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Copyright 0 1989 by the Genetics Society of America

Yeast

merl

Mutants Display Reduced Levels

of

Meiotic Recombination

JoAnne Engebrecht and

G.

Shirleen Roeder

Department of Biology, Yale University, New Haven, Connecticut 0651 1

Manuscript received August 5 , 1988 Accepted for publication November 5 , 1988

ABSTRACT

Mutations at the MERl locus were identified in a search for meiotic mutants defective in chromo- some segregation. merl strains show decreased levels of inter- and intrachromosomal meiotic recom- bination and produce inviable spores. The MERl gene was cloned by complementation of the spore inviability phenotype. Strains carrying disruptions of the MERl gene are mitotically viable. The epistatic relationships between MERl and previously characterized meiotic genes are described.

M

EIOSIS is a special type of cell division whereby

a diploid cell gives rise to four haploid progeny. Two important events occur during meiosis: genetic recombination and chromosome segregation. These events lead to the generation of new genotypes and ensure the faithful transmission of genetic informa- tion during sexual reproduction.

Two nuclear divisions occur during meiosis. At meiosis I, the reductional division, homologous chro- mosomes disjoin from each other. At meiosis 11, the equational division, sister chromatids separate from each other and four haploid cells are generated. T h e morphological events that occur during meiosis have been well characterized (for a recent review, see MOENS 1987); however, very little is known about the underlying mechanism of chromosome segregation.

Chromosome pairing and meiotic recombination are essential for proper chromosome segregation at meiosis I (BAKER et a l . 1976). T h e pairing of homol- ogous chromosomes is mediated by a protein network called the synaptonemal complex (SC) (VON WETT- STEIN, RASMUSSEN and HOLM 1984). Exchange may be mediated by recombination nodules, proteinaceous structures observed along the paired homologues in association with the SC. A correspondence between the distribution and frequency of recombination nod- ules and those of meiotic exchange events indicates that nodules may be the sites of localization of recom- bination enzymes (CARPENTER 1975).

Many mutants have been isolated that are defective in pairing, recombination or both. T h e c ( 3 ) G muta- tion in Drosophila melanogaster abolishes meiotic cross- ing over and causes a high degree of first division nondisjunction (HALL 1972). Furthermore, there is no SC observed in oocytes of c(?)G females (SMITH and KING 1968). Other recombination-defective mu- tants of Drosophila (mei-218, mei-41; reviewed in BAKER et al. 1976) appear to make normal SC (CAR- PENTER and BAKER 1974) but the distribution of cross- overs is altered such that some chromosomes fail to recombine and consequently undergo nondisjunction.

Genetics 121: 237-247 (February, 1989)

Meiotic mutants defective in pairing and recombi- nation have also been isolated in the yeast, Saccharo-

myces cerevisiae. T h e HOPI gene is believed to encode

a component of the SC; hop1 mutants fail to pair homologous chromosomes, display reduced levels of interhomologue recombination and produce inviable spores (HOLLINGSWORTH and BYERS 1989). T h e

rad50 (MALONE and ESPOSITO 198 1) and s p o l 1 (KLA-

PHOLZ, WADDELL and ESPOSITO 1985) mutants fail to initiate meiotic recombination. FARNET et al. (1988) failed to detect SC in nuclear spreads from rad50

mutants. DRESSER, GIROUX and MOSES (1986) also observed no SC in nuclear spreads from strains car- rying a disruption of the SPOl I gene. However, KLA-

PHOLZ, WADDELL and ESPOSITO ( 1 985) have reported

the appearance of SC in thin sections prepared from s p o l l - 1 mutants. Other yeast mutants initiate but cannot complete genetic exchange (rad52, rad57;

CAME 1983) and, at least in the case of rad52, appear to make normal SC (GIROUX 1988). All of these yeast mutants produce inviable meiotic products.

Pairing and recombination do not ensure the proper segregation of homologous chromosomes. In maize (desynaptic; MAGUIRE 1978), Drosophila (ord, meiS??2, G 6 7 ; BAKER et al. 1976) and yeast (DZSI,

ROCKMILL and FOGEL 1988; red I , ROCKMILL and ROE- DER 1988), mutants have been isolated that are recom- bination-proficient but defective in meiotic chromo- some segregation.

T o further elucidate the molecular events occurring during meiosis, a search for new yeast spore inviability mutants was undertaken. Such a mutant screen is expected to detect mutants defective in chromosome pairing, recombination and/or segregation. We re- port here the isolation and characterization of mutants defective in meiotic recombination. T h e gene respon- sible for the mutant phenotype is called MER1 (for MEiotic Recombination).

MATERIALS AND METHODS

Strains: Yeast strains are listed in Table 1. karC2-4 was obtained from NANCY HOLLINGSWORTH and Y20 from

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238 J. Engebrecht and G . S. Roeder

TABLE 1

Yeast strains

BR1824-3B

J 1

J9

BR1824-5B

BR2171-7B

BR2265-6B

514

5 1 5

J'S

JE77-4C

JE103-1B

593

594

Y20

J l O O

5102

5104

MATa HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 MATa HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1

"

-

lys2

MATa HO trpl-I arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 merl-1 MATa HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 marl-1

-

""

M A T a H O t r p l - I arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 merl-2 M A T a H O t r p l - 1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 merl-2

MATa leu2 his4-290 HO ar~4-8 CUPl ade2-1

""

MATa leu2 his4-290 HO arg4-8 CUPl ade2-1

MATa leu2 HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 MATa leu2 HO trpl-1 arg4-8 thrl-4 CUP1 ura3-1 ade2-1

M A T a his4-280 spol3::URA3 CUPl trpl-289 ura3-1

M A T a his4-290 spol3::URAjr thrl-4 CUPl trpl-1 ura3-1 MERl ade2-1 MATa HIS4 spol3::URA3 thrl-4 CUP1 trpl-289 ura3-1 MERl ADEZ

"

""

M A T a his4-290 sbo13::URA3 thrl-4 CUP1 trbl-1 ura3-1 merl-1 ade2-1 LYS2

1""

MATa HIS4 spol3::URA3 thrl-4 CUPl trpl-289 ura3-1 merl-1 ADEZ lys2

M A T a his4-290 spol3::URA3 thrl-4 CUPl trpl-1 ura3-1 MERl ade2-1 LYS2

MATa HIS4 spol3::URA3 thrl-4 CUP1 trpl-289 ura3-1 merl-1 ADE2

"

