Molecular and Genetic Analysis of REC103, an Early Meiotic Recombination Gene in Yeast

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CopyTight 0 1997 by the Genetics Society of America

Molecular

and

Genetic

Analysis

of

RECl03,

an

Early Meiotic

Recombination Gene

in

Yeast

Jack

M. Gardiner, Steven A Bullard, Chris Chrome

and

Robert

E. Malone

Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242 Manuscript received January 27, 1997

Accepted for publication May 6, 1997

ABSTRACT

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A

new early recombination gene RECl03 is described in this paper. It was initially defined by the reclO3-l

mutation found in a selection for mutations overcoming the spore inviability of a rad52 spol3 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. reclO3 spol3 strains produce viable spores; these spores show no evidence of meiotic recombination. reclO3 SP013 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., RECI02, REC104, REC114, MEI4,

hfER-2,

and SPOII). REC103 maps to chromosome VI1 between ADE5 and RAD54. Cloning and sequencing of

REC103 reveals that REC103 is identical to SK18, a gene that depresses the expression of yeast double- stranded (“killer”) (ds)RNA viruses. REClO3/SKI8is transcribed in mitotic cells and is induced -15-fold in meiosis. RECI03 has 26% amino acid identity to the Schizosaccharomycespombe recl4+ gene; mutations in

both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.

M

EIOSIS may be thought of as an intracellular “de- velopmental” process that is essential in most eukaryotes. During meiosis high levels of recombination, chromosome pairing, and a reductional division take place to ensure that new combinations of genes are produced and accurately packaged into viable haploid gametes (e.g., BAKER et al. 1976). Failure to undergo these processes in an orderly manner leads to missegre- gation of chromosomes, resulting in aneuploid and inviable meiotic products. The budding yeast Sacchare myces cerevisiae has played an important role in elucidat- ing many of the key events in meiosis (ROEDER 1995). Clever selections and screening schemes, a relatively synchronous and easily inducible meiosis, extensive genetic markers, and efficient transformation have all contributed to making yeast an excellent system for studying meiosis. These features have allowed a detailed characterization of many of the events in yeast that occur during prophase I, including genetic recombination.

The sf1013 mutation is a useful and unique tool for the study of meiotic recombination in yeast. Mutations in the SP013 gene cause a “single division meiosis” (KLAPHOLZ and ESPOSITO 1980; HUGERAT and SIMCHEN 1993) generating two diploid spores (a “dyad”) instead of the usual four haploid spores. Dyads produced in

spol3 diploids display normal levels of recombination

Corresponding authm: Robert Malone, Department of Biological Sci- ences, University of Iowa, Iowa City, IA 52242.

E-mail: [email protected]

(KLAPHOLZ and ESPOSITO 1980). In strains containing a mutation in a gene required early in recombination

( . g . ,

R A D S U , SPOII) essentially every division (>99%) is equational, and viable spores are produced (e.g., MA- LONE and ESPOSITO 1981; MALONE 1983; KLAPHOLZ et al. 1985). The genes involved in meiotic recombination can be placed into two classes based on double mutant phenotypes in combination with $013 (MALONE 1983; PETES et al. 1991). Mutations in “early” recombination genes produce viable spores in combination with a

spol3 mutation, whereas mutations in genes required “later” in recombination (e.g., RAD52) produce inviable spores whether a spo13 mutation is present or not. It is possible to divide the early class of genes into two subclasses, which have been referred to as Early Ex-

change (“EE”) and Early Synapsis (“ES”) genes (MAO-

DRAAYER, et al. 1996). Mutations in EE genes completely eliminate intra- and interchromosomal meiotic recombination and are epistatic to rad52 mutations; mutations in ES genes reduce interchromosomal recombination only an average of 10-fold, do not affect intrachromoso- mal recombination, and are not epistatic to rad52 mutations. Included in the EE class are RAD50 (GAME et al. 1980; MALONE and ESPOSITO 1981), SPOlI (KLAPHoLz et al. 1985), ME14 (MENEES and ROEDER 1989), REC102 (MALONE et al. 1991; COOL and MALONE 1992), REC104 (MALONE et al. 1991; GALBRAITH and MALONE 1992),

MEYE (ENCEBRECHT et al. 1990; COOL and MALONE 1992), REC114 (MALONE et al. 1991; PITTMAN et al. 1993), XRS2 (IVANOV et al. 1992), and MREl1 (AJIMURA et al. 1993). Included in the ES class are R E D 1 (ROCK-

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1266 G a r d i n e r et al.

