Copyright Q 1992 by the Genetics Society of America

Meiotically Induced rec7 and rec8 Genes of Schixosaccharomyces Pombe

Yukang Lin, Karen L. Larson,' Russell Dorer' and Gerald R. Smith Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

Manuscript received March 2, 1992 Accepted for publication May 15, 1992

ABSTRACT The Schixosaccharomyces pombe rec7 and reed genes, which are required for meiotic intragenic

recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the Tee genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the recd gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and recd gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.

H OMOLOGOUS recombination involves a series of steps, including chromosome pairing, DNA strand exchange and resolution of intermediates. These multiple steps suggest that multiple enzymes catalyze the process. One approach to understanding the mechanism of homologous recombination is to isolate recombination-deficient mutants and thereby identify the genes required for recombination. Study- ing the activities of these gene products can help to elucidate the mechanism of recombination. As ex- pected from the multistep nature of recombination, many genes encoding functions required in recombi- nation have been identified both in prokaryotes and eukaryotes (SMITH 1988; PETES, MALONE and SYM-

Mutants of Schirosaccharomyces pombe deficient in meiotic recombination have been isolated (PONTI- CELLI and SMITH 1989; DEVEAUX, HOAGLAND and SMITH 1992). Forty-three recessive mutations, includ- ing the previously isolated m i 5 mutations (GUTZ and SCHMIDT 1985; SCHMIDT, KAPITZA and GUTZ 1987), have defined 17 complementation groups, which fall into three discrete classes based on their degree of reduction of meiotic intragenic recombination at the a d e 6 locus (DEVEAUX, HOAGLAND and SMITH 1992). Mutations in the six class I genes may totally eliminate meiotic intragenic recombination: ade6 intragenic re- combinant frequencies are reduced about 1 000-fold, to a residual level that may result from mitotic recom- bination. Mutations in the three class I1 genes reduce a d e 6 recombination 100-fold, and mutations in the

INGTON 1991).

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eight class I11 genes decrease ade6 recombination about 5-fold.

Mutations in the class I rec7 gene not only abolish intragenic recombination at the ade6 and ura4 loci but also reduce intergenic recombination between the p r o 2 and arg3 loci at least 30-fold (PONTICELLI and SMITH 1989). The rec7 gene product, therefore, ap- pears to be required for high levels of both gene conversion and crossing over.

Unlike rec7 mutations, mutations in the class I rec8 gene appear to eliminate ade6 intragenic recombina- tion, but intergenic recombination between the pro2 and arg3 loci is reduced only about 3-fold (L. DE- VEAUX, personal communication). These observations suggest that the rec8 gene product may be stringently required for gene conversion but much less so for crossing over. From the results of studies in fungi and Drosophila, CARPENTER (1 987) and HASTINCS (1 988) suggested that gene conversion and crossing over may occur by distinct mechanisms. The behavior of rec8 mutations supports the idea that meiotic gene conver- sion and crossing over proceed by different mecha- nisms. Other explanations for the rec8 phenotype, such as regional specificity of the rec8 gene product, have not been ruled out (DEVEAUX, HOACLAND and SMITH 1992).

To search for the biological function of the rec7 and rec8 genes and the activities of the gene products, we have cloned and analyzed these two rec genes. We report here their nucleotide sequences and their tran- sient expression early in meiosis, which suggests that the rec7 and rec8 gene products may be required only in the early steps of meiotic recombination.

MATERIALS AND METHODS Strains: S. pombe strains and their genotypes are described

in Table 1 according to the genetic nomenclature given by

76 Y. Lin et al.

TABLE 1

S. pornbe strains

Strain Genotype Sourcea or referenceb

GP19 h+ 1 GP32 h+ ade6-M26 leul-32 A. S. PONTICELLI~ GP48 h+ endl-458 patl-114 GP265

A. S. PONTICELLI' hPO ade6-M26 ura4-294 rec7-I14 2

GP27 1 hPO ade6-M26 ura4-294 reed-1 I 5 2 GP277 h- ade6-52d rec7-102 2 GP290 h- ade6-52 rec8-110 2 GP350 h+ pro2-1 ura4-595 2 GP363 h+ ade6-M26 ura4-294 arg3-124 2 GP369 h- ade6-52 ura4-595 pro2-1 2 GP377 h+ ade6-52 rec7-114 GP381 GP42 1 h- ade6-52 ura4-595 pro2-1 rec7-102 2 GP426 h+ ade6-52 ura4-595 pro2-1 reed-110 GP350 X GP290 GP43 1 h+ ade6-M26 ura4-294 arg3-124 rec7-102 2 GP436 h- ade6-M26 ura4-294 arg3-124 rec8-110 3 GP442 h- ade6-52 ura4-294 pro2-l GP663 h+ ade6-M26 ura4-294 arg3-124 rec7-102 (pKL1) T of GP43 1 GP664 h- ade6-M26 ura4-294 arg3-124 reed-110 (pKL2) T of GP436 GP665 hPo ade6-M26 ura4-294 rec7-I 14 (pKL1) T of GP265 GP666 hP0 ade6-M26 ura4-294 rec8-I15 (pKL2) GP7 13

T of GP27 1 h- ade6-M26 ura4-294 arg3-124 rec8-110 (pYL3) T of GP436

GP7 14 h- ade6-M26 ura4-294 arg3-124 rec8-I10 (pYL4) GP7 15

T of GP436 h- ade6-52 ura4-595 pro2-l red-102 (pYL1) T of GP43 1

GP7 16 h- ade6-52 ura4-595 pro2-1 rec7-102 (pYL2) GP720

T of GP43 1 h- ade6-52 ura4-294 leul-32 GP32 X GP442

GP722 h+ ade6-52 leul-32 rec7-102 GP32 X GP277 GP723 h+ ade6-52 leul-32 rec8-110 GP32 X GP290 GP8 19 h+ ade6-M26 ura4-294 arg3-124 rec7-102 (pYL45) T of GP43 1 GP820 h+ ade6-M26 ura4-294 arg3-124 rec7-102 (pYL46) T of GP43 1 GP821 h- ade6-52 ura4-294 leul-32 rec7-146::TnIO-LLK T of GP720' GP838 h- ade6-M26 ura4-294 arg3-124 rec8-I10 (pYL53) GP855f