"

-

lys2

MATa leu2 HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 merl::LEU2 MATa leu2 HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 merl::LEU2

"

MATa HO trpl-1 arg4-8 thrl-4 CUPl ura3-1 ade2-1 lys2 mer1::LYSZ M A T a H O t r p l - 1 arg4-8 t h r l - 4 CUP1 ura3-1 ade2-1 lys2 merl::LYS2

-

"

-MATa leu2 HIS4 sbol3::URA3 thrl-4 CUP1 trbl-289 CYHlO lvs2 M E R l MATa leu2 his4-280 spol3::URA3 THRl CUP1 trpl-289 cyhIO-100 L Y S 2 M E R l

MATa leu2 HIS4 spol3::URA3 thrl-4 CUPl trpl-289 CYHlO lys2 merl::LEU2 MATa leu2 his4-280 spo13::URA3 T H R l C U P l t r p l - 2 8 9 cyh10-100 LYS2 merl::LEU2

M A T a C R Y l leu2-I 12 his4-260,34

M A T a cryl leu2-3 his4-280 spol3::TRPl trpl ura3-52 ade2-1 lys2

MATa CRYl leu2-112 his4-260,34

MATa cryl leu2-3 his4-280 spol3::TRPI trpl ura3-52 ade2-1 lys2 mer1::LYSZ

MATa CRYl leu2-112 his4-260,34

MATa' y y l leu2-3 his4-280 spol3::TRPI trpl ura3-52 ade2-1 1y52 spo1l::ADEP

MATa CkY1 leu2-112 his4-260.34

M A T a cryl leu2-3 his4-280 spol3::TRPl trpl ura3-52 ade2-1 lys2 rad5O::ADE2

TOM MENEES. BR1824-3B, BR1824-5B, BR2171-7B and BR2265-6B were obtained from BETH ROCKMILL. Bacterial strains YMClOrecA (MCCARTER and SILVERMAN 1987) and R895 (YMClOrecA transduced to pyrF::KanR with P1; this study) were used.

Genetic procedures: Media were prepared and yeast ma- nipulations were carried out according to SHERMAN, FINK and HICKS (1986). Copper-containing medium has been described (ROCKMILL and ROEDER 1988). Mutagenesis and ether screen for spore viability were performed as described by ROCKMILL and ROEDER (1988). Yeast transformations were carried out according to SHERMAN, FINK and HICKS (1986). All transformants were verified by SOUTHERN (1 975) blot analysis. J114, a lys2 derivative of karC2-4 (HOL-

LINGSWORTH and BYERS 1989) was obtained by selection on

a-aminoadipate as previously described by CHATTOO et al.

( 1 979). This allele was designated lys2-99.

Plasmid constructions: Plasmids were constructed by standard procedures (MANIATIS, FRITSCH and SAMBROOK

1982). T h e original M E R 1 -complementing plasmid was des- ignated pME1. pME4 was constructed by inserting a 2.5- kbp EcoRI-BglII fragment from pMEl into the EcoRI and

BamHI sites of the vector pHSS6 (SEIFERT et al. 1986). The

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Yeast mer1 Mutants TABLE 1-Continued

239

Strain Genotype

J l 0 6

J l 0 8

J110

J112

karC2-4"

J114"

5115"

5117"

J119"

JE24-18C

5121

5122

5123

5124

5125

J126

5127

5128

MATa CRYl leu2-112 his4-260,34 MATa cryl leu2-3 his4-280

MATa CRYl leu2-112 his4-260,34 MATa cryl leu2-3 his4-280

MATa CRYl leu2-112 his4-260,34 M A T a cryl leu2-3 his4-280

MATa CRYl leu2-112 his4-260,34 MATa cryl leu2-3 his4-280

MATa CDClO leu2 his4 MATa cdcl0 LEU2::pNHIB-I HIS4

MATa CDCl0 leu2 his4 MATa cdcl0 LEU2::pNH18-1 HIS4

MATa CDCl0 leu2 his4 MATa cdcl0 LEU2::pNH18-1 HIS4

spol?::TRPl trpl ura3-52 ade2-1 lys2 mer1::LYSZ spol1::ADEZ

spol3::TRPl trpl ura3-52 ade2-1 lys2 merl::LYS2 rad5O::ADEZ

spol3::TRPl trpl ura3-52 ade2-1 1y52 hop1::ADEZ

spol3::TRPl trpl ura3-52 ade2-1 lys2 mer1::LYSP hopl::ADE2

ura? trpl canl cyh2 ade2-1 spol3-1 sap3

ura? trpl canl cyh2 ade2-1 spo13-1 sap3 lys2-99

ura? trpl canl cyh2 ade2-1 spol3-1 sap3 lys2-99 merl::LYS2

MATa CDC1O leu2 ura? t r p l c a n l cyh2 ade2-1 spol3-1 sap3 lys2-99 hop1::ADEZ MATa cdcl0 LEU2::pNH18-1 HIS4

MATa CDCl0 leu2 his4 MATa cdcl0 LEU2::pNHlB-1 HIS4

MATa his4-1176,864 pBR313 his4-39,260 leu2-3,112 merl-1

MATa leu2 his4 spol3::URA? thrl-4 trpl-289 lys2 M A T a l e u 2 his4 spol3::URA3 THRl trpl-289 lys2

MATa leu2 his4 spol3::URA? thrl-4 trpl-289 lys2 merl::LYS2 MATa leu2 his4 spol3::URA? T H R l t r p l - 2 8 9 lys2 merl::LYS2

MATa leu2 his4 spol1::TRPI spol3::URA3 thrl-4 trpl-289 lys2 MATa leu2 his4 spol1::TRPI spol3::URA3 THRl trpl-289 lys2

MATa leu2 his4 spol?::URA3 thrl-4 trpl-289 lys2 rad52:TRPl M A T a l e u 2 his4 spol3::URA3 THRI trpl-289 lys2 rad52::TRPI

MATa leu2 his4 sPol3::URAJ thrl-4 trpl-289 lys2 mer1::LYSZ rad52::TRPI M A T a l e u 2 his4 spo13::URA3 T H R l t r p l - 2 8 9 lys2 merl::LYS2 rad52::TRPI