TABLE I

Yeast strains, plasmids, and lambda clones

Yeast strain Genotype Reference

RM69

JMG2-15

RM217

RM218

MATa spol3-1 Iys2-2 tyrl-l his7-1 canl ura3-13 ade5 metl3-d CYH2 trp5-2 leul-12

e

MALONE and Esposno (1981) MATa spol3-l lys2-I tyrl-I his7-2 CANl ura3-1 ade5 metl3-c q h 2 trp5-c leul-c ade2-l

-

RM69 except reclti3::URA3 rec103:rURA3

JMG2-15 except SP013::LYS2

SP013::LYS2

JMG2-15 except rad52::ADE2 rad52::ADEZ

This study

RM186

" MATa spol3-l lys2-2 tyrl-l his7-1 canl ura3-13 ade5 metl3-d CYH2 trp5-2 leul-12 This study

MATO spo13-I lys2-I tyrl-I his7-2 CAN1 ura3-1 ade5 metl3-c cyh2 trp5-c leul-c ade2-l

RM219 MATa ade5 reclti3-l metl3-d CYH2 trp5-2 ade6 lys2-2 tyrl-1 HIS7 c a n l u r d 1 ade2-1 This study MATa ade5 reclti3-l m~tl3-c ryh2 tq5-c ADE6 LYS2 tyrl-2 his7-2 CANl ura3-I3 ade2-l

MATa spol3-1 ADE5 reclO3-1 metl3-c cyh2 trp5-c leul-c ade6 tyrl-l canl ura3-52

ade2-l

This study MATa spol3-l d e 5 recl03-l metl3-d CYH2 tq5-c leul-c ADE6 F 2CANl ura3-52 a&-1

cc1-8

"

CCl-10

" MATa spol3-l ADE5 recl03-I metl3-c cyh2 trp5-c leu]-c tyrl-l CAN1 ura3-52

ade.2-1 This study

MATa spol3-1 add reclti3-1 metl3-d CYH2 trp5-2 LEU1 F 2CANl ura3-1 ade2-1

C3-16 MATa $1013-l metl3-c q h 2 tvp5-c leul-c ade6

tyrl-l

CANl ura3-52

e

MATO spol3-1 metl3-d CYH2 trp5-2 leul-12 ADEG tyrl-2 CAN1 ura3-1 ade2-1

" Cool. and IMAI.ONE (19%)

JMG2-13 rad52::UR43

md52::URA3

CCl-IO except This study

JMG2-9 M4Ta reclO3-l metl3-d cyh2 trp5-2 tyrl-2 his7-1 ALE1 MtTa reclO3-I metl3-c CYH2 tq5-c tyrl-2 his7-2 adel

JMG2-5 MA7i a d d recIO3-I RAD54 metl3--d CYH2 tq5-2 lys2-2 tyrl-1 ura3-1 canl MATa ADE5 REC103 rad54-3 MET13 CYH2 TRP2 LYS2 TYRl ura3-1 CANl

JMG2-17 MATa rad50::URA3 lys2-2 tyrl-2 his7-l CANl ura3-l ade2-l

MATa rad50::URAj lys2-1 tyrl-l his7-2 canl ura3-1 ade2-1

This study

Plasmids Description References

pRS3 16 E N 6 ARSH4 URA3 Amp' SJ~CORSKI and HEWER (1989)

pSportl Amp' BRL (Bethesda, MD)

pSM22 pBR322 containing rad52::URA3 for one step gene replacement of RAD52 MALONE (1983)

pBM2240 CEN6 ARSH4 URA3 Ampr (gap rescue plasmid for rescuing A clones with MG3 backbone) ERICKSON and JOHNSTON

pBM2906 CEN6 ARSH4 U R 4 3 Amp' (gap rescue plasmid for rescuing A clones with MG14 backbone) ERICKSON andJOHNSrON pMJG6.8H3/H3R pRS316

+

6.8 kb Hind111 fragment from A 3141 (contains RECl03) This study

pJMG6.8H3/H3L Identical to pJMG6.8H3/H3R but inserted into pRS316 in opposite orientation This study pJMG4.3H3/Sp pRS316

+

4.3 kb HindIII-SpeI fragment from pJMG6.8H3/H3R (contains IUXlti3) This study

pJMG2.1H3/ER pi6316

+

2.1 kb EcoRI-Hind111 fragment from pJMG4.3HS/Sp This study

pJMG2.2 Sp/ER pRS316

+

2.2 kh EcoRI-SpeI fragment from pJMG4.3H3/Sp This study

pJMGREClOSD1-1 pJMG4.3H3/Sp with the 1.2 kb BsaBI-BgZII fragment deleted This study pJMGREC103Dl-2 pSportl

+

4.3 HzndIII-SpeI fragment with a 1.2-kb BsaBI/BglII deletion and 1.1-kb URA3 This study

pRM272 pJMG4.3H3/Sp gap rescue plasmid of the reclO3-l allele (BsaBI-BglII fragment). Does not This study (1993)

( 1993)

fragment inserted to create the reclti3::UR43 disruption plasmid.

complement a reel03 mutation.

Lambda clones Description Source

m a 5 A T C C n o . 70103 A c l o n e c o n t a i n i n g ADE5 ATCC (Rockville, MD)

x4703 ATCC no. 70302 X clone contigous and c e n t r o m e r e p r o x i m a l t o X2985 ATCC (Rockville, MD)

X3141 A T C C n o . 70127 X clone contigous and centromere proximal to h4703. ATCC (Rockville, MD)

X1207 ATCC no. 70010 X c l o n e c o n t i g o u s a n d c e n t r o m e r e p r o x i m a l to X3141. ATCC (Rockville, MD)

Contains R E C l 0 3

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REClO? and Meiotic Recombination 1267

MILL and ROEDER 1988), HOPl (HOLLINGSWORTH and BYERS 1989), and h4EKl (ROCKMILL and

ROEDER

1991). Antibodies to both HOPl and €?.ELI1 have been shown to interact with the synaptonemal complex (HOLLING- SWORTH et al. 1990; cited in NAG et al. 1995).