T of GP436 h- ade6-52 ura4-294 leul-32 re~8int::pYL7~

GP857h T of GP720

h- ade6-52 ura4-294 leul-32 rec7int::pYLSB T of GP720 GP860 h- ade6-M26 ura4-294 arg3-I24 reed-110 (pYL55) T of GP436

GP946 h- ade6-M26 ura4-294 arg3-124 rec8-110 (pRD3) T of GP436 GP947 h- ade6-M26 ura4-294 arg3-124 rec8-110 (pRD4) T of GP436

A. S. PONTICELLI' h+ ade6-52 rec8-I15 A. S. PONTICELLI'

N. M. HOLLINGSWORTH'

GP945 h- ade6-M26 ura4-294 arg3-124 rec8-I 10 (pRD2) T of GP436

a T, transformation; X meiotic cross.

' Genealogies are available upon request. 1, PONTICELLI, SENA and SMITH (1988); 2 , PONTICELLI and SMITH (1 989); 3, DEVEAUX, HOALAND and SMITH (1992).

This allele was formerly designated ade6-L52, but has been redesignated ade6-52 (SZANKASI et al. 1988). The transforming DNA was pYL54 cut with BanHI .

The symbol indicates that the plasmid is integrated at the rec locus, duplicating but not inactivating it. Strain GP858 was constructed similarly and has an identical genotype (see Table 4).

fStrains GP856 and GP948 were constructed similarly and have identical genotypes (see Table 4).

KOHLI (1987). Escherichia coli strains MC1066 (hsdR hsdM trpC9830 leuB600 pyrF::Tn5 strA galU galK lacAX74) (from V. ZAKIAN), V66 (hisG4 met argA2l recFl43 rpsL3l galK2 xyl-5 rac- X- F-) (SCHULTZ, TAYLOR and SMITH 1983), C600 (thr-1 leuB6 thi-1 supE44 lacy1 tonA2l X- F-) (APPLEYARD 1954) and their derivatives were used for transformation by the CaC12-treatment method (SAMBROOK, FRITSCH and MANIATIS 1989) and for phage infections. E. coli transform- ants were selected on LB agar plates containing an appro- priate drug [ampicillin (100 pg/ml) or kanamycin (25 pg/ ml)]. Additional E. coli strains are described in the text.

Plasmids: The plasmids used in this study are described in Table 2. Plasmid pSP2 (SIKORSKI and HIETER 1989) was used as a vector for all subclones, except for those specified in the text.

S. pombe media: YEA is yeast extract agar and YEL is yeast extract liquid (GUTZ et al. 1974). SPA and NBA are synthetic sporulation agar and minimal medium agar, re- spectively (GUTZ et al. 1974; PONTICELLI and SMITH 1989). EMM2* medium (SZANKASI and SMITH 1992) was used as a liquid minimal medium. These media were supplemented with the required nutrients (1 00 pg/ml).

Transformation of S. pombe: The method of ITO et al. (1983) produced about 100-1000 transformants per pg of transforming DNA. The selective NBA plates were incu- bated at 30" for 5 days, and colonies were purified by streaking to obtain isolated colonies on the same medium.

Mutagenesis with TnlU-Kan and TnZO-LLK E. coli strain V66 containing the plasmid to be mutagenized was infected with XNKllO5. This phage contains a derivative of

Meiotically Induced rec Genes 77

TABLE 2

Plasmids

Plasmid Description ~ource' or reference b

pSP2 pPOCYC(O.54) YIp32 pFL2O pKLl pKL2 pRDl pRD2 pRD3 pRD4 pRD5 pYL 1 pY L2 pY L3 pYL4 pYL5 pYL7 pY L45

pYL54 pYL55

pY L46

arsl URAP ampR ampR cyc ampR LEU2 ars-stb URA3 ampR pFL20 + 1 1.1-kb Sau3A rec7 fragment pFL20 + 8.5-kb Sau3A rec8 fragment pKL1::TnlO-Kan' pKL2 rec8-147::TnIO-Kan pKL2 1x8-148::TnlO-Kan pKL2 rec8-149:TnlO-Kan pKLl rec7-150::TnIO-Kan pSP2 + 2.8-kb BamHI rec7 fragment pSP2 + 2.8-kb BamHI rec7 fragment pSP2 + 3.9-kb SacI rec8 fragment pSP2 + 3.9-kb SacI recd fragment YIp32 + 2.8-kb BamHI rec7 fragment YIp32 + 3.9-kb SacI rec8 fragment pSP2 + 1.6-kb BamHI-MluI rec7 fragment pSP2 + 1.6-kb BamHI-MluI rec7 fragment pYL45 rec7-146::TnIO-LLK pSP2 + 2.1 kb NdeI-Nhel recd fragment

D. BEACH; 1 B. HALL; 2 K RUNGE; 3 J. KOHLI; 4 J. CARBON; 5 J. CARBON; 5 pKLl + XNKllO5 pKL2 + XNKllO5 pKL2 + XNKl105 pKLP + XNK 1 105 pKLl + XNKllO5 pSP2 + pRDl pSP2 + pRDl pSP2 + pKL2 pSP2 + pKL2 YIp32 + pYLl YIp32 + pKL2 pSP2 + pYLl pSP2 + pYLl pSP2 + pYL4 pYL45 + X1747

b For details, see text. 1, SIKORSKI and HIETER (1989); 2, RUSSELL and HALL (1 982); 3, BOTSTEIN et al. ( 1 979); 4, LOSSON and LACROUTE (1 983); 5, ELLIOTT et

The symbol indicates that the TnlO-Kan element is inserted near but not within the rec7 gene (data not shown). al.11986).

transposon TnlO, designated element 9, with the kanamy- cin-resistance (KanR) determinant of Tn903 flanked by 78 bp inverted repeats of the ends of TnlO (WAY et al. 1984). Infection, expression of KanR and selection of KanR tran- ductants and transformants was as described by WAY et al. (1984).