MATa leu2 his4 spol1::TRPl spol3::URA3 thrl-4 trpl-289 l& rad52::LEU2 MATa leu2 his4 s p o l 1 : : T R P l s p o l 3 : : U R A 3 T H R l t r p l - 2 8 9 lys2 r a d 5 2 : L E U 2

MATa leu2 his4 spol1::TRPl spol3::URA3 thrl-4 trpl-289 lys2 mer1::LYSZ M A T a l e u 2 his4 s p o l 1 : T R P l s p o l 3 : : U R A 3 T H R l t r p l - 2 8 9 lys2 merl::LYS2

MATa leu2 his4 spol1::TRPl spol3::URA3 thrl-4 trpl-289 lys2 mer1::LYSZ rad52::LEU2 M A T a l e u 2 his4 s p o l 1 : : T R P l s p o l 3 : : U R A 3 T H R l t r p l - 2 8 9 lys2 merl::LYS2 rad52:LEU2

ura3 trpl canl cyh2 a d e 2 - 1 4 0 1 3 - 1 5ap3 lys2-99 mer1::LYSP hop1::ADEZ

"

"

"

"

"

"

a In strains karC2-4, J114, J115, J117 and J119, pNH18-1 refers to the plasmid containing URA3 and CYH2 which was integrated into chromosome I l l to create an 1 1.4-kbp duplication of sequences that lie between H I S 4 and LEU2 (HOLLINGSWORTH and BYERS 1989). Strains are heterothallic unless otherwise indicated.

(FLEIG, PRIDMORE and PHILIPPSEN 1986) to create pME6. ADE2 fragment from Yp3.6 Ade to create pME303 and pGB324 contains the s p o l l ::TRPl allele and was obtained pME301, respectively. T h e centromere plasmid containing from CRAIG GIROUX. pME302, which contains the M A T a , pA2, was obtained fromJEFF STRATHERN.

s p o l l : : A D E 2 allele, was constructed by digesting pCB324 with EcoRI and BglII and inserting the BamHI-EcoRI frag-

ment of the ADE2 gene from Yp3.6 Ade (obtained from RESULTS JEFF LEMONTT). T h e rad50::ADEB and the hopl::ADE2 al-

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240 J. Engebrecht and G. S. Roeder

chromosome pairing, recombination or segregation should result in the random segregation of homolo- gous chromosomes at the first meiotic division. In organisms with a large number of chromosomes (in yeast, n = 16), the aneuploidy resulting from random segregation is lethal to the majority of gametes. Therefore, mutants defective in meiotic chromosome disjunction should produce inviable spores.

Spores from a homothallic ( H O ) strain, BR1824- 3B, were mutagenized with UV to 50% survival. T h e spores were allowed to form diploid colonies, homo- zygous for any induced mutations. These colonies were sporulated and then exposed to ether vapors. Vegetative cells are more sensitive to ether vapors than spores (DAWES and HARDIE 1974); therefore, colonies in which 90% or more of the spores are inviable are ether-sensitive. T h e spore inviability phe- notype was verified by tetrad dissection. Of approxi- mately 20,000 colonies screened, 12 meiotic lethal mutants were recovered.

T h e phenotypes of two of the mutants isolated are shown in Table 2. J1 and J9 produce spores with a reduced frequency and most of the spores are inviable. T o determine whether the spore inviability phenotype is due to random segregation of chromosomes during meiosis, the degree of aneuploidy among the rare viable spores was measured. BR1824-3B has one copy of the CUPl gene on each chromosome VZZZ homolog. The level of copper resistance is a function of CUPl gene dosage; therefore, spores that inherit an extra chromosome VZZI are resistant to higher concentra- tions of copper than euploids (ROCKMILL and FOCEL

1988). Chi-square tests showed that there is a signifi-

cant difference in chromosome VZZZ aneuploids among

wild-type spores compared to the rare viable spores

derived from strains J1 and J9 at P 0.005 (Table

Many mutants defective in DNA repair produce inviable spores (rad50, 51, 52, 53, 54, 55, 57; GAME

1983). These mutants are sensitive to gamma rays and

MMS because they are unable to repair the DNA

damage induced by these agents. Strains J1 and J9

were tested for their sensitivity to the DNA damaging agent, MMS. BR1824-3B, J1 and J9 are resistant to concentrations of MMS that inhibit the growth of rad52 strains (data not shown).

T o determine if the meiotic phenotypes of strains

J1 and J9 are due to single mutations, spores from J1

and J9 were mated to spores from a wild-type HO strain, BR1824-5B; the resultant diploids were spor- ulated and tetrads dissected. T h e individual diploid spore colonies were sporulated and tested for ether sensitivity. In ten four-spore-viable tetrads from each cross, ether sensitivity segregated 2:2, indicating that single mutations are responsible for the spore invia- bility phenotypes in both mutants.

Haploid (ho) tester strains carrying the mutations

2).

TABLE 2

Phenotypes of mer1 strains

Percent

Relevant Percent spore Percent CuR Strain eenotvpe soorulation" viabilitvb colonies'

BR1824-3B M E R l

MER 1

J1

-

m e r l - 1

J9

-

merl -2

merl-1

merl -2

514 M E R l spo13

M E R l spol3

515 merl-1 spol3 merl-1 spol3

JE77-4C merl::LEU2

merl::LEU2

JElO3-IB merl::LYS2

merl::LYS2

90 92 0.1 (1/1120)

22 2 4 (23/587)

20 1 6 (37/622)

52 63 ND

31 75 ND

24 1 ND

22 2 ND

Percent sporulation was determined by light microscopy. Four-

spored asci were rarely formed in mutant strains.

* Percent spore viability was determined by dissectinga minimum of 25 tetrads or 50 dyads.