The rescue of a mutation in a gene required late in exchange (i.e., an “LE” gene such as RAD52) by an EE mutation (in the presence of $1013) was used to isolate

177

mutants defective in the initiation of meiotic recombination (MALONE et al. 1991). An examination of 56 of these mutants revealed mutations in all the EE genes known at the time, as well as three new early meiotic recombination genes (MALONE et al. 1991). In this paper, we present a characterization of a new recombination gene isolated from this selection for mutations in EE genes. This new gene RECl03 is required for meiotic gene conversion and crossing over and is not required for mitotic recombination. In the process of restriction mapping and sequencing RECl03, we found it to be identical to Sm8, a gene required for the suppression of yeast killer dsRNA expression (MAT- SUMOTO et al. 1993). Recently, the SKI8 gene product has been discovered to play a role in suppressing the translation of non-polyA mRNAs (MASISON et al. 1995). One possible connection between this observation and early meiotic recombination events is presented in the DISCUSSION.

MATERIALS AND METHODS

Strains, plasmids, and lambda clones: All strains, plasmids, and lambda clones used in this study are listed in Table 1. All yeast strains used were constructed in this laboratory. Yeast strains were either grown on rich medium (YPD) or GURA (synthetic complete medium lacking uracil) (SHERMAN et al.

1979). YPA and sporulation media have been previously described by MALONE et al. (1991).

Lambda clones A2985, A4703, A3141, and A1207 (ATCC nos. 70103, 70302, 70127, and 70010, respectively) were ob-

tained from the American Type Culture Collection in Rock- ville, MD, as were the cotransformation plasmids pBM2240 and pBM2906 (ATCC nos. 87021 and 87022). Lambda DNA was prepared according to the method of AUSUBEL et al.

(1995). Subcloning was performed according methods described in SAMBROOK et al. (1989). Mating, diploid isolation, and tetrad analysis were carried out as described by SHERMAN and WAKEN (1991). Transformation was done by either the spheroplast (HINNEN et al. 1978) or by the lithium salts (ALJSU-

BEL et al. 1995) method. All integrations into yeast were verified by Southern analysis and by genetic tests.

Recombination measurements: Meiotic crossing over in

spol? diploids was initially measured at the heterozygous drug- resistance markers CAN1 and CYH2 by the frequency of drug- resistant colonies. Although drug-resistant colonies may arise by several mechanisms, crossing over accounts for the majority of these in a spol? meiosis (MALONE 1983). Crossing over was

also measured in detail by ascospore dissection from dyads. Meiotic gene conversion was measured by allowing diploids

to sporulate and measuring the frequency of prototrophs at heteroallelic loci, which is predominantly due to gene conversion (PETES et al. 1991). Mitotic recombination was measured as previously described (e.g., COOL and MALONE 1992).

Cloning and molecular analysis of REG103 RECIO? was

cloned by complementation of reclO3-1 and direct extraction of the 17-kilobase (kb) insert contained in A3141 using the method described by ERICKSON and JOHNSTON (1993).

REC103 was also contained in A1207, which shared at least a 2.2-kb region of overlap with A3141. A 6.8-kb Hind111 fragment from A3141 inserted into the Hind111 site of pRs316 (pJMG6.8H3/H3R or pJMG6.8H3/H3L) was able to complement the reclO3-1 mutation. The pJMG4.3H3/Sp plasmid was derived from pJM6.8H3/H3R by digesting with SpeI to remove a 2.5-kb fragment and recircularizing the vector. This repre- sented the minimal restriction fragment that could complement the reclO3-l mutation. The pJMGP.lHS/ER plasmid was constructed by inserting the EcoRI/HindIII fragment from pJMG.8H3/H3L into the EcoRI/HindIII sites of pRS316. The pJMG2.2Sp/ER plasmid was constructed by inserting the 2.1-kb EcoRI/SpeI fragment from pJMG4.3H3/Sp into the HindIII/SpeI sites in pRS316. pJMGREClO3DI-1 was created by digesting pJMG4.3H3/Sp with BglII, filling in with Klenow, digesting with BsaBI to remove a 1221 base pair (bp) fragment, and recircularizing the vector. This 1221-bp deletion removed 119 bp upstream from the ATG start site and all but the last 90 bp (30 amino acids) of the RECIO? open reading frame. The plasmid for one-step gene disruption was created by first inserting the 4.3-kb HindIII-SpeI fragment from pJMG6.8H3/H3L into pSportl. The resulting pJMGREClO3 was then cut with BglII, filled in with Klenow fragment to create a blunt end, and digested with BsaBI to remove the REG103 open reading frame. A 1-kb SmaI URA3 fragment was then ligated into the BsaBI-BglII sites (converted in to blunt ends by Klenow polymerase treatment) to create pJMGREC103Dl-2. This vector was used to create

reclo?:: URA? strains.

