.g., rad50, Alani et al., 19901. Other genes have also been identified in yeast that are required for meiosis. One of these, SP013, is a gene required for the reductional division. In spol3 mutant diploids, recombination occurs at wild-type levels and the two diploid spores are viable and usually result from a single equational division [Klapholz and Esposito, 19801. The only defect seen is the absence of a reductional division. Since recombination is required for proper segregation during the reductional division, and since there is no reductional division in a spol3 diploid, the normal meiotic requirement for recombination is abolished [Malone and Esposito, 1981; Malone, 1983; Klapholz et ul., 19851. Using mutations in the SP013 gene, mutations in the exchange class of

Received for publication October 16,1992;accepted December 3,1992. Address reprint requests to Robert E. Malone, Department of Biological Sciences, Iowa City, IA 52242-1324.

AN EARLY MEIOTIC RECOMBINATION GENE IN YEAST meiotic recombination genes have been found formally to fall into two classes: “early” or “late” [Petes et al., 19911. Mutations in early genes produce viable spores in the presence of a spo13 mutation. Therefore, early genes are thought to act a t or before the initiation of strand exchange. (Interestingly, mutations in genes in the pairing class are also rescued by spol3 mutations.) Mutations in genes in the late exchange class, such a s rad52, rad57, and dmcl, are not rescued by the spo13 mutation [Malone and Esposito, 1981; Game, 1983; Bishop et al., 19921; e.g., rad52 spol3 diploids still produce inviable meiotic products. It has been proposed that diploids containing these later blocks have already begun strand exchange and the tangled chromosomes cannot segregate properly in the second equational division [Malone, 1983; Borts et al., 19861. An early block such as a rad50 mutation can prevent the inviable spore phenotype of a rad52 spo13 diploid, presumably by preventing initiation before strand exchange has begun [Malone, 19831. Malone and colleagues [19911 isolated 177 mutants specifically defective in the initiation of meiotic recombination (early blocks) by selection for the meiotic rescue of rad52 spol3. Examination of 56 of these mutants revealed that they contained mutations in most known early Rec genes of the exchange class, and defined four new Rec genes. Detailed analysis of three of these new genes has been described elsewhere [MER2, Engebrecht et al., 1990; REC102 and MER2, Cool and Malone, 1992; RECl02, Barghava et al., 1992; REC114, Pittman et al., 19921. The other gene defined by this mutant search, REClO4, has been studied in some detail, and the results of this characterization and analysis are presented here. This gene is in the early exchange class and is absolutely required for meiotic recombination with no detectable role in mitosis.

MATERIALS AND METHODS Strains, Media, and Plasmids The yeast strains used in this report are listed in Table 1. The furl strains were a gift from Duane Jenness (University of Massachusetts Medical School). The rad5OS-K181 strains were created by transforming haploid wild-type yeast with the 7.5-kb BamHI-EcoRI fragment containing the rad5OS-K181 mutation. This mutation on plasmid pNKY349 was kindly provided by Nancy Kleckner (Harvard University). The yeast strains were grown on either YPD medium or C-URA (synthetic medium with all auxotrophic nutrients except uracil) [Sherman et al., 19791. YPA and sporulation media are described in Malone et al. [1991]. Escherichia coli RK1448 was used for plasmid amplification as described [Hoekstra and Malone, 19851. The media and yeast transformation protocol are described by Hoekstra and Malone [1985]. The plasmids pRS306, pRS316, and pRS315 were provided by P. Hieter [Sikorski and Hieter, 19891.

393

Subclones were constructed by the method of Sambrook et al. [19891. The plasmids pRM211 and pRM212 were created by inserting the 3.4-kb HpaI-BamHI fragment into the SmaI-BamHI sites of pRS306 and pRS316, respectively. The plasmid pAMG403 was constructed by inserting the 2.4-kb BgZII-PstI fragment into pRS315 a t BamHI-PstI. An insertion allele of REClO4, rec104-T5, was obtained by transforming haploids with a 12.8-kb BglII-BgZII fragment containing the 6.1-kb TnlOLUK construct within the RECl04 gene. The exact location of this particular insertion was determined to be position + 61 of the REC104 coding region by sequence analysis [Sanger et al., 19771 using a primer to the lacZ gene. Since only 20 amino acids of the Recl04 protein can at most be made from strains containing the rec104-T5 allele, we presume it is a null allele.