Spores were isolated (LAMBIE and ROEDER 1988) and plated onto rich solid medium. After 2-3 days of growth at 30", the

resultant colonies were replicated to synthetic complete medium containing 0.1 mM CuS04. The number o f spore colonies examined and the number of C U R segregants are indicated in parentheses. N o mitotic CuR colonies were detected. N D = not determined.

were constructed to facilitate further genetic analysis. Spores from J1 and J9 were mated to the haploid (ho) strain, BR2265-6B; the diploids were sporulated and tetrads dissected. ho segments were scored for the mutant phenotype by crossing M A T a segregants to M A T a segregants. T h e resultant diploids were spor- ulated and the spores tested for viability. These crosses showed that the mutations in J1 and J9 are recessive. Complementation tests performed with hap- loid mutant segregants from each cross showed that the mutations are allelic. T h e mutations in strains J1

and J9 are designated m e r l - 1 and m e r l - 2 , respectively. spol3 rescue: spol3 mutants have previously been shown to bypass the first meiotic division and complete a single, primarily equational division resulting in the production of two diploid spores (KLAPHOLZ and ES-

POSITO 1980). Many mutants defective in processes

necessary for the first reductional division ( i e . , spol

I,

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Yeast merl Mutants 24 1

0.005). Therefore, it seems likely that the defect in mer1 strains is at or prior to the first meiotic division.

Complementation tests: T o determine whether the

mutations in J 1 and J9 define a previously undescribed gene, complementation tests with other meiotic lethal mutants that are s p o l 3 rescued ( s p o l l , r a d 5 0 , h o p l , r e d l ) were performed. In all cases, the mutations complemented, indicating that a new meiotic gene has been identified.

Cloning the MERl gene: MERI was cloned from a

yeast genomic library in a centromere-containing plas- mid marked with the U R A 3 gene (ROSE et al. 1987) by complementation of the spore inviability pheno- type of strain J l . O n e Ura+ transformant of approxi- mately 3000 screened produced viable meiotic prog- eny. Spontaneous mitotic Ura- segregants n o longer generated viable spores. Plasmid DNA was recovered in Escherichia coli and found to contain a 15-kbp insert. This plasmid (pME1) was re-transformed into the original mutant strain, J1, and all of the resulting transformants produced viable spores. Complement- ing activity was localized to a 2.5-kbp EcoRI-BglII fragment by subclone analysis. A restriction map of this fragment is shown in Figure 1A.

Further localization of the MER1 complementing activity was achieved by transposon mutagenesis (SEI- FERT et al. 1986). Insertions of m-Tn3(URA3) or m- T n 3 ( L E U 2 ) were generated in E. coli and plasmids containing these insertion mutations were introduced into the HO strain BR2171-7B by substitutive trans- formation (ROTHSTEIN 1983). T h e resulting hetero- zygous diploids were sporulated and tetrads dissected. Ura+ and Leu+ segregants were sporulated and the resultant spores were examined for viability. These insertion mutations delimit the MERI gene to an interval of approximately 1 kbp (Figure 1A).

Figure 1B shows the maps of two deletions con- structed in vitro using the cloned MERI gene. T h e LEU2 deletion was constructed from a transposon insertion (pME162) and results in the loss of approx- imately 600 bp of MERI DNA. A LYS2 fragment was used to replace approximately 300 bp of MER1 se- quences (see MATERIALS AND METHODS). These dele- tions were used to substitute for the wild-type se- quences in the yeast genome by transformation of HO strains BR2 17 1-7B (with pMEAl62, containing merl::LEU2) and BR1824-3B (with pME6, containing merl::LYS2). T h e heterozygous diploids were sporu- lated and tetrads dissected. T h e tetrads segregated 2+:2- for either leucine or lysine prototrophy. Segre- gants containing the disruptions have no apparent mitotic defect, indicating that the MER1 gene product is not essential for vegetative growth. Furthermore, when the LEU2 and LYS2 segregants were sporulated, they produced dead spores. Thus, the phenotype of strains carrying the deletions is similar to that of the original mutant isolates (Table 2).

A.

1 7 0 1 1 5 1 3 9 1 6 5 1 6 2 1 3 0 1 0 1

R A C H H X S M G

B.

H

200 bp FIGURE 1 .-A, Restriction map of the MER1 gene. Closed circles indicate transposon insertions (SEIFERT et al. 1986) which confer a Mer- phenotype; open circles indicate transposon insertions which confer a Mer+ phenotype. The numbers above the circles corre- spond to the insertions; plasmids containing transposon insertions are identified by the same number. R, EcoRI; A, AatII; C, ClaI; H , HindIII; X , XbaI; S, Sad; M, MluI; G, BglII. B, Maps of M E R l deletion/disruption alleles constructed in vitro. The endpoints of the deletions are shown. The LEU2 and LYS2 genes are not drawn

to scale.

Strains carrying the deletion alleles were mated to a strain containing the original mutant allele, m e r l - I , and the resultant diploids produced dead spores. This verifies that the gene cloned and defined by the LEU2 and LYS2 mutations is the MERI gene.

Intergenic recombination: A defect in pairing and/

or exchange could cause meiotic nondisjunction; therefore, it was of interest to measure meiotic recom- bination in merl strains. This was made possible by the high spore viability exhibited by merl strains in the presence of the $1013 mutation. Isogenic s p o l 3 strains with and without the merl::LEU2 null allele were used to assay crossing over in two intervals, the CYHIO-LYS2 interval on chromosome ZZand the HZS4- MAT interval on chromosome

ZZZ.

Figure 2 shows the possible patterns of recombination and segregation of chromosome

ZZ

in s p o l 3 strains. T h e data in Table 3

show the calculated map distances in Mer+ and Mer- strains. Crossing over is reduced to approximately

10% of wild-type levels in Mer- strains in both inter- vals examined. Intergenic recombination in merl-1 strains is also reduced to approximately 10% of wild- type levels, suggesting that this allele is a null muta- tion.