T o have an additional selectable marker for DNA manip- ulations, we constructed a phage analogous to h TnlO-LUK (HUISMAN et al. 1987) with the S. cerevisiae LEU2 gene in place of the S. cerevisiae URA3 gene of X TnlO-LUK. Phage X NK780 (gt7-his cI857 susP80 nin-5; HUISMAN et al. 1987) was grown by lytic infection in E. coli strain C600 (pTnlO- LLK). Plasmid pTnlO-LLK, a derivative of pBR322, con- tains the major part of the E. coli lacZ gene, the LEU2 gene, and the KanR determinant from Tn903 (R. REENAN and R. KOLODNER, submitted). These genes are flanked by 78 bp- inverted repeats of the ends of TnlO, which in turn are flanked by the Salmonella typhimurium his DNA present in XNK780 (N. KLECKNER, personal communication). With the lysate, E. coli strain C600 (XcI857) was transduced to KanR. One of six transductants contained X Sus- phage that trans- duced the leuB6 strain C600 (XcZ857) to KanR and to Leu+. After purification and retesting, one phage was designated X1747 (gt7-his::TnlO-lacZ-LEU2-k~n cI857 susP80 nin-5). The transposable element in this phage is designated TnlO- LLK.

Plasmids in E. coli were mutagenized with TnlO-LLK by infection with X1747 as described by HUISMAN et al. (1987). TnlO transposase with an altered target specificity was pro- vided by plasmid pNK2882 (KLECKNER, BENDER and GOTTESMAN 199 1). S. pornbe genomic library: LOUISE CLARKE and JOHN

CARBON (University of California at Santa Barbara) kindly provided a mixture of E. coli cells containing derivatives of plasmid pFL2O (LOSSON and LACROUTE 1983) with about

10-kb partial Sau3A digestion products of S. pombe DNA. A sample (0.5 ml) was grown to saturation in 500 ml of LB broth containing ampicillin (100 pg/ml), and plasmid DNA was extracted and purified in a CsCl gradient as described by SAMBROOK, FRITSCH and MANIATIS (1 989).

Meiotic crosses and rec complementation assay: Meiotic crosses were made on SPA solid medium, and spores were harvested and analyzed as described (PONTICELLI, SENA and SMITH 1988). Meiotic recombinant frequencies were meas- ured by intragenic recombination between ade64426 and ade6-52 or by intergenic recombination between arg3-124 and pro2-1 as described (PONTICELLI and SMITH 1989). For qualitative assays, the cells were mated on an SPA plate for 3 days, replicated to an NBA plate and incubated for several days. Rec+ crosses produced more than 50 white Ade+ papillae, and Rec- crosses produced none. Strains with plasmids harboring the S. cerevisiae URA3 gene, which com- plements S. pombe ura4 mutations (GRIMM et al. 1988), were grown to saturation in EMM2* liquid medium lacking uracil at 30" before crossing with the tester strain.

Gene subclones: A 2.8-kb BamHI fragment carrying rec7 and 78 bp of TnlO DNA (WAY et al. 1984) was isolated from pRD1, a ret+ derivative of pKLl containing TnlO- Kan. This fragment was ligated in one or the other orien- tation into the BamHI site of pSP2 to yietd pYLl and pYL2. A 1.6-kb BamHI-MluI fragment was isolated from pYL1, the MluI site was converted to a BamHI site by ligation of a linker, and the modified fragment was ligated into the BamHI site of pSP2 to form pYL45 and pYL46.

A 3.9-kb SacI fragment of pKL2 containing the rec8 gene was ligated into the Sac1 site of pSP2 to generate pYL3 and pYL4 by the method described for the rec7 subclone. A 2.1- kb NdeI-NheI fragment was isolated from pYL4, and its ends were filled out with DNA polymerase I (SAMBROOK, FRITSCH

78 Y. Lin et al.

and MANIATIS 1989). This fragment was ligated into the SmaI site of pSP2 to form pYL55.

Southern blot analysis: S. pombe DNA was prepared as described by BEACH and KLAR (1984), except lyticase (0.3 mg/ml, Sigma) was used instead of zymolyase. A 1-pg sample of such DNA was digested with a restriction enzyme over- night and electrophoresed on a 1% agarose gel containing ethidium bromide (1 pg/ml). After transfer onto a nitrocel- lulose filter (SAMBROOK, FRITSCH and MANIATIS 1989), the filter was hybridized with a desired DNA fragment that had been labeled using the random ?rimer DNA labeling kit (Boehringer Mannheim) and [CY-' PIdCTP and [a-'*P]TTP (FEINBERG and VOGELSTEIN 1983).