Expression of REG103 in mitosis and meiosis: To determine the expression pattern of RECl 03 in mitosis and meiosis, RNA was prepared from meiotic and mitotic cultures as described by COOL and MALONE (1992). RNA concentrations were determined by spectrophotometry, and the concentra- tion was checked on test gels by ethiduim bromide staining of ribosomal bands. If necessary, concentrations were readjusted and verified on a second gel. A 1492-bp BsuBI-Ssp1 fragment encompassing the entire 1191 bp open reading frame (- 119 to +1373) was labeled by random priming (Bethesda Re- search Laboratories) and used as a REClO? probe. All North- ern filters were analyzed on a model 445SI Molecular Dynam- ics Phosphor Imager according to instructions specified by the manufacturer.

RESULTS

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1268 Gardiner et al.

TABLE 2

Sporulation and viability of diploids containing recZO3 mutations

Diploid

name Genotype

Percentage sporulation Spore viability No. of spores

(%)

(%I

examined

RM219 reclO3-1 1 <1.8 14 asci

RM21'7 reclO3::URA3 8 <0.5 54 asci

C3-16 REG1 03 ~p013-1 54 64 399

JMG2-15 recl 03::URA3 $1013-1 75 81 80

cc1-8 reclO3-1 spol3-1 35 61 160

CCl-10 reclO3-1 spol3-1 56 65 156

RM186 REG1 03 rad52-1 spol3-1 9 0 160

RM218 reclO3::URA3 rad52::ADE2 spol3-1 63 68 100

JMG2-13 reclO3-1 rad52::URA3 spol3-1 39 65 108

At least 200 cells of three independent clones of each diploid were counted to determine sporulation percentage. Spore viability was determined by dissection of spores except for RM219 and RM217. For these two Rec- diploids whole asci were placed on the agar and no growth from any of the asci was detected. The percent viability is calculated as <[1 + (4 X no. of asci)

1.

ous points along the meiotic pathway like cells containing other EE- mutations such as reclO4. To verify that spol3 diploids with reclO3 mutations can generate live spores, double mutant strains were sporulated and dissected (Table

2).

The data clearly indicate that reclO3 spol3 strains produce live spores. To verify that reclO3 can rescue rad52 spol3 diploids, reclO3-1 spol3 rad52 and reclO3:: URA3 spol3 rad52 diploids were examined. The data in Table 2 demonstrate that reclO3 is epistatic to rad52 for both sporulation percentage and for spore viability. We conclude that mutations in the REClO3 gene act like typical mutations in other known EE genes (see Introduction).

Mitotic phenotypes: To test whether REClO3 is required for recombination during mitosis, the frequency of spontaneous mitotic recombination was measured at several loci on four chromosomes. The data presented in Table 3 show that neither reclO3-1 nor reclO3 :: .!IRA3

affect mitotic recombination. The values observed are not significantly different from the wild-type control.

The early exchange genes RADS0 (MALONE 1983), M R E l 1 (AJIMURA et al. 1993), and XRS2 (IVANOV et al. 1992) are required for normal mitotic growth rates and for DNA repair; we therefore tested whether RECl03 is required in these processes. Figures 1 and 2 demonstrate that neither reclO3-1 nor reclO3:: URA3 strains are sensitive to U V or to the radiomimetic agent methyl methanesulfonate (MMS). Likewise, the reclO3-1 and reclO3::URA3 mutations do not affect mitotic growth rate; the generation times in W D medium at 30" of strains with reclO3-1, reclO3::URA3, or REClO3were 83, '78, and 80 min/generation, respectively. Taken to- gether, these data argue that REClO3 is not required for normal rates of DNA repair, mitotic recombination, or growth. This is similar to what has been observed for the EE genes REClO2, REC104, RECl14, m I 4 , SPOll, and MER2 (see Introduction).

Meiotic recombination: Mutations in the EE genes previously studied in this laboratory [REC102and MER? (COOL and MALONE 1992), REClO4 (GALBRAITH and

TABLE 3

Spontaneous mitotic recombination in reclO3 diploids

Drug-resistant colony

frequency [ ~ 1 0 ~ 1 Prototroph frequency [ X lo5] Diploid Relevant No. of

name genotype cultures - cyh2 __ can1 his7-1 tyrl-1 lys2-I uru3-1 metl3-c leul-c tq5-c

CYHZ CAN1

-

has7-2 tyrl-2 lys2-2 ura3-13 metl3-d leul-d tq5-d

-

- -

RM69 REC103spol3-1 5 2.4 % 1.2 4.9 t 4.9 0.5 2 0.2 0.3" 0.6 2 0.2 0.9 L 0.2 2.5 ? 0.7 12.4 t 3.7 10.3 i- 3.3