Spore viability is increased (P

<

0.0 1) and the levels of aberrant and reductional segregation are reduced

(P

<

0.005) in the merl spol3 strain compared to the

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242 J. Engebrecht and G . S. Roeder

No Recombination

Reductional Equational

cy# LYS* CyhS Lys- C y P P L y s + C y P Q L y s +

+ Intergenic Recombination

Reductional Equational

00

-11111 , 0 1 1 1 1 1 1 1 1 1 1 1

@

1 1 . 1 1 1 1 1 1 1

f

,.I,,*,,,,,,,,,

J

cyff LYS+ CyhS Ly: C y P P L y s C C y P Q L y s -

FIGURE 2,“Patterns of chromosome I1 segregation and recom- bination in $013 dyads. Chromosome II is marked with CYHlO and

LYS2 as shown. The different homologs are designated by the solid and dashed lines. The centromere is represented by the closed circle. The chromosomes shown at the top have completed pre- meiotic DNA replication; each line represents a sister chromatid. The products of segregation in s p o l 3 dyads are shown below in the large open circles, with phenotypes indicated. CYHlO is very tightly centromere-linked; therefore, it is possible to determine which dyads result from reductional division and which dyads result from equational division. Furthermore, because the dominant CYHlO

allele is sensitive and the recessive allele is resistant to cyclohexi- mide, it is possible to differentiate between spore colonies which are homozygous or heterozygous at the CYHlO locus by measuring the amount of papillation on medium containing cycloheximide. Heterozygotes give rise to CyhR papillae due to mitotic recombi- nation; CYHlO homozygotes only rarely, if ever, give rise to CyhR papillae. Reductional segregation generates CyhR Lys+ and CyhS Lys- spore colonies. A recombination event followed by a reduc- tional division produces CyhR Lys+ and CyhS Lys+ spore colonies. Equational segregation produces two CyhS Lys+ spore colonies which papillate to CyhR, represented by CyhPP. Half of the time that a recombination event is followed by an equational division, CyhPp Lys+ and CyhPP Lys- spore colonies are generated. The alternative pattern of chromatid segregation gives rise to two CyhPP Lys+ spore colonies which are not detected as recombinants.

s p o l 3 (KLAPHOLZ, WADDELL and ESPOSITO 1985) and hop1 s p o l 3 (HOLLINGSWORTH and BYERS 1989) strains. Chromosome synapsis and recombination may hold homologous chromosomes together in a config- uration which impairs the segregation of sister chro- matids during a s p o l 3 equational division. However, in the presence of a mutation that disrupts pairing and/or recombination ( i e . , spol

I,

h o p l , merl ), sister chromatids can apparently separate without difficulty.

Intragenic recombination: Intragenic recombina-

tion was examined in haploid spol? strains disomic

for chromosome

ZZZ

that contain his4 and leu2 heter- oalleles. Mitotic and meiotic levels of histidine and leucine prototrophs were determined for isogenic Mer+ and Mer- strains and the results are presented in Table 4. Like intergenic recombination, intragenic recombination is substantially decreased, but not elim- inated, in Mer- strains. This is in contrast to both spol

I

and rad50 strains, where meiotic recombination is completely abolished (KLAPHOLZ, WADDELL and ESPOSITO 1985; MALONE and ESPOSITO 1981; Table

Intrachromosomal recombination: Exchange be-

tween duplicated elements on the same homolog (in- trachromosomal recombination) occurs at wild-type levels in hop1 strains even though interchromosomal recombination is reduced to approximately 10% of wild-type levels (HOLLINGSWORTH and BYERS 1989). T o determine if the recombination defect observed in Mer- strains affects only interhomolog exchange, intrachromosomal recombination was measured.

Strain J114, a derivative of karC2-4 (HOLLINGS- WORTH and BYERS 1989), was used to measure ex- change between duplicated elements on the same homolog. This haploid spol? strain is disomic for chromosome

ZZZ.

One of the chromosome

ZZZ

homo- logs contains a duplication of sequences between LEU2 and HZS4; these repeats flank inserted vector

DNA including the URA? and CYH2 genes (Figure 3).

Intrachromosomal recombination between the dupli- cated elements results in loss of the duplication. When this event is followed by equational division, recom- binants can be selected on medium lacking histidine and leucine and containing cycloheximide (Figure 3).

T h e pattern of segregation (equational or reductional) must be taken into account when determining the level of intrachromosomal recombination and can be monitored by following the centromere-linked chro- mosome

IZZ

marker, CDCIO, in dyad spore products.

T h e data in Table

5A

show the levels of intrachro- mosomal recombination in isogenic Mer+ and Mer- strains. T h e corrected fold induction normalizes for the levels of equational and reductional division in Mer+ and Mer- strains. T h e level of intrachromoso- mal recombination in the Mer- strain is approximately 8% of wild-type levels. Therefore, Mer- strains are defective in both inter- and intrachromosomal ex- change.

T h e effect of a merl mutation on intrachromosomal gene conversion was also measured. A haploid MATa spol? mer1 strain carrying a duplication of HIS4 genes, as described by JACKSON and FINK (1 98 l), was trans- formed with a centromere plasmid containing MATa

(7)

Yeast merl Mutants

TABLE 3

Intergenic recombination in spolf strains

243

A. Chromosome I1

Relevant geno- Percent spore aberrant CYHIO-LYSZ dis-

Percent equational

Percent re- ductional

Percent

Strain type viabilitf segregationb segregationb segregation' tance ( c M ) ~

593

-

MER I MER 1

53 (341/640) 77.4

594 merl::LEU2 66 (292/440) 96.6

merl::LEU2

13.6 9

0.9 2.5

38

4.5

B. Chromosome III

Strain

Relevant geno. type

Percent

aberrant HIS4-MAT dis- segregation' tance ( c M ) ~

593 MER

-

I 9 42

MER I

594 merl::LEU2 mer1::LEUZ

516

-

merl-1 MER 1

5 2.7

8 32

515

-

merl-1 5 2.9

merl-1

The following numbers of two-spore viable dyads were scored: 103 forJ93, 114 for 594, 59 for 516 and 105 for 515.593 and 594 are

a The numbers of viable spores and the total numbers of spores examined are given in parentheses.

*

Equational and reductional segregation were determined by examining the Cyh and Lys phenotypes of the dyad spore colonies as illustrated in Figure 2. Equational and reductional segregations of chromosome III cannot be distinguished due to the absence of a tightly centromere-linked marker.

Dyads in which one spore colony was CyhS (CYHIO monosome) and the other spore colony papillated on medium containing cycloheximide to a greater extent than CYHIO heterozygotes (CYHlO/cyh10-Z00/cyhlO-lOO trisome) were scored as aberrant segregations. Dyads in which one spore colony was a nonmater and the other spore colony mated as either an a or an a were assumed to result from aberrant segregation of chromosome III. Dyads displaying aberrant segregation were disregarded in calculating map distances.