Northern blot analysis: RNA was prepared from strain GP48 (h+ patl-114 endl-485). A 20-ml sample grown to saturation in YEL at room temperature was added to 3 liters of EMM2* medium and grown at room temperature (22- 25") until the OD596 reached 0.3. The temperature of the culture was increased to 34" by swirling the flask in hot water for about 5 min, and the culture was shaken for another 6 hr at 34". Samples (200 ml) were taken every hour. Microscopic examination showed that the cells formed multinucleate asci at about 6 hr. The cells were centrifuged and washed with RNA buffer (50 mM Tris-HCI, pH 7.4, 100 mM NaCI, 10 mM EDTA) and frozen at -80" for future use. The samples were thawed at room temperature, and the cells were broken at 4" with glass beads in a 2-ml tube using a Biospec Product Mini-beadbeater (model 13 1 10 BX) at high speed for 10 X 30 sec with 1 min on ice between agitations. The supernatant, combined with a 1-ml wash of the glass beads, was extracted once with an equal volume of phenol/CHCls/isoamyl alcohol (25:24: 1) and once with CHCls/isoamyl alcohol (25:l). RNA was precipitated with ethanol at 4" for 30 min and stored in 400 pl of diethylpy- rocarbonate-treated H 2 0 at -80". The yield was about 7 pg of nucleic acid per ml of EMM2* culture. Samples of 15 pg of nucleic acid were electrophoresed on a 6% formalde- hyde- 1 % agarose gel overnight and transferred onto a nitro- cellulose filter (SAMBROOK, FRIT~CH and MANIATIS 1989). The filter was hybridized at 42" with the StyI-BstNI 590-bp fragment internal to the rec7 open reading frame (ORF) (see Figure l) , the BamHI-Hue11 616-bp fragment internal to the rec8 ORF (see Figure 2), or plasmid pPOCYC(O.54) containing the S. pombe cyc gene (RUSSELL and HALL 1982) as an internal control. The probes were labeled with "P using random DNA primers. The filter was washed at 55" in SDS buffer (1.75% NaCI, 0.8% sodium citrate pH 7.0, 0.1 % sodium dodecyl sulfate) and exposed at -80" for 3 days using Kodak XAR5 film and a Du Pont Cronex Light- ning Plus intensifying screen. Ex0111 deletions: ExoIII-generated deletions were iso-

lated as described (HENIKOF'F 1984); 2 pg of DNA from pYLl, pYL2, pYL3 or pYL4 were linearized with KpnI and SmaI, treated with ExoIII, and sampled every 30 sec. The DNA was then treated with S1 nuclease and the Klenow fragment of DNA polymerase I. After ligation, the plasmids were introduced into E. coli strain MC1066 by transforma- tion and ampicillin-resistant transformants were selected. Plasmid DNA was extracted (SAMBROOK, FRITSCH and MAN- IATIS 1989) and analyzed for the size of the deletion. Dele- tion candidates were introduced into appropriate S. pombe rec strains to assay for rec complementation.

Nucleotide sequence determination and analysis: Nu- cleotide sequences were determined by the dideoxy chain- termination method (SANGER, NICKLEN and COULSEN 1977) using '5S-labeled deoxyadenosine triphosphate. Double- stranded DNA was denatured with dimethyl sulfoxide and sequenced directly (D. CLARK, personal communication),

using an M13 reverse primer or oligonucleotide primers synthesized on an Applied Biosystem 350B DNA synthe- sizer. Some sequences were determined on templates gen- erated by Ex0111 deletion of pYL1, pYL2, pYL3 and pYL4, or by TnlO-Kan insertion into pKLl and pKL2. Sequences were compared with the GenBank (version 68), EMBL (version 27) and protein data bases (PIR and SWISS-PORT) (BAIROCH and BOECKMANN 199 1 ; BARKER et al. 199 1) using computer program PATMAT 2.1 (WALLACE and HENIKOFF 1992).

Plasmid integration: Plasmid pYL5, containing the rec7 gene, was constructed by ligation of the 2.8-kb BamHI fragment from pYLl into the BamHI site of the YIp32 vector (BOTSTEIN et al. 1979), which contains the S. cereuisiae LEU2 gene. pYL5 DNA was linearized at the MluI site located near the rec7 gene (see Figure 1) and introduced into strain GP720 by transformation to Leu+. The Leu+ transformants were purified once on supplemented NBA without leucine, grown in YEL to allow segregation of the plasmid and plated on YEA. All 10 independent transform ants tested were stably Leu+.

Plasmid pYL7, containing the rec8 gene, was constructed by ligation of the 3.9-kb SacI fragment from pKL2 into the PuuII site of YIp32. The SacI 3' protruding ends were made blunt with T4 DNA polymerase before ligation (SAMBROOK, FRITSCH and MANIATIS 1989). pYL7 DNA was linearized at the MluI sites near the rec8 gene (see Figure 2), and stable Leu+ transformants were isolated as described above.

rec7 gene disruption: Plasmid pYL45 was mutagenized with TnlO-LLK to generate plasmid pYL54 (rec7- 146::TnIO-LLK). pYL54 DNA was digested with BamHI and introduced into strain GP720 by transformation to Leu+. The transformants were isolated and purified as de- scribed above.

Nucleotide sequence accession numbers: GenBank accession numbers for rec7 and rec8 are M85297 and M85298, respectively.

RESULTS

Isolation of rec7 and rec8 clones: We isolated DNA clones bearing the rec7 (or rec8) gene from a genomic S. pombe library (ELLIOTT et al. 1986). Since direct selection for these clones is not possible, we took advantage of the enrichment for ret' clones among viable recombinant spores issuing from a meiotic cross of two appropriately marked rec7-102 (or rec8-110) strains, one of which was transformed with an S. pombe plasmid library. The rec7-102 (or rec8-110) parents differed at the ade6 locus and at the arg3 and pro2 loci in such a way that Ade+ intragenic recombinants and Arg+ Pro+ intergenic recombinants could be se- lected on NBA minimal medium. Since the rec7-102 mutation reduces recombination between ade6-M26 and ade6-52 about 1000-fold and recombination be- tween arg3-124 and pro2-l about 30-fold (PONTICELLI and SMITH 1989), we expected that rec7+ clones would be enriched about 30,000-fold among Ade+ Arg+ Pro+ recombinants. An additional &fold enrichment was expected from the 4-fold higher viability of spores from a rec+ cross than from a rec7 mutant cross (PON- TICELLI and SMITH 1989). Similar arguments apply to the rec8 mutant.