JMG2-15 recIO3::URA3 5 9.4 ? 3.3* 9.4 t 3.8 0.2 % 0.1 ND 0.2 2 0.1 ND 2.1 t 0.8 8.3 t 4.9 5.2 t 5.5 CC1-8 reclO3-1 spol3-1 3 3.2 ? 0.5 3.8 t 1.3 ND 0.2 t 0.1 ND ND 4.0 ? 1.4 ND ND

CCl-10 reclO3-1 spol3-l 3 3.9 t 2.1 ND ND 0.1 ? 0.1 ND 2.3 t 0.2 4.7 t 1.9 ND 2.0 t 0.1*

(1) (1) (1) (1) (1) (1) (1) (1) (1)

spol3-1 (3.9) (1.9) (0.4) (0.3) (0.8) (0.7) (0.5)

(1.3) (0.8) (0.7) (1.6)

(1.6) (0.3) (1.3) (1.9) (0.2)

~~ ~~

The values shown are the means t SD. Values given in parentheses represent the relative value of the Ret- strain as compared to the Rec+ RM69 strain. *Significant difference from RM69 at the 95% confidence level using the unpaired Student's t test. ND, not determined. RM69 data is from MAO-DRAAYER et al. (1996).

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RI.:C103 and Meiotic Recombination 1 2 69

0

-1

h

0

t

=

-2

d.

L

a

0

2 -3

v, -4

.-

3

-5

0 50 100 150 200

UV

Dose

(Joules/m2)

FI(X.RE I.-RI.:ClO3 is not needed for UV-excision repair. Five haploid strains were irradiated at 2.2 J/m'/sec to give the LR' dose U/m') indicated: m d l - 2 ( + ), Rec' (W), Rec'

(V),

r r c l O 3 : : l ~ l w 3 ( O ) , rrc103-I

(A).

Thc two Rec' llaploids are the " A a ( W ) and MATa (V) parent of RM69, the rr-

c103::URA3 haploid is the M A 7 9 parent ofJMG'2-1.3, and the

rrclO3-1 haploid is the A4A'la parent o f CC1-8. The m d l - 2 haploid is a U\'-sensitive control and is strain RM143-9R; its genotype is the same as the MATa parent o f RM69 except that i t is SP013.

h%\I.OXE 1992), and M C l I 4 ( P I I T M A N et nl. 1993) ] dra- matically decrease meiotic recombination at all loci tested. To determine if this was also the case for

RECI03, we examined meiotic gene conversion at six loci on several chromosomes (Table 4). Both the rerl03-I and rec103:: URA3 mutations reduce meiotic gene conversion to a degree similar to that seen for mutations in the other EE genes [e.g., RECl04 (GAL.-

BR.\ITH and MALONE 1992)l. Since the heterozygous

recessive drug-resistance markers cnnl' and qh2' moni- tor crossing over between the resistant locus and the centromere, the data in Table 4 also suggest that meiotic crossing over in recl03::URAJ and rec103-I strains is reduced several hundredfold. The meiotic recombination frequencies measured in the r ~ c l 0 3 strains are, in fact, not significantly different that the normal spontaneous mitotic recombination values (compare Tables

3 and 4). To obtain a more direct measurement of meiotic crossing over, recl03-1 sp013 diploids were sporulated, dyads were dissected, and recombination was measured at several loci on four chromosomes (Table

5 ) . The results clearly demonstrate no evidence of meiotic crossing over in rerl03-I s p o l 3 diploids. Addition- ally, \'en/ few aberrant dyads were observed, which is consistent with the proposal that high levels of recombination preceding an equational division increase the frequency of aberrant segregation (MALONE and ESPOS

ITO 1981 ; MALONE 1983). All these results are exactly what would be expected for a mutation in an EE gene.

Mapping and cloning of

RECZO3

During the genetic analysis of rec103-I, w e noticed that the mutation was linked to ADZ3 on chromosome VII. To verifi this, a diploid heterozygous for RI.:C103, A D f 3 , and liAD54

was analyzed. The double recombinants from the three- factor analysis of this cross indicated that RI:'C103 was located behveen A I X 5 and liAD54. Tetrad analysis indicated that Rl~CI03was 11.9 cM from ADfi3 (10 tetratype, 42 parental, and 0 nonparental tetrads) and 50.2 CM from RAD54 (29 tetratype, 13 parental, and 2 nonparental tetrads).

Determination of a precise genetic location for

xI:'CI03 allowed u s to clone RI:C103 b y gap rescue of yeast genomic inserts contained in appropriate A clones (Table 1). M'e took advantage of the yeast physical map (RIIXS PI 01. 1993) and the method described by ERI(:K-