Recombinants for the CYHIO-LYS2 interval were identified as illustrated in Figure 2. Four-strand double crossovers could be detected only when followed by reductional division (CyhR Lys-:CyhS Lys'); therefore, the map distance is a minimum estimate. The failure to detect the majority of 4-strand double crossovers could explain the discrepancy between the map distance observed (38 cM) and the published map distance (45-50 cM; MORTIMER and SCHILD 1985). Chromosome III recombinant dyads had the following phenotypes: His+ nonmater:His- nonmater or His'a:His+ a. Four-strand double crossovers segregated His+a:His-a when the two crossovers occurred on opposite sides of the centromere; this represents the majority of 4-strand double crossovers. (Nonrecombinants segregated His+ nonmater:His+ nonmater or

His+a:His-a.) Map distances were calculated using a derivation of PERKIN'S (1949) formula as follows: map distance = [single crossovers

+

6(4-strand double crossovers)/total] X 100. This equation accounts for the fact that half of the recombination events that are followed by equational chromosome segregation escape detection.

isogenic; 594 was derived by transformation ofJ93.

Table 5B. As expected, the Mer- strain also shows a decreased frequency of intrachromosomal gene con- version.

Recombination in multiple mutants: Interchro-

mosomal recombination was measured in merl s p o l l $1013 and merl rad50 $1013 strains (Table 4). Recom- bination is completely abolished in the triple mutants as it is for s p o l l $ 0 1 3 and rad50 s f 0 1 3 strains. There- fore, both s p o l l and rad50 are epistatic to m e r l .

Inter- and intrachromosomal recombination was also examined in merl hop1 s p o l 3 mutants. Intra- chromosomal exchange in merl hop1 spo13 strains occurs at the same level as in merl $ 1 0 1 3 mutants (Table 5 ) . However, interchromosomal recombina- tion in the merl hop1 s p o l 3 mutant is reduced to a lower level than that observed in either of the double

mutants (Table 4). This synergistic effect suggests that the MER1 and HOPI gene products effect different processes, both of which are necessary for normal levels of meiotic recombination.

Spore viability in multiple mutants: Spore viability

(8)

244 J. Engebrecht and G. S. Roeder

TABLE 4

Intragenic recombination in s p o l 3 strains

His+ Leu+

Relevant

Strain EenotvDe M T ME -Fold M T ME -Fold

Y20 2.5 X 1 0 - ~ 9.1 X 10-3 36 4.3 x l o + 1.3 X 10+ 302

JlOO merl 4.0 X 10-4 1.3 X 1 0 - ~ 3.2 3.4 x IO+ 8.5 X I O + 25

5102 sp011 4.3 x 1 0 - ~ 4.2 X 1 0 - ~ 0.9 4.0 X 3.8 X 0.9

J106 m e r l s p o l l 2.7 X 10-4 4.2 X 1 0 - ~ 1.5 2.9 x 3.5 x 1.2

5104 rad50 2.9 x IO+ 2.5 X 1 0 - ~ 0.9 1.6 X 1.2 x 0.8

JlOS merl rad50 4.0 X 10-5 2.8 X 1 0 - ~ 0.7 1.1 x 1.2 x l o + 1.1

J l l O hop 1 4.3 x 1 . 8 X 4.1 4.2 X 1.2 X 29

J112 merl hop1 6.6 X 6.2 X 10-4 0.9 3.6 X 1O"j 3.3 x I O + 0.9

M T = mitotic mean frequency; ME = meiotic mean frequency; -Fold = -fold meiotic induction of His and Leu prototrophs over mitotic levels. Duplicates of two independent transformants for each strain were grown to stationary phase in rich media. Prior to transfer to sporulation medium, cells were plated onto complete medium, medium lacking histidine and medium lacking leucine to determine the mitotic frequencies of intragenic recombination. One milliliter of cells was washed in water, resuspended in 10 ml of 1 % potassium acetate and aerated at 30" for 4 days. The frequency of meiotic intragenic recombinants was then determined by plating onto complete medium, medium lacking histidine and medium lacking leucine. All of the above strains are isogenic and were derived by transformation of strain Y20.

HIS4 URAWCYH2 LEV2 cdclO MATa

+

:

-

c

I I C

x

h i s 4 leu2 CDCIO MATO

No Recombination

Reductional Equational

His+Le$ CyhS Hi< Leu CyhR His+Le$CyhS His+Le$ CyhS

+

Intrachromosomal Recombination

Reductional Equational

His+Le; CyhS Hi< Leu CyhR His+Le$ CyhR His+Le$CyhS

FIGURE 3,"Patterns of segregation and intrachromosomal recombination in a spo13 disome. Chromosome III homologs are marked with

H I M , LEU2, CDClO and MAT alleles as shown. The duplicated sequences are represented by open boxes and flank inserted vector sequences including the URA3 and CYHZ genes (closed box). There is a CyhR allele at the CYH2 locus on chromosome VI1 and a Ura- allele at the URA3

(9)

Yeast merl Mutants 245

TABLE 5

Intrachromosomal recombination in spol3 strains

A. lntrachromosomal exchange

His+ Leu+ CyhR Percent

Relevant equational

Percent re- ductional

Strain genotype segregation" segregation" MT ME -fold Corrected

-

J114 25 75 7.4 X 1 0 - ~ 4.3 x lo-' 23

5115 merl 98 2 4.2 X 10-4 6.9 x 1.7

5117 hop 1 95 5 4.4 x 9.2 X IO-' 22

1119 merl hob1 95 5 2.5 X 1 0 - ~ 4.0 X 1 0 - 4 1.7

B. lntrachromosomal gene conversion

His+ Strain

Relevant

eenotvDe MT ME -Fold

JE24-18C/pMEI MER I 8.7 X 1 0 - ~ 5.6 X IO-' 64

JE24-18C m e r l - 1 1.5 X 10-4 3.2 x 2

M T = mitotic mean frequency; ME = meiotic mean frequency; -Fold = -fold meiotic induction of His prototrophs over mitotic levels. Corrected -fold = -fold meiotic induction of His+ Leu+ CyhR recombinants over mitotic levels normalized for the amount of equational division occurring in these strains; this was calculated by multiplying the -fold meiotic induction by (loo%/% equational segregation). Duplicates of two independent transformants from each strain were grown to saturation in rich medium and plated onto complete medium and cycloheximide medium lacking histidine and leucine in the case of strains J114, J115, J117, J119 or medium lacking histidine for strains JE24-18C/pMEI and JE24-18C. One milliliter of each culture was then washed with water, resuspended in 10 ml of 1% potassium acetate and aerated for four days at 30". Meiotic intrachromosomal recombination was then measured. Strains J114, J115, J117 and J119 are isogenic and derived by transformation of J 1 14.