Measurements of Recombination Mitotic crossing over was measured a s the frequency of drug-resistant colonies in diploids that were heterozygous at loci conferring recessive drug resistance. Although drug-resistant colonies may be the result of crossing over, gene conversion, or chromosome loss or missegregation, crossing over occurs at a much greater frequency than the other events [Roman, 19561. Mitotic gene conversion was monitored in diploids by measuring the frequency of prototroph formation from heteroalleles at five different loci. Meiotic gene conversion frequencies were determined by allowing diploids to go through meiosis and measuring the frequency of prototroph formation at five heteroallelic loci; this prototrophy results mainly from gene conversion [Roth and Fogel, 1971; Petes et al., 19911. Crossing over was monitored by measuring the frequency of drug resistant colonies in spold diploids as well as by dissection. Production of drug-resistant colonies in spo13 diploids heterozygous for a recessive drug resistance marker can occur by gene conversion or aberrant segregation, but the predominant cause is crossing over between the locus and the centromere [Malone, 19831. Cloning and Analysis of REC104 The REClO4 gene was cloned as previously described [Malone et al., 19911. The gene was more precisely localized by ATnlOLUK mutagenesis. The strains and plasmids necessary to carry out the mutagenesis were generously provided by J a n Fassler (University of Iowa). The method used to create the TnlOLUK insertions is a modified version of the protocol described in Huisman et al. [1987]. E. coli strain NK5830 [Huisman et al., 19871 was transformed with pKMC16, the original l l - k b YCp50-based clone and pNK629, a pACYC184 derivative containing the transposase gene necessary for TnlOLUK to insert randomly into DNA. These bacteria were concentrated 10-fold and infected with ATnlOLUK a t a multiplicity of infection of 0.3.

394

GALBRAITH AND MALONE TABLE 1. Yeast Strains and Plasmids

Strain or plasmid Strains: C3-16

Relevant genotype

Reference or source

MATa spol3-l lys2-2 CANl ura3-1 metl3-c cyh2 trp5-c leul-c Cool and Malone, 1992 ~-~~~~~ MATa spol3-1 lys2-1 canl ura3-52 metl3-d CYH2 trp5-d leul-12 MATa spol3-1 lys2-2 CANl u r d - 1metl3-c cyh2 trp5-c leul-c his7-1 ~ Malone et al., 1991 -reclO4-l ~ ~ MATa reclO4-l spol3-l lys2-1 canl ura3-13 metl3-d CYH2 trp5-d leul-12 his7-2 MATa reclO4-1 lys2-2 CANl ura3-1 metl3-c cyh2 trp5-c leul-c Malone et al., 1991 MATa reclO4-1 lys2-1 canl u r d - 1 3 metl3-d CYH2 trp5-d leul-12 MATa spol3-l lys2-2 ura3-13 metl3-c trp5-c leul-c ~ Malone et al., 1991 - reclO4-l ~ -CANl - cyh2 ~ -~ MATa reclO4-l spol3-1 lys2-1 canl ura3-1 metl3-d CYH2 trp5-d leul-12 MATa rad52-1 spol3-1 Malone and Esposito, 1981 MATa rad-52-1 spol3-l MATa rec104-T5 lys2-2 CANl cyh2 leul-c This paper MATa rec104-T5 lys2-1 canl CYH2 leul-12 This paper MATa rec104-T5 spol3-l lys2-2 --canl CYH2 -trp5-d -leul-12 MATa rec104-T5 spol3-l lys2-1 CANl cyh2 trp5-c leul-c MATa lys2-2 CANl cyh2 leul-c This paper MATa lys2-1 canl CYH2 leul-12 MATa reel 04 -1 rad52 -1 spol3 -1 This paper -~~~ MATa reclO4-1 rad-52-1 spol3-1 This paper MATa reel 04-1 rad-5OS-KI81spol3-l MATa reclO4-l rad5OS-KI81 spol3-1 MATa rad5OS-KI81 spol3-1 This paper MATa rad5OS-KI81 spol3-1 ~~

RM161

~~

RM163

~~~~~~~

RM179 RM186

~

~~

~~~

A3-1

~~~

A13-4 A13-5

~~~~

A4-7 A7-2

~

~

~~

A7-7 Plasmids pRS306 pRS316 pRS315 pKMC16 vRM211 pRM212 pAMG403 pA6HOP5 pNK629 pNKY349