SON andJ0HSSTOS (1993). A region centromere proximal to ADK5 contained in the contiguous A clones A2985, A4703, A3141, and A1207 was examined by cotransformation (ERICKSOS and JOIISSTOS 1993) into a

rec103-I diploid (RM219) and tested for complementation of the rec103-1 mutation. The DNA present in hvo

contiguous A clones, A3141 and A1207, complemented the recl03-1 mutation equally well. The minimal region of overlap for these two clones was a 2.2-kb fihR1- HindIII fragment. This is the minimal overlap because the A clones were constructed from SmAA partial di- gests and can contain additional DNA up to, but not including, the adjacent fi;coRI o r HindIII site. The minimal fragment capable of complementing the rec103-1

mutation (Figure 3 ) was a 4.3-kb SpI-Hind111 fragment

YPD

MMS

RM69

JMG2-15

I

JMG2-17

CC1-8

FIGURE 2.-IU*X,'/O? is n o t nccdcd 1 0 1 . SIllS rcsistallcc. Patches of diploids RM69 (lire*), JMG'L-15 (rrc103::1'1L43), JMGP-I7 (rad5O-l), and CC1-8 (rrc103-I) wcre grown on YPD and replica plated to either YPD (left) or YPD

+

0.024% Mh4S (right). For each diploid (the middle patch) the MA'la parent is o n the left and the MATa parent is on the right. Note that

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1270 Gardiner et al.

TABLE 4

Meiotic recombination in red03 diploids Drug-resistant colony

frequency [x104] Prototroph frequency [X105]

Kelevant N O . O i

name genotype cultures

-

cyh2 __ canl ““” his7-1 tyrl-l lys2-1 ura?-1 metl?-c h l - r

-

trp5-c

CYH2 CAN1 his7-2 tyrl-2 lys2-2 ura3-13 metl?-d leul-d t1p5-d

RM69 spo13-1 5 2400 3400 117 ND 14 102 4300 1600 3400

JMG2-15 reclO?::URA3 5 8.3 16.8 0.4 ND 0.3 ND 1.8 6.5 4.8

CC1-8 reclO3-1 spo 13-1 3 5.9 5.6 ND 0.2 ND ND 4.7 ND ND

CC1-10 reclO?-1 spol?-l 3 2.4 ND ND 0.7 ND 1.3 2.2 ND 1.1

(78) (1954) (3090)

Recombination values shown are the mean of the number of cultures shown. All SDs were <35% of the mean. The numbers in parentheses represent the reduction in recombination relative to the Rec+ (RM69) strain. Values for Rh469 were taken from MAO-DRAAYER et al. (1996). The recombination frequencies shown for the reclO3 strains are not significantly different from the

(1) (1) (1) (1) (1) (1) (1) (1)

spol?-l (289) (202) (292) (47) (2388) (246) (708)

(406) (607) (914)

(1000)

mitotic recombination frequencies shown in Table

3.

(pJMG4.3H3/Sp) containing a centrally located EcoRI

site. This site was used to create the two subclones pJMG2.1H3/ER and pJMG2.2Sp/ER, neither of which was able to complement a reclO3 diploid for meiotic recombination, sporulation, or spore viability (Figure 3). These results imply that the centrally positioned

EcoRI site in pJMG4.3H3/Sp is within REClO3.

Sequence analysis of REClO3: To obtain sequence information on RECIO3, sequences were obtained for the two regions flanking the EcoRI site described above (see also Figure 3). A GenBank search of the sequenced region surrounding the EcoRI site revealed a match for SKI8, a gene that suppresses the propagation and expression of dsRNA viruses (MATSUMOTO et al. 1993).

S I u 8 genetically maps to the REClO3 region on chromosome VII. The restriction enzyme map (for BgYII, BamHI, EcoRI, and X b d ) generated for REClO3 is the same as that reported by MATSUMOTO et al. (1993) for

SKIS. In addition, a subclone (pJMGREC103Dl-1) that removed 119 bp upstream from the ATG and all but the last 30 amino acids (see MATERIALS AND METHODS) of the SKIS open reading frame does not complement a reclO3 mutation (Figure 3). MATSUMOTO et al. (1993)

noted the presence of a second, smaller possible open reading frame (“cORF1”) on the antisense strand of

SKI8 that is transcribed in the opposite direction as

SKI8 To exclude the possibility that this small open reading frame might be RECIO?, the entire cORFl and its promoter region were subcloned into the plasmid pJMG2.2Sp/ER. This plasmid could not complement a reclO? mutation (Figure 3). Taking all the data to- gether, we conclude that R E C l O? and SKI8 are the same gene.

Mutational analysis: To determine the nature of the reclO3-1 mutation, a 1221-bp BsaBI-BgZII fragment was gap rescued (ROTHSTEIN 1991) from a reclO3-1 strain. The resulting plasmid (pRM272) was unable upon re- transformation to complement the reclO?-I mutation, indicating that the reclO3-I mutation was contained within the 1221-bp BsaBI-BgZII fragment. Sequencing revealed a change at amino acid 352 (TTA to TAA), changmg a leucine into a stop codon (ochre); we pre- sume this is the relevant mutation in reclO3-I. This results in the loss of 46 amino acids from the carboxy terminus and suggests that this region is important for REClO? function. Three other sequence changes were

TABLE 5

Analysis of meiotic crossing over in red03 spol3 diploids

No. of dyads (P:RA)