Percent equational and reductional segregation was determined by dissecting and scoring a minimum of 50 two-spore viable dyads for growth at 37" (CDClO) and mating type. Equational segregation gives rise to two Cdc+ nonmaters, while reductional segregation gives rise to one Cdc- colony and a Cdc+ colony. Dyads with one nonmater and one mater were classified as aberrant segregants and disregarded in determining the percent reductional and equational segregation. Aberrant segregation occurred at approximately the same frequency (12%) in all strains.

regation observed in the s p o l 3 single mutant. merl is not as effective as s p o l 1 in alleviating spore inviability

(P

<

0.005) and reducing aberrant segregations ( P

<

0.005) in s p o l 3 strains. This may be due to the low levels of recombination that do occur in merl strains.

In contrast to the s p o l l and merl mutants, the rad52 mutant is not rescued by s p o l 3 . This is believed to be due to recombination intermediates that are inefficiently resolved in the absence of the RAD52 gene product (MALONE 1983; BORTS, LICHTEN and HABER 1986). s p o l l and rad50 rescue the lethality of rad52 in the presence of s p o l 3 because recombination is not initiated in s p o l l and rad50 strains (MALONE

1983). merl also rescues rad52 but not to the same extent as spol

I

(P

<

0.005; Table 6). T h e decrease in viability in merl strains in the presence of the rad52 mutation is probably due to the low frequency of recombination events initiated in Mer- strains.

In a s p o l 3 background, the viability profile of the s p o l l merl strain is similar to the merl strain and different from the s p o l l strain (P

<

0.005). T h e viability of the s p o l 1 merl rad52 mutant is interme- diate between and significantly different from both merl rad52 ( P

<

0.005) and spol

I

rad52 (P

<

0.005) mutants (Table 6). T h e effect of a merl mutation in a spol

I

background was unexpected because meiotic recombination in s p o l 1 merl strains is abolished (Ta- ble 4). Therefore, it appears that spore viability in s p o l 3 strains is determined by a number of factors, only one of which is recombination.

Mapping the MER1 gene: T h e cloned MERI gene

was used to probe yeast chromosomes that had been electrophoretically separated (CHU, VOLLRATH and DAVIS 1986) and the gene hybridized to chromosome XZV (data not shown). A strain containing the merl::LEU2 deletion was crossed to strains containing the chromosome XZV markers met2, petx, rad50, and lys9 and the location of MERI was determined by tetrad analysis. MER1 maps to the left arm of chro- mosome XZV, less than 1 cM from PETX. In 69 four- spore-viable tetrads examined, no crossovers between MERI and PETX were detected.

DISCUSSION

We have identified a new yeast gene, M E R I , which is necessary for meiotic recombination. merl strains recombine at only 10% of wild-type levels, resulting in the random segregation of homologues at the first meiotic division. Both inter- and intrachromosomal recombination are reduced, indicating that the MERI gene product is required for recombination between and within homologous chromosomes.

(10)

246 J. Engebrecht and G . S. Roeder

TABLE 6

Spore viability in multiple mutants

Percent

Percent small Percent spore colon- aberrant

- Strain Relevant genotype viability" ies' segregation'

5121

5122

5123

5124

5125

5126

5127

5128

72 (144/200)

merl s p o l 3 82 (1 64/200) merl s p o l 3

sp011 sp013 sp01 I sp013

rad52 s p o l 3 rad52 s p o l 3

93 (93/100)

4 (4/100)

m e r l rad52 s p o l 3 6 1 (1 22/200) merl rad52 s p o l 3

s p o l l s p o l 3 rad52 90 (90/100) s p o l l s p o l 3 rad52

merl s p o l l s p o l 3 85 ( 1 70/200) merl s p o l l s p o l 3

merl s p o l l s p o l 3 rad52 78 (156/200) merl s p o l 1 s p o l 3 rad52

39 17

16 8

1 0

50 0

52 8

25 0

2 1 6

46 2

a The number of viable spores over the total number of spores

examined is given in parentheses.

Percent small colonies refers to the percentage of the total viable sporers that gave rise to observable colonies only after 3 days on rich medium.

Percent aberrant segregation of chromosome III was measured by scoring for mating type. Dyads containing one nonmater and an a or an a were classified as aberrant segregants.

All of the above strains are isogenic and derived by transforma- tion ofJ121.

gene product, recombination cannot proceed and the presence or absence of the MER1 gene product is of no consequence. Like RAD50, S P O l l is believed to act at an early step in meiosis and is essential for recombination (KLAPHOLZ, WADDELL and ESPOSITO

1985). It seems likely that both RAD50 and SPOl

I

act

before the MERI gene product.

T h e phenotype of the merl hop1 mutant suggests that MERI and HOPI are involved in different proc- esses, both of which are necessary for meiotic recom- bination. Interhomolog recombination is approxi- mately 10% of wild-type levels in both the merl and hopl mutants. In contrast, the merl hopl mutant is completely defective in interhomolog recombination. HOLLINGSWORTH and BYERS ( 1 989) suggest that the HOP1 gene product is a structural component of the SC because hopl mutants are unable to pair homolo- gous chromosomes. However, the recombination ma- chinery appears to be normal in hop1 strains since intrachromosomal recombination occurs at wild-type levels. On the other hand, merl strains are reduced for both inter- and intrachromosomal recombination. Taken together, these results suggest that the MERI gene product is most likely a component of the meiotic

recombination machinery. However, the possibility cannot be ruled out that the MER1 gene product is involved in chromosome pairing, possibly as a struc- tural component of the SC. Alternatively, MERI may control the expression of one or more genes encoding components of the recombination apparatus or the

sc.