Meiotically Induced rec Genes 79

The isolation of the rec7 clone proceeded as follows. Strain GP431 (h+ rec7-102 ade6-M26 ura4-294 arg3- 124) was transformed to Ura+ with the S. pombe library plasmid DNA; the library vector pFL2O (LOSSON and LACROUTE 1983) contains the S. cerevisiae URA3 gene, which complements the S. pombe ura4-294 mutation (GRIMM et al. 1988). About 13,000 Ura+ transformant colonies were pooled, and a 1 % sample was grown for about seven generations in EMM2" liquid medium supplemented with adenine and arginine. About 3 X 10' cells were mixed with an equal number of cells of strain GP421 (h- rec7-102 ade6-52 ura4-595 pro2-1) grown in YEL and mated on supplemented SPA solid medium. After 4 days, spores were harvested and plated on NBA solid medium, to select Ade+ Arg+ Pro+ recombinants that still harbored the plasmid. A total of 443 colonies were obtained, purified on the same medium and streaked on YEA solid medium. Nineteen were stably Ade+ (produced only white col- onies on YEA), while the rest were not stably Ade+ [produced white and red (Ade-) colonies on YEA] and were presumed to contain ade6+ clones from the library. Ten of these 19 stably Ade' colonies were diploids, since they sporulated upon starvation, and were set aside; they were presumably Arg+ Pro+ due to complementation rather than recombination. Plas- mid DNA from 6 of the 9 stably Ade+ colonies was recovered in E. coli and reintroduced into strain GP431. Matings with strain GP421 showed that two of these plasmids complemented the recombination deficiency of rec7-102. Analysis of these plasmid DNAs after cleavage with Hind111 and Sal1 showed that these two plasmids were indistinguishable and contained 1 1. l-kb DNA inserts (data not shown); only one, designated pKL 1, was analyzed further. We show below that pKLl contains the S. pombe rec7+ gene.

The rec8 clone was isolated similarly from 28,000 Ura+ transformants of strain GP436 (h- ade6-M26 ura4-294 arg3-124 rec8-110). Mating of the pooled transformants with strain GP426 (h+ ade6-52 ura4- 595 pro2-1 rec8-110) resulted in 1 13 Ade+ Arg+ Pro+ recombinants, of which 5 were haploids and stably Ade+. Plasmid DNA was recovered in E. coli, and plasmid from one of them transformed strain GP436 to Ret+. This plasmid, designated pKL2, contained an 8.5-kb DNA insert (data not shown) with the rec8+ gene (see below).

Locus-specific complementation by the rec7 and rec8 clones: As a first step in determining whether the plasmids pKLl and pKL2 contained the rec7 and rec8 genes rather than suppressors of the rec muta- tions, we assessed their ability to complement rec7 and rec8 mutations. As expected, plasmid pKLl comple- mented the rec7-102 and rec7-114 mutations (Table 3, lines 3 and 6 compared to control ret+ and rec7 strains in lines 1, 2 and 5) but not the rec8-115 or

rec8-110 mutations (lines 13 and 15). Similarly, plas- mid pKL2 complemented both rec8 mutations (lines 10 and 14, compared to lines 1 , 9 and 12) but neither rec7 mutation (lines 7 and 8). Where tested, pKLl restored to a rec7 mutant strain proficiency for both intragenic recombination (Ade+ recombinants) and intergenic recombination (Arg+ Pro+ recombinants) (line 3), whereas pKL2 restored neither (line 8). Coin- cident loss of the rec+ and Ura+ properties (lines 4 and 11) gave additional evidence that each rec+ character was plasmid-borne. The observed locus-specific, allele- nonspecific complementation suggested that the cloned fragments contained the rec7 and rec8 genes rather than suppressors that bypass the requirement for the rec gene functions. This suggestion was further supported by homologous integration of the plasmids, described later.

Localization and subcloning of the rec7 and rec8 genes: We isolated derivatives of pKLl and pKL2 containing insertions of the TnlO-Kan transposable element described by WAY et al. (1984). These deriv- atives were analyzed physically, to determine the po- sition of the insertion, and genetically, to determine their ability to complement the appropriate S. pombe rec mutation (data not shown). These analyses sug- gested that the rec7 gene was located on a 2.8-kb BamHI fragment of one of the ret' derivatives of pKL1; one of the BamHI sites defining this fragment is in TnlO, and the fragment contains 78 bp of TnlO DNA (data not shown; WAY et al. 1984). Similar analyses suggested that the rec8 gene was located on a 3.9-kb SacI fragment of pKL2.

Based on this information, a 2.8-kb BamHI frag- ment from pRDl, a ret+ TnlO-Kan derivative of pKLl, was cloned into the BamHI site of the pSP2 vector (SIKORSKI and HIETER 1989) to generate pYL 1 and pYL2, containing the fragment in opposite ori- entations. Both subclones fully complemented rec7- 102 (Table 3, lines 16 and 17), confirming that the complementing gene was located within the 2.8-kb fragment. Based on information gained during the determination of the rec7 nucleotide sequence (see below), a 1.6-kb MluI-BamHI fragment was isolated from the 2.8-kb fragment and inserted into the BamHI site of pSP2 to form pYL45 and pYL46, containing the fragment in opposite orientations. Both subclones complemented rec7-102 (Table 3, lines 18 and 19). This 1.6-kb fragment was sequenced and shown to contain the rec7 coding region (see below). Subclones such as the 1.2-kb BglII fragment (see restriction sites in Figure 1) failed to complement rec7-102 (data not shown); because the deduced rec7 open reading frame is contained within this 1.2-kb fragment, we conclude that this fragment lacks essential control regions at one or both ends of the gene.