URA3 Amp' (integrating plasmid) CENG ARSH4 URA3 Amp' CENG ARSH4 LEU2 Amp' YCp50 + REC104 (original 11-kb clone) vRS306 + REC104 (3.4-kb HDaI-BamHI f r a m e n t from ~ K M c 1 6 ) pRS316 + REC104 (3.4-kb HiaI-BamHI fra-gment from pKMC16) pRS315 + REC104 (2.4-kb BglII-PstI fragment from pKMC16) pKMC16 + TnlOLUK a t position +61 of REClO4 coding region pACYC184 + ptac-transposase gene for TnlOLUK pSP65 + rad5OS-KI81 (vector containing rad5OS-KI81 mutation for transforming yeast)

Sikorski and Hieter, 1989 Sikorski and Hieter, 1989 Sikorski and Hieter, 1989 This paper This paper This paper This paper This paper Huisman et al., 1987 Alani et al., 1990

The bacteria were then plated on LB medium contain- termine when the RECl04 gene was expressed. The ing sodium pyrophosphate, ampicillin (100 pg/ml), and 900-bp ClaI-EcoRV fragment was end-labelled with kanamycin (50 pg/ml). Plasmids obtained from a pool y A T F 2 a t the EcoRV site, denatured for 10 minutes at of the bacterial colonies were retransformed into 85", and then hybridized overnight at 49" to 100 pg of RK1448 and then analyzed by restriction digests to total RNA from mitotic and meiotic cells. These reactions were then treated with S1 nuclease (BRL) for 30 determine the locations of the insertions. The plasmid pAMG403 (described above) was used to minutes, denatured, and run out on a 0.9% agarose gel. generate fragments for sequencing by using the Erase- The gel was then blotted to a nylon filter and exposed a-Base kit (Promega). Dideoxy sequencing was per- to film. formed as described [Sanger et al., 19771 with the SeNucleotide Sequence Accession Number quenase kit (U. S. Biochemicals). The search for The nucleotide sequence data reported in this report homology of the DNA and protein sequences was performed with the GenBank and EMBL data bases using have been submitted to the EMBL Data Library and assigned the accession number 215007. Fasta and Tfasta programs [Devereux et al., 19851.

Regulation of Expression of REC104 Total RNA from mitotic and meiotic cells was extracted by the method of Elder et al. [1983] using 100 ml samples. S1 analysis was then done strictly to de-

RESULTS Mitotic recombination and repair. Since it was previously shown that the reclO4-1 mutation had no effect on mitotic growth [Malone et al., 19911, it was of

AN EARLY MEIOTIC RECOMBINATION GENE IN YEAST

395

TABLE 2. Spontaneous Mitotic Recombination in Rec- diploids*

Prototroph frequency ( x lo5) No. leul-c trp5-c metl3-c urd-13 lrS2-2 Diploid name Genotype cultures leul-12 trp5-d metl3-d ura3-1 lvs2-1 C3-16 REC104 spol3-la 5 4.3 ? 4.0 5.2 t 38 1.9 2 0.2 O.€Bb 0.15 t 0.07 (1.0) (1.0) ( 1.O) (1.0) (1.0) RM161 reclO4-l spol3-1 4 5.1 i 3.5 3.7 t 2.3 1.7 t 0.4 0.59 t 0.04 0.27 2 0.07 (1.2) (0.71) (0.89) (0.67) (1.8) RM163 reclO4-l SP013 5 4.1 i 4.9 1.4 t 0.51 1.2 i 0.3 1.5 1.3 0.14 t 0.03 (0.95) (0.27) (0.63) (1.7) (0.93) 5 2.6 t 0.8 ND ND ND 0.21 t 0.05 A13-1 rec104-T5 SP013 (0.60) ( 1.4)

*

Drug resistant colony frequency ( X lo4) cyh2 can1 CYH2 CAN1 5.2 i 3.1 4.7 t 0.7 (1.0) (1.0) 3.1 2 0.44 12 t 2.1 (0.60) (2.6) 4.2 ? 7.3 2.7 t 0.98 (0.81) (0.57) 23 t 4.4 9.6 ? 1.5 (4.4) (2.0)