V I 1 V I1 111

Relevant

Diploid genotype met 13 cyh2 v 5 canl ura? 571 MAT

C3-16 REClO? spol?-l ND 50:18:13 (22) 5617:8 (21) 53:22:4 (28) 61:13:7 (16) ND 59:13:15 (15) CC1-8 redo?-l spol?-1 44:0:2 (<2.1) 44:0:2 (<2.1) ND 46:O:O (<2.1) ND 46:O:O (<2.1) 44:O:O (<2.1) CC1-10 reclO?-1 spol3-1 54:O:O (<1.8) 54:O:O (<1.8) 540:O (C1.8) ND 540:O (<1.8) 54:O:O (<1.8) 540:O (<1.8)

Diploids were sporulated and dyads were dissected and the spore segregants were tested. The chromosome on which the markers are located is indicated by a roman numeral above the locus name. Dyads may be classified as parental (P), recombinant (R), or aberrant (A) (see MALONE 1983). Numbers in parentheses represent the percent R type asci. If no R type asci were observed, the percent R asci is calculated as <1 f (total number of dyads examined). The data for C3-16 was taken from COOI.

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REG103 and Meiotic Recombination 1271

H X B X E A G _{%Sporulation %Viability Recombination}

-

REC103

H s 34

pJMG4.3H3ISp

H

I

d

E

pJMG2.1 HWER 1

.o

E

I

1

S

.o

pJMG2.2SplER

H

u

B

A

us

1

.o

pJMGREC103A1

I

1 K b I

noted upstream of the reclO3-1 mutation that we attri- bute to strain polymorphisms. (We have noted polymorphisms between our strains and the yeast genome data- base for other REC genes.) The three changes are an alanine to threonine (GCT to ACT) at amino acid 73, a glutamic acid to glycine (GAA to GGA) at amino acid 180, and a glycine to glycine (GGT to GGC) at amino acid 268.

of RECl03 expression: Mutations in REC103 affect meiotic recombination and REC103 is identical to SKI8, which affects killer dsRNA in mitosis. These observations suggest that RECl03 should be expressed in both mitosis and meiosis. Northern analysis of RNA isolated from diploids grown exponentially in YPA and in sporulation media indicate that RECl03 is expressed in mitosis and in meiosis (Figure 4). To estimate the degree of induction during meiosis, the URA3 and RECl03 bands were quantitated and the RECl03/URA3 normalized value was calculated. Induction begins at

-2

hr after transfer to sporulation medium, reaching a peak of -15-fold induction at 8 hr, consistent with REC103 playing a role in early meiotic recombination.

DISCUSSION

The reclO3-1 mutation was isolated in a selection for mutations that could rescue the meiotic lethality of a haploid rad52 spol3 strain (MALONE et al. 1991). This selection originally resulted in the identification of three new early meiotic recombination genes in addition to the isolation of new alleles of all other known EE genes. Both the reclO3-1 and the reclO3:: URA3 null mutation confer the same phenotypes. The data presented here clearly demonstrate that REC103 is a gene that may be placed in the early exchange class along with SPOll, ME14, REC104, etc. (see Introduction and

71

+++

FIGURE 3.-Restriction map for a 4.3-kb fragment that complements the

reclO3-1 mutation. The meiotic phenotype (percent sporulation, viability, and recombination) is shown for the various subclones in pRS316. In all cases the recombination phenotype (+

+

+,

normal levels; -, no induction of meiotic recombination was observed) was determined by examination of at least five different loci. The arrow represents the direction of transcription and the coding region of REC103. S, SpeI; X, XbaI; B, BsaBI; E, EcoRI; A, BumHI;

G, BgnI; H, HzndIII.

MAO-DRAAYER et al. 1996). Mutations in REC103 confer the phenotypes that define a gene as a member of the EE class. (1) Diploids with a reclO3 mutation have reduced sporulation and produce inviable spores.

(2)

red03 spol3 strains produce viable spores. (3) Both reclO3-1 and reclO3::URA3 spol3 diploids are completely deficient in both meiotic gene conversion and crossing over at numerous loci on several chromosomes. (4) r e d 0 3 rad52 spol3 diploid strains produce viable spores ( i e . , r e d 0 3 is epistatic to rad52). We have detected no defects in mitotic recombination, growth rate, or DNA repair in r e d 0 3 strains. The failure of r e d 0 3 mutations to affect these mitotic processes exclude REC103 from the subgroup of EE genes [RAD50

(MALONE 1983), XRS2 (IVANOV et al. 1992), and M R E l l

(AJIMURA et al. 1993)] that are also required for recombination-repair in mitosis. Mutations in all three genes in this subgroup also result in elevated levels of spontaneous mitotic recombination and slow growth rates.

The evidence presented in this paper indicates that RECl03 is identical to SKI8. For example, a plasmid containing a 1221-bp deletion that removes the SKZ8

promotor and all but the last 30 amino acids of the SKI8

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1 2 i 2 Gardiner 111.