The 3.9-kb SacI fragment of pKL2, containing the

80 Y. Lin et al.

TABLE 3

Genetic complementation by plasmids with the rec7 and rec8 genes

ade6-M26 parent ade6-52 parent Recombinants/lO' viable sporesa

Line GP# rec Plasmid GP# rcc Ade+ Arg' Pro'

1 363 + - 369 + 950-2600(5) 45,000-1 10,000(4) 2 43 1 7-102 - 42 1 7-102 1 1400 3 663 7-102 pKL 1 42 1 7-102 2600-4000(4) 75,000-88,000(2) 4 663Wb 7-102 - 42 1 7-102

Meiotically Induced rec Genes 81

181 " U I C A ~ T C T I T T A l ? " A T A ~ ~ TMC N K S S A Q ~ P N L L L ~ P S Y I P M T Q T A T T A V N N S PO

211 A ~ A ~ ~ T G X A ~ T Q C I C A " T " C U 2 A ~ T N Y V N P A P L Q H V Y P N A E I P S N T P P L K R F U G 120

D A G Y T ~ Y P L R S D T S ~ E ~ ~ T A S Q ~ P T ~ D E N ~ 150

V I T S S P F N P N R N A Y S Y Q A N S ~ Y P I I A A T P L ISO

N S Q T Q A S W V A Q P E N ~ A Y A N L I P S P P T T S ~ I 210

301 G A ~ T U K C C M A ~ T A C A T C A A " T ~ TaMKwm

451 l ? l U A T ~ ~ A A T I X C M ~ A A T G C i T ~ ~ T ~ l T A ~ ~

541 1 T A C G C A M T P N A ~ P T C

121 ~ A ~ T G T ~ C A C M X C " A A ' I C C C A M T P P ~ A U I X M " A M T S L E R V W N K Y . 240

811 C . A ~ T C G G M ~ A ~ ~ " l n W X M ~ ~ l T ~ ~

901 T A ~ T ~ ~ n l T C A C A T T A ~ P T A T Q A ~ l T A

PO1 ~~""~""

FIGURE 1.-Nucleotide sequence of the rec7 gene and the deduced amino acid sequence of its product. Numbers on the left refer to the nucleotide sequence, 1 being the first base of the rec7 open reading frame.. Numbers on the right refer to the amino acid sequence. The nucleotide sequence was determined independently from both strands, except for nucleotides 529 and 530 (GC), which were determined only from the encoding strand. The TAG corresponding to the translational stop is indicated by an asterisk. The MluI site in the putative 5' regulatory region (see DISCUSSION), the BglII sites defining a noncomplementing fragment (data not shown), and the possible 3' polyadeny- lylation signal 5'AATAAA3' (HUMPHREY et al. 1991) are underlined. The 9-bp repeat of the rec7-150::TnlO-Kan insertion in pRD5 is doubly underlined.

TABLE 4 Leu+ Rec- integrant was designated GP82 1 (h- adeb Genetic linkage of integrated rec7 and e& LEU2 plasmids 52 urd-294 leul-32 rec7-146::TnlO-LLK). The rec7-

the reef and rec8 genes 146 insertion mutation, like the previously isolated

Number of meiotic segregap with indicated phenotype

Gene integrated" Isolate Rec+Leu+ Rec-Leu- Rec-Leu+ Rec+Leu- Plasmid

rec7 pYL5 GP857 7 9 0 0 GP858 3 13 0 0

rcc8 pYL7 GP855 7 9 0 0 GP856 7 9 0 0 GP948 5 3 0 0

" Described in Table 2 and MATERIALS AND METHODS. Strain GP720 (h- ade6-52 leul-32 ura4-294) was transformed

with the indicated linearized plasmids, derivatives of plasmid YIp32. Stable Leu+ transformants were purified and mated with strain GP722 (h+ ade6-M26 led-32 red-102) or GP723 (h' ade6-M26 led-32 rec8-110). Meiotic segregants were tested for Rec and Leu its described in MATERIALS AND METHODS.

insertions in or near the rec7 gene were isolated based on restriction digestions and failure to complement rec7-102 (data not shown). From one derivative, pYL54, a 10.8 kb BamHI fragment containing the 8- kb TnlO-LLK insert was isolated and used to trans- form strain GP720 (rec' leul-32) to Leu+. A stable

nitrosoguanidine-induced rec7 mutations (PONTICELLI and SMITH 1989), abolished intragenic recombination at the ade6 locus and was recessive (Table 3, lines 24 and 25). Southern analysis of genomic DNA prepared from strain GP82 1 demonstrated the restriction diges- tion patterns expected for a homologous substitution (data not shown). Corresponding experiments with rec8 have been hindered by the lack, in the pYL55::TnlO-LLK derivatives, of appropriate restric- tion sites needed to generate a linear fragment for homologous replacement.

Nucleotide sequences of the ree7 and rec8 genes: The 1.6-kb rec7 fragment and the 2.1-kb rec8 frag- ment were sequenced on both strands. The templates were produced by ExoIII-generated deletions or from TnlO insertions. On each fragment, there was a single large ORF (Figures 1 and 2). These ORFs correspond to polypeptides of 249 amino acids with a molecular mass of 28 kD for rec7 and 393 amino acids with a molecular mass of 45 kD for rec8, respectively. TnlO- Kan insertions within these ORFs (see Figures 1 and

82 Y. Lin et al.

1 A - P - T A P T I C l ' A C T R X T A l l l U A ~ T A W A W . M S S F T 4 P K C N P N I B V L C T L P D S T S Y L I N T S 30

91 C " T I ' A T " Q M A C M ~ ~ " 4 N Y S L R N N V S S F V Y E D S R A F S T C C P L D F E F 60

181 Q A ~ T A T C C M C 3 M ~ A T A M ~ ~ P T A Q C " D E N Q D I Q E L T K Q T I N S D P S L 4 A A S 4 H S N L Q 90

211 120

381 T W % A ~ A T W A ~ T M W T G U ~ P -m L 4 S V M D S E H N E N E P R A L K R R K V 4 K L L C P D C 150

451 180

541 O ~ I ' ~ ~ ~ " ~ ' W X U ~ ~ ~ T C I ~ A ~ R R Q A S S A K K K E L N K F F D W E S P H P L L K P I I E 210

691 ~ ~ T ~ ~ T Q T A " I ' I U A T P CGGGAm K L K P S N N T P S C I D D V L R N I D T S C V C V Q R D V 240

I21 " l T " A C A T ~ T " l X 3 C Q A ~ T A M " M W C A ~ ~ 4 Q E L G L N I P W N T S S R S N S A I N S K S H S 4 T Q S 210