*Each 5 ml culture was grown from 100 cellsiml to - 2 x lo7 cellsiml. The values given are the geometric mean ? t h e standard deviation. Numbers in parentheses represent the relative mitotic recombination frequency for the Rec- strain compared to wild-type. ND = not determined. aData for the wild-type strain taken from Cool and Malone [19921. bThe value for the ura3 locus for the Rec+ diploid was taken from Malone and Esposito [1981].

interest to determine what effects, if any, r e d 0 4 mutations had on either mitotic recombination or repair. Initial results suggested that mitotic recombination occurred at wild-type levels in a reclO4-1 strain [Malone et al., 19911, but only two loci were examined. We continued the analysis and measured recombination at, five different loci on three chromosomes; in addition, a null mutation was examined. As the data in Table 2 demonstrate, neither the r e d 04-1 mutation nor the null insertion allele rec104-T5 (see Materials and Methods for the construction of this allele) has a significant effect on the levels of mitotic recombination. Since mutations in several genes involved in meiotic recombination have mitotic repair phenotypes as well (e.g., RAD50, RAD52), i t was important to determine whether a reclO4 mutation would confer a defect in DNA repair. Figure 1 indicates that UV repair is not affected by a recl 04-T5 mutation. Similar results were seen with the reclO4-1 allele, and we observed no defect in UV repair in recl 04 haploids (data not shown). Furthermore, reclO4 mutant strains are not sensitive to the radiomimetic methyl methanesulfonate (MMS) (see Fig. 2). We conclude that RECl04 is not required for mitotic recombination or repair. Meiotic recombination. Initial results from meiotic recombination tests of recl 04-1 indicated that i t did indeed confer a defect in meiotic recombination a t two loci [Malone et al., 19911. To determine whether this was a general effect, we measured meiotic recombination a t five loci on various chromosomes. As depicted in Table 3, the wild-type control has the typical 1,000-fold increase in meiotic recombination; however, both the reclO4-1 and rec104-T5 mutations reduce the normal level of meiotic recombination by 400- to 13,000-fold (Table 3). Crossing over was also examined in reclO4 spol3 diploids. Once again, the reclO4-1 and rec104-T5 mutations greatly reduced the frequency of drug resis-

tant colonies (Table 3). In fact, we observed no significant induction of meiotic recombination above the background mitotic levels in a reclO4 mutant diploid (compare Tables 2 and 3). To verify that crossing over was eliminated in reclO4 miitant, s h i n s , dyad asci from r ~ c 7 0 4sp07,’3-1diploids were dissected after meiosis and recombination was assayed. No recombinant-type dyads were found among the spores examined from the r e d 04 -1 and recl 04-T5 diploids (Table 4 ) . In addition, no aberrant type dyads were observed (see Discussion). Rescue of rad52 spol3 diploids. The recl 04-1 mutation was originally isolated in a rad52 spol3 haploid strain [Malone et ul., 19911. In order to confirm the expectation that reclO4-1 would be able to rescue rad52 in a diploid, a r e d 0 4 rad52 spol3 diploid was tested. Sporulation of the triple mutant was similar to that for both RECl04 spol3 and reclO4-1 spol3 diploids, and dissection of the dyads resulted in 79.2% viable spores after meiosis compared to 78% spore viability for the reclO4-l spo13 diploid (Table 5). We conclude that the reclO4-1 mutation can rescue the rad52 mutation in diploids a s well as in haploids, and r e d 0 4 mutations are epistatic to rad52 mutations in meiosis. Epistatic relationship with rudSOS-KI81. An allele of RAD5O (rad50S-KISl)has been isolated, which confers a different phenotype on cells than most rad50 alleles (Alani et al., 19901. A null rud50 mutation produces inviable spores after meiosis, and this defect can be overcome by the presence of the spol3-1 mutation, a s expected for a n early mutation [Malone and Esposito, 19811. The rad5OS-KIS1 defect cannot be rescued by the spo13-1 mutation [Alani et al., 19901. We took advantage of this unique phenotype, and created the triple mutant diploid reclO4-1 rad5OSKIS1 spol3-1 to determine the relationship of the