A)

RECI

03 EA 2 4

6

8 10 12 24

u . 3

EA 2 4

6 8 10 12 24

e RECIO3lURA3

rn RECI03

0 5 10 15 20 25

Time in Meiosis

FICCRF: 4.-RI:CIO? is expressed in mitosis and meiosis. Total RNA was isolated from a RM69 (Rec') yeast diploid after exponential growth in YPA immcdiately before exposure to sporulation medium (EA lane, 0 hr) or at various times after transfer from W A to sporulation media (2, 4, 6, 8, 10, 12, and 24 h r ) . All lanes contained 5 pg of total RNA (see \l.ATERIAIS ASD wrrrons) except for lane EA, which was over- loaded threefold (-15 p,g) in order to obtain a detectable signal for REClO?/SKT8. A 1492-bp BsnBI-.%pI fragment con- taining the entire RI;ClO?/SK18 open reading frame was used as a probe. A 1.1-kb Hind11 Ufi\? fragment containing the entire coding region was used as a prohc for URA?. (A) North-

ern blot probed with RKCIO3or UlU3. The RECIO? transcript is -1.6 kb (markers not shown). (D) Induction of R I X l O ? / SKI8 during meiosis. The left axis is the ratio (e) of RECIO? counts divided by the URA? counts at the same time (see \l;\TERIAI.S ASD \fETIIODS). The right axis gives the number of actual counts detected for W;CIO? ( W ) at each time point.

tion occur normally or raclO3 spo13 strains would not produce viable euploid spores. Second, reclO3 is epistatic to r(rd52, indicating not only that the defect conferred is in recombination, but also is in the early part of meiotic recombination.

IECIO3is expressed in both meiotic and mitotic cells.

This finding differs from other genes in the EE class that are only expressed in meiosis [P.R., ME14 (MENEES and ROEDER 1989), mc102 (COOI. and MALONE 1992), RI:'C104 (GALRRAITH and MALONE 1992), XEC114 (PITT- M A N et nl. 1993), and

SPOII

(ATCHESON at 01. 1987)l. xI:'cI03 expression appears to peak at -8-10 hr into meiosis and is induced -15-fold. Expression, relative to mitosis, remains elevated throughout the 24hr meiotic time course. The expression pattern is consistent with the induction of meiotic recombination in our strains, which begins at -4 hr, as well as with the induction patterns of other genes required early in meiotic recombination [a.g., xI.:CIO2 (COOL and MALONE 1992)].

The recent cloning and molecular analysis of the .Schizosncchnromvces p o m h rac14+ gene (EVANS et nl. 1997) has revealed that there is 26% amino acid indentity between

XECIO?

and rac14'. The racl4' gene was defined by one of several very tight Rec- mutations isolated by PONTECELLI and SMITH (1989) and DEVEAUX and SMITH (1992). Like REClO3, S. pomha rac14' mutations essentially eliminate meiotic recombination and spore viability. Also like RECIO3, racl4' is expressed in mitotic cells, although at lower levels than in meiosis. The conservation of amino acid sequence, in addition to the similar meiotic mutant phenotypes, suggest that rec14' and RECIO? may play similar roles in meiotic recombination.

The discovery that

IECIO3

is identical to SKI8 raises a number of interesting questions as to the function of XECl03 in meiotic recombination. Mutations in SKI genes were first isolated by their ~ u p e r ~ l l e r phenotype (TOH-E et al. 1978). Superkiller strains secrete increased levels of a toxin that kills sensitive cells (M'ICKNER 1996).

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REC103 and Meiotic Recombination 1273

indication of any meiotic recombination in recl03 mutants. Of course, it is also possible that the R E C l 0 3

gene product acts directly in the initiation of meiotic recombination.

Finally, we note that there have been hints of interac- tions between the SKIgenes and genes potentially involved in meiotic recombination. The SEPl (also known as

XRNI,

DSTZ, KEMI, S K I l , and

RAR5)

gene codes for a multifunctional protein that includes within its complex repertoire of activities DNA strand exchange activity; BAEHLER et al. (1994) have shown that cells with a mutation in SEPI arrest in pachytene in meiosis. In a recent mitotic screen for mutations that were syntheti- cally lethal in combination with mutations in SEPI, mutations in SKI2 and SKI3 were identified (JOHNSON and KOLODNER 1995). The lethality of sepl ski2 and sepl ski3

strains was independent of the M dsRNAvirus. JOHNSON and KOLODNER (1995) argued that these observations support the idea SKI2 and SKI3 have important roles in cellular metabolism other than the repression of dsRNA virus levels, consistent with the proposal of MASISON

et al. (1995). The finding that recl03/ski8 strains are

specijically deficient in meiotic recombination provides additional evidence that the SKIgenes in general, and

SKI8 in particular, may have essential roles in cellular events not directly related to the dsRNA killer virus replication and expression.

We thank WILLIAM ALLEN who provided technical assistance in the early phases of this project. We express our thanks to members of our lab who read and commented on the manuscript, including MAUREEN

CANTWELL, GEORGE HALLEY, KAI JIAO, YANG MAO-DRAAYER, JOHN NAU, and LAURA SALEM. This research was supported by National Institutes of Health (NIH) grant ROI-GM-36846 and March of Dimes grant 1- 1099 to R.E.M. J.M.G. was supported by NIH postdoctoral fellowship F32-GM-17160. C.C. was supported by a Howard Hughes summer undergraduate fellowship administered by the Department of Biologi- cal Sciences.

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