811 lztaXP A M T A T A ~ T N X C C A T C G A ~ A Q W T A Q " E H S T P L L D T K Y R K R L P H S P S M P S R V C F L P A 300

901 TI'AGh4"" T I ' A V " T A T ~ T A C ' E " L E S S 4 F H C T L N S E L S L 4 L S D D F V L Y K N T 4 E 330

991 380

1081 A T M C A ~ A l T A ~ ~ ~ ~ ~ ~ l T T A ~ l T T ~ ~ A ~ ~ ~ I T F S S L L P N D L K R P V V A Q A F S H L L C K Y H N Q 390

1111 A ~ ~ ~ M ~ T ~ ~ ~ ~ ~ A ~ ~ T A M ~ T ~ T I ' ~ T T N * 993

1261 ~ ~ ~ ~ ~ ~ ~ C l T ~ ~ ~ ~ T l T C Q M A A ~

1351 T G U ~ ~ ~ ~ A ~ ~ ~ ~ A M T ~ A T ~ A T ~ ~ T I ' M T G U ~ T M ~ ~

1441 T A M T M ~ - ~ M l T ~ A ~ ~ ~ A ~ M T I ' A ~ A T A ~ - ~ A T A ~ A ~ A C A T

1531 A A ~ T A ~ T A ~ T M ~ ~ ~

FIGURE 2.-Nucleotide sequence of the rec8 gene and the deduced amino acid sequence of its product. Numbers on the left refer to the nucleotide sequence, 1 being the first base of the ret8 open reading frame. Numbers on the right refer to the amino acid sequence. The nucleotide sequence was determined independently from both strands. The TAA corresponding to the translational stop is indicated by an asterisk. The MluI sites in the putative 5' regulatory region (see DISCUSSION) and the possible 3' polyadenylylation signals 5'A(T/A)TAA(T/ C/A)3' (HUMPHREY et al. 1991) are underlined. The 9-bp repeats of three rec8::TnlO-Kan insertions in pRD3, pRD4, and pRD2 located at nucleotides -245 to -253, 508-516, and 1007-1015, respectively, are doubly underlined. All of these insertions abolished the r e d gene function (data not shown).

2) abolished complementation of the appropriate rec mutation (data not shown), verifying that these ORFs are required for rec gene function.

Induction of the rec7 and recd genes in meiosis: Identification of DNA segments within the rec7 and rec8 genes allowed preparation of hybridization probes to assay rec7 and re68 mRNAs. RNA was prepared from cells of strain GP48 (pa t l -114 end l - 458) before and after induction of meiosis by temper- ature-shift; the pat l -114 allele encodes a temperature- sensitive repressor of meiosis (IINO and YAMAMOTO 1985). The RNA was analyzed by Northern hybridi-

zation (Figure 3), using as probes DNA fragments within the rec7 and rec8 ORFs or a plasmid bearing the S. pombe c y gene (RUSSELL and HALL 1982); the latter was a control for yield and integrity of mRNA.

For both rec7 and rec8, mRNA was most abundant at 3 hr after meiotic induction; lower levels were detected at 2 h, but no mRNA was detectable at 0 , 4, 5 or 6 hr. Neither mRNA was detectable in RNA prepared 0-6 hr after temperature shift of the pa t l+ strain GP19 (data not shown), indicating that the induction was not due to heat shock. The c y mRNA level increased slightly during meiosis, but this RNA

Meiotically Induced rec Genes a3

Tlme (h) 0 2 3 4 5 6 Time (h) 0 2 3 4 5 6 A ” -,-. . .. , .. . . B - I , . . . . I

*5 kb rec7

1.3 kb rec8 - 0.7 kb cyc * I

FIGURE 3.-Northern analysis of the rcc7 and the rcc8 mRNAs. RNA was extracted from strain GP48 (pat1-114 end1-458) at the indicated number of hours after shift to 34’ to induce meiosis, fractionated by gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with radioactive probes as described in MATERIALS AND METHODS. (A) Hybridized with the 590-bp BstNI- Sfyl fragment internal to rcc7 ORF (Figure 1). (B) Hybridized with the 61 6-bp BamHI-HaeII fragment internal to the rcc8 ORF (Figure 2) and a plasmid pPOCYC(O.54) containing the S. pornbe cyc gene (RUSSELL and HALL 1982). The upper bands crossing the width of the filter in both panels are presumably due to hybridization be- tween chromosomal DNA and the gene probes. The approximate sizes of the mRNAs, determined from ’*P single-stranded DNA markers (not shown), are indicated.

was readily detectable and apparently intact at each time point. The apparent sizes of the S. pombe cyc and rec8 mRNAs were a few hundred nucleotides longer than their ORFs, but that of the rec7 mRNA was, unexpectedly, about 4 kb longer than its ORF. Hy- bridization with a radioactive probe extending from about 0.5-2.5 kb 3’ of the rec7 ORF revealed an RNA with a size and meiotic induction pattern indistin- guishable from that revealed by the rec7 ORF probe (Figure 3A), whereas this RNA was not detected with a probe extending from about 0.5-1.0 kb 5’ of the rec7 ORF (data not shown). These results suggest that the rec7 mRNA has an unexpectedly long 3‘ end.

DISCUSSION

We have cloned the S. pombe rec7 and rec8 genes using a procedure for enrichment of rec+ clones among selected prototrophic recombinants. Since there is no known condition under which S. pombe meiotic rec genes are essential for growth, a direct selection for these clones was not possible. Although the enrichment procedure successfully yielded the desired rec clones, these were outnumbered by ade6 clones about 100 to 1. We expect that, using a library made with DNA from an ade6 deletion strain, the enrichment procedure described here will efficiently yield clones for the seven other rec genes in which mutations strongly reduce recombination (classes I and 11; DEVEAUX, HOACLAND and SMITH 1992).