396

GALBRAITH AND MALONE I

'0

UV DOSE (J/m2) Fig. 1. REClO4 is not required for UV resistance. Isogenic diploid strains were either wild-type (A13-5) or rec104-2'5 A (Al3-1). The strains were UV-irradiated at 2.4 J/m2/s. Survival was calculated as the log (concentration surviving cellshnitial concentration of cells), or log (NIN,). Shown is the average of two trials for each diploid. The 260-nm UV lamps (General Electric) were calibrated with a Blak-Ray Ultraviolet Intensity Meter (UVP).

reclO4-1 and rad5OS-K181 mutations in the recombination pathway. After sporulation and dissection of the resulting dyads, viable meiotic products were produced (Table 5) indicating that reel 04 mutations are epistatic to radSOS mutations. Analysis of the cloned REC104 gene. The selection scheme used to clone the wild-type REC104 gene by complementation has been described [Malone et al., 19911. The plasmid pRM211 was used to verify that the cloned DNA did indeed contain the REC104 gene and not a n extragenic suppressor [Malone et al., 19911. Figure 3 shows the restriction map for part of the original l l - k b clone, which was shown to complement the reclO4-1 phenotype, and the ability of various subclones, Ex0111 deletions, and TnlOLUK insertions to complement the sporulation, recombination, and meiotic viability defects conferred by the recl 04-1 allele. From these data, the REClO4 gene was localized to the region indicated in Figure 3 and was then sequenced. The sequence of the REC104 gene is shown in Figure 4. Searches of the GenBank and EMBL data bases [Devereux et al., 19851 revealed no homologies to any published gene; thus, REClO4 is a previously unknown yeast gene. Examination of the promoter region of the nucleotide sequence reveals a potential URSl at position -93, which is identical to the URSl found within

the coding region of SPOl1 [Buckingham et al., 19901. There is also a putative UAS at position -139, which contains 616 bases identical to the UAS upstream of the SPOll coding region [Vershon et al., 19921. The nucleotide sequence predicts a protein of 182 amino acids with a molecular weight of 21 kD, a net charge of -7, and a n isoelectric point of 4.6. The hydropathicity plot suggests that the protein is soluble (data not shown). There are no putative DNA-binding motifs present, nor is there a n ATP-binding domain or conserved kinase motifs [Devereux et al., 19851. There are, however, four consensus protein kinase C phosphorylation sites [Devereux et al., 19851 at residues 8, 24,37, and 76, and two consensus casein kinase I1 phosphorylation sites [Devereux et al., 19851 at amino acids 2 and 113. Finally, there is significant similarity (33% identity) from residues 85-105 with region VI of Saccharomyces CDC2 DNA polymerase I11 (see Fig. 6 and Discussion). Genetic mapping of REC104. Using the 3.4 kb HpaI-BamHI fragment containing RECl04 as a probe, a Southern blot of yeast chromosomes separated by pulse-field gel electrophoresis showed that REClO4 was located on chromosome VIII (data not shown). Analysis of genetic crosses places REC104 on chromosome VIII, 16 cM distal to the FUR1 gene (Table 6).

-

AN EARLY MEIOTIC RECOMBINATION GENE IN YEAST

397

lation medium, and no message is detectable in mitotic cells. These data suggest that the RECl04 gene is transcribed specifically in meiosis.

DISCUSSION

A

B

1- wild-type haploid

1

1

2

2- wild-type diploid

3

3- rud50 haploid

4

4-rad50 diploid

5- rec104-T5 diploid

6- rec104-T5 haploid 7-recl04-T5/rec104-1diploid

5

5

5

6

7

7

7

8

8

9

6

8- recl04-l diploid 9- recl04-1 haploid

Fig. 2. REC104 is not needed for MMS resistance. Patches of cells were grown on YPD plates. These were replica-plated to another YPD plate (A) and a YPD plate containing 0.012%MMS (B). The results of these two platings are shown; a description of the various patches is under the figure.