The clones described here contain the rec genes. This was demonstrated by the locus specificity of their complementation (Table 3), by genetic linkage of homologously integrated plasmids with the rec gene

in question (Table 4), and, for rec7, construction of a chromosomal gene disruption based on the cloned gene (Table 3).

The nucleotide sequences of the smallest comple- menting subclones contained single large ORFs. These ORFs are the coding regions of the rec genes, since transposon insertions within them abolished rec gene complementing activity. The ORFs predict poly- peptides of 249 amino acids for rec7 and 393 amino acids for rec8, respectively (Figures 1 and 2). We found no significant homology between these polypeptides and others previously reported, including those of numerous S. cerevisiae genes involved in meiotic re- combination, nor did we find significant homologies with protein blocks (HENIKOFF and HENIKOFF 1991). These results suggest that the S. pombe rec7 and reed gene products have novel biochemical activities.

The rec7 and rec8 genes contain MZuI sites (5’ACGCGTS’) in the region 5‘ of the ORFs (Figures 1 and 2). In S. cerevisiae these sequences are the sites of action of transcriptional activators of DNA metab- olism genes that are under mitotic celkycle control (ANDREWS and HERSKOWITZ 1990; GORDON and CAMPBELL 199 1 ; LOWNDES, JOHNSON and JOHNSTON 199 1 ; L. BREEDEN, personal communication). These sites may regulate the S. pombe rec7 and rec8 genes, which are transiently expressed during the first meiotic division (Figure 3; SZANKASI and SMITH 1992). rec8 contains three MluI sites, located 330,601 and 630 bp 5’ of the ORF; a transposon insertion 300 bp 5’ of the ORF abolished complementation of a rec8 mutation (Figure 2; data not shown). Therefore, nucleotide sequences in the neighborhood of the three MZuI sites appear to be required for expression of the rec8 gene. The single MZuI site near rec7 appears not to be required for the expression of the rec7 gene, since destruction of the MZuI site to form plasmids pYL45 or pYL46 left these plasmids able to comple- ment rec7 mutations (Table 3).

The apparent size of the rec8 mRNA (about 1.3 kb; Figure 3) was about the size of its ORF (1 179 nucle- otides; Figure 2), but the apparent size of the rec7 mRNA (about 5 kb; Figure 3) was much larger than its ORF (747 nucleotides; Figure 1) or of the smallest complementing subclone (1.6-kb fragment in pYL45 and pYL46; Table 3). The rec7 mRNA appears to have an unexpectedly long 3’ end (data not shown). We saw no evidence of mRNA splicing (Figure 3), but low levels of spliced mRNA may have escaped detec- tion. There is a precedent for meiotic-specific splicing of mRNA from a yeast meiotic recombination gene: the S. cerevisiae MER2 mRNA, whose product is re- quired for meiotic recombination, is spliced by the MER2 gene product (ENGEBRECHT, VOELKEL-MEIMAN and ROEDER 1991). The meiotically induced MER2 mRNA extends about 2 kb beyond the 3’ end of its

a4 Y. Lin et al.

ORF (ENGEBRECHT and ROEDER 1990), but the role of this RNA at the 3’ end, like that of the rec7 mRNA, is unknown.

The rec7 and rec8 genes were only transiently ex- pressed during meiosis: mRNAs for both genes were abundant at 3 hr after induction of meiosis but were undetectable before induction or at 4 hr and later (Figure 3). This meiotic-specific expression can ac- count for the meiotic-specific phenotype of rec7 and rec8 mutants: meiotic intragenic recombination is re- duced about 1 000-fold (PONTICELLI and SMITH 1989), but mitotic intragenic recombination is not signifi- cantly affected (N. HOLLINGSWORTH, personal com- munication; K. L. LARSON, unpublished data). The rec7 and rec8 genes are maximally expressed slightly after premeiotic DNA replication, which occurs at maximal rate 2-2.5 hr after meiotic induction, but well before induction of exonuclease I, at 4.5 h, or nuclear divisions, at 5-7 hr (SZANKASI and SMITH 1992). It is noteworthy that exonuclease I activity, which has been postulated to be involved in meiotic recombination, and the rec7 and rec8 mRNAs are apparently unstable and return to their premeiotic levels within about 1 hr after being induced (SZANKASI and SMITH 1992; Figure 3). These observations sug- gest that the meiotic developmental program is largely intact in the temperature-shifted p a t l mutant (IINO and YAMAMOTO 1985) used here. Meiotic recombi- nation, including action of the M26 recombination hotspot (GUTZ 1971), is induced in the pat1 mutant (BAHLER et al . 1991; A. S. PONTICELLI and H. CLARKE, personal communications). Thermally induced pa t l mutant cells therefore may be a fruitful source to investigate the temporal control of recombination ac- tivities during meiosis. The transient expression of rec7 and rec8 early in the meiotic program suggests that their gene products may be involved primarily in the early steps of meiotic recombination.

We especially thank LOUISE CLARKE and JOHN CARBON for a sample of the S. pombe DNA library, NANCY KLECKNER and RICH- ARD KOLODNER for phage and plasmids and advice on their use, and HOWARD CLARKE for a pool of cells transformed with the S . pombe library. We thank PHILIPPE SZANKASI for helpful advice on experiments; DAVID BEACH, LINDA BREEDEN, LINDA DEVEAUX, HOWARD CLARKE, BEN HALL, NANCY HOLLINGSWORTH, J*G KOHLI, FRED PONTICELLI and JEFF VIRGIN for unpublished data, plasmids, and strains; and SUE AMUNDSEN, DENISE CLARK, STEVE HENIKOFF, SUE HOLBECK, KURT RUNGE, ANDREW TAYLOR and WAYNE WAHLS for helpful discussions. R.D. was supported by a Medical Scientist Training Program fellowship (GM 07266) from the National Institutes of Health, and Y.L. by funds provided to the Fred Hutchinson Cancer Research Center from the Edel Peter- son Baker Guild. This research was supported by grants from the National Institutes of Health to G.R.S.

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Communicating editor: F. WINSTON