Analysis of REC104 expression. Since mutations in RECl04 appeared to affect only meiosis and since other genes involved in meiotic recombination have been shown to be transcribed only in meiosis [e.g., REDl, Thompson and Roeder, 1989; REC102, Cool and Malone, 19921, i t seemed reasonable to propose that the REC104 gene might also be expressed only in meiosis. To test this hypothesis, total RNA was isolated from wild-type cells growing exponentially and after various times in sporulation medium. REClO4 expression was then examined by S1 analysis a s described in Materials and Methods. As shown in Figure 5, the RECl04 gene appears to be expressed after 4 and 7 hours in sporu-

High levels of recombination and pairing of homologous chromosomes during meiosis are necessary in Saccharomyces cerevisiae for the successful production of viable spores; mutations that affect either of these two processes result in decreased spore viability. In addition, all early exchange mutants examined so far are able to rescue the inviable spore phenotype of a rad52 mutation in a spol3 background. This suggests that these early mutations block recombination at or before initiation, presumably before strand exchange has begun. A pathway was presented by Malone et al. l19911 extending a proposal by Engebrecht and Roeder [19901, which subdivides the known early mutants into two groups: those involved in the initiation of recombination per se (i.e., the exchange group) and those primarily involved in chromosome pairing and SC formation (i.e., the pairing group). The data for the RECl04 gene presented here strongly suggest that it is in the exchange group and is a gene directly involved in the initiation of exchange. Unlike HOP1 and REDl, two genes proposed to be involved in SC formation and chromosome pairing [Hollingsworth and Byers, 1989; Rockmill and Roeder, 19881, mutations in the REClO4 gene completely eliminate meiotic recombination; the hop1 and red1 mutations only reduce meiotic recombination by 2- to 15-fold. Not only is crossing over abolished in r e d 04 strains, but aberrant-type dyads are also completely eliminated (Table 4). Similar results have been observed in other Rec- spo13 mutant strains, and it has been suggested that exchange is deleterious to a n equational division [Malone and Esposito, 1981; Malone, 19831. Therefore, a Rec- mutation t h a t eliminates exchange increases the chance of proper segregation at the second meiotic division. Unlike the early recombination mutation rad50 [Malone and Esposito, 19811, reclO4 mutations have no apparent mitotic phenotype; mutations in the REClO4 gene confer no defects in mitotic growth [Malone et al., 19911, DNA damage repair, or mitotic recombination. Like mutations in other genes of the early exchange group, however, recl 04 mutations do rescue rad52 mutations in a spol3 background [ s p o l l , Klapholz et al., 1985; recll4, Doug Pittman and Robert Malone, personal communication; reclO2 and mer2, Cool and Malone, 1992 and unpublished data; reclO2, Barghava et al., 1992; mer2, Engebrecht et al., 1990; mei4, Menees and Roeder, 1989; rad50, Malone, 19831. Although i t is certainly possible that the eight early recombination genes (REC104, REC102, REC114, S P O l l , MER2, MEI4, RADSO, and MER1) act at several different points in the recombination pathway prior to the rad52 mutant block, we as yet have no evidence supporting

398

GALBRAITH AND MALONE TABLE 3. Examination of Meiotic Recombination in s ~ o 1 3DiDloids*

Diploid name C3-16

leul-c leul-12 6,200 (1.0) 0.76 (8,200) 2.3 (2,700)

Prototroph frequency" ( X lo5) metl3-c ura3-13 trp5-d metl3-d ura3-1 9,200 ND 77d (1.0) (1.0) 2.1 0.64 0.19 (4,400) (400) 0.7 ND ND (13,000)

trp5-c

-

Drug resistant colony frequencyb (X

lo4)

&

cJ@ canl Genotype lys2 -1 CYH CAN REC104" 300 9,600 7,900 (1.0) (1.0) (1.0) RM179 recl 04 -1 0.06 2.3 44 (5,000) (4,200) (180) A13-4 recl 04-T5 0.03 2.1 2.7 (10,000) (4,600) (2,900) *Each diploid was sporulated and the frequency of recombination was measured. The values given are the geometric mean of at least three cultures. The numbers in parentheses represent the fold reduction in meiotic recombination frequency for each locus measured for the Rec- strain as compared to wild type. ND = not determined. "Prototrophs represent predominantly gene conversion events. bDrug-resistant colonies represent crossing over between the locus and the centromere, gene conversion, and missegregation. "Wild-type data taken from Cool and Malone [19921. dThe value for the ura3 locus for the Rec+ diploid was taken from Malone and Esposito I198ll. TABLE 4. Analysis of Crossing Over in spol3 Diploids* VIP

Diploid name C3-16

Genotype REClO4"

met13 ND

RM161

r~rl04-1

A13-4

recl 04-T5

58:O:O (