If the MERI gene product is a component of the recombination machinery, it seems likely that it is involved at an early step, possibly initiation. merl is rescued by $ 1 0 1 3 whereas mutants defective in resolv- ing recombination intermediates (e.g., rad52, rad57; MALONE 1983; BORTS, LICHTEN and HABER 1986)

are not. Furthermore, merl rescues rad52 in a s p o l 3 background, indicating that the MER1 gene product acts prior to the RAD52 protein. MERI may encode an endonuclease which forms nicks or breaks in the DNA which initiate recombination. Other possible functions for the MER1 gene product include strand displacement and assimilation. Proteins catalyzing strand transfer have been implicated in the early stages of recombination (COX and LEHMAN 1987).

Approximately 90% of recombination is eliminated in merl strains, indicating that MERI represents the major recombination pathway during meiosis. How- ever, the fact that merl mutants display some meioti- cally induced recombination suggests the existence of a MER1 -independent pathway. T h e existence of mul- tiple recombination pathways is not without prece- dent. Both RAD52-dependent and RAD52-independ- ent pathways of mitotic recombination have been demonstrated (MALONE and ESPOSITO 1980; JACKSON and FINK 198 1 ; HABER and HEARN 1985). T h e recom- bination that does occur in Mer- strains could be initiated by other meiotically induced enzymes. Alter- natively, paired chromosomes may serve as better substrates for mitotic recombination enzymes which are present during meiosis. In fact, many proteins implicated in recombination are present during both mitosis and meiosis (BAKER, CARPENTER and RIPOLL

1978; GAME 1983). Finally, lesions in the DNA accu-

mulated during premeiotic DNA synthesis and pairing may serve as the substrates for enzymes involved in later steps in recombination.

T h e hypothesis that merl mutants are defective at an early step in recombination can be tested by assay- ing extracts from mer1 strains for their ability to

catalyze recombination in vitro (SYMINGTON, MORRI- SON and KOLODNER 1984). It will also be of interest to examine merl strains cytologically to determine whether a normal SC is formed. Further characteriza- tion of the MERI mutant may help to elucidate the interactions between functions involved in pairing, recombination and chromosome segregation.

(11)

Yeast mer1 Mutants 247

prior to publication. This work was supported by National Institute of Health grant GM28904 and National Science Foundation grant PCM-8351607. J.E. is a Jane Coffin Childs Fellow.

LITERATURE CITED

BAKER, B. S., A. T. C. CARPENTER, M. S. ESPOSITO, R. E. ESPOSITO and L. SANDLER, 1976 The genetic control of meiosis. Annu. Rev. Genet. 10: 53-1 34.

BAKER, B. S., A. T . C. CARPENTER and P. RIPOLL, 1978 The utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster. Ge- netics 9 0 531-578.

BORTS, R. H., M. LICHTEN and J. E. HABER, 1986 Analysis of meiosis-defective mutations in yeast by physical monitoring of recombination. Genetics 113: 551-567.

CARPENTER, A. T. C., 1975 Electron microscopy of meiosis in

Drosophila melanogaster females. 11. The recombination nod- ule-a recombination-associated structure at pachytene? Proc. Natl. Acad. Sci. USA 72: 3186-3189.

CARPENTER, A. T. C., and B. S . BAKER, 1974 Genic control of meiosis and some observations on the synaptonemal complex in Drosophila melanogaster, pp. 365-375, in Mechanisms in Re- combination, edited by R. F. GRELL. Plenum, New York. CHATTOO, B. B., F. SHERMAN, D. A. AZUBALIS, T . A. FJELLSTEDT,

D. MEHNERT and M. OGUR, 1979 Selection of lys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of a-

aminoadipate. Genetics 93: 51-65.

CHU, G., D. VOLLRATH and R. W. DAVIS, 1986 Separation of large DNA molecules by contour-clamped homogeneous elec- tric fields. Science 234: 1582-1585.

Cox, M. M., and I. R. LEHMAN, 1987 Enzymes of general recom- bination. Annu. Rev. Biochem. 56: 229-262.

DAWES, I. W., and I. D. HARDIE, 1974 Selective killing of vege- tative cells in sporulated yeast cultures by exposure to diethyl ether. Mol. Gen. Genet. 131: 281-289.

DRESSER, M. E., C. N. GIROUX and M. J. MOSES, 1986 Cytological analysis of meiosis using synaptonemal complexes in spread preparations of yeast nuclei. Yeast 2: S96.

FARNET, C., R. PADMORE, L. CAO, W. RAYMOND, E. ALANI and N. KLECKNER, 1988 The RAD50 gene of S. cereuisiae, in Mecha- nisms and Consequences of DNA Damage Processing, edited by E. FRIEDBERG and P. HANAWALT. Alan R. Liss, New York, pp 201-215.

FLEIG, U. N., R. D. PRIDMORE and P. F'HILIPPSEN, 1986 Construction of LYS2 cartridges for use in genetic manipula- tion of Saccharomyces cereuisiae. Gene 4 6 237-245.

GAME, J. C., 1983 Radiation-sensitive mutants and repair in yeast,

pp. 109-137, in Yeast Genetics, edited by J. F. T . SPENCER, D. M. SPENCER and A. R. W. SMITH. Springer-Verlag. New York. GIROUX, C. N., 1988 Chromosome synapsis and meiotic recom- bination, in Genetic Recombination, edited by R. KUCHERLAPATI and G. R. SMITH. American Society for Microbiology, Wash- ington, D.C., pp. 465-496.

HABER, J. E., and M. HEARN, 1985 RAD5Z"independent mitotic gene conversion in Saccharomyces cerevisiae frequently results in chromosomal loss. Genetics 111: 7-22.

HALL, J., 1972 Chromosome segregation influenced by two alleles of the meiotic mutant c(3 )G in Drosophila melanogaster. Ge- netics 71: 367-400.

pairing gene. Genetics (in press).

duplicated genetic elements in yeast. Nature 292: 306-31 1.

KLAPHOLZ, S . , and R. E. ESPOSITO, 1980 Recombination and chromosome segregation during the single division meiosis in HOLLINGSWORTH, N., and B. BYERS, 1989 H O P I : a yeast meiotic

JACKSON, J. A,, and G. R. FINK, 1981 Gene conversion between

spoZ2-I and spoZ3-1 diploids. Genetics 96: 589-61 1.

KLAPHOLZ, S., C. S. WADDELLand R. E. ESPOSITO, 1985 The role

of the SPOZl gene in meiotic recombination in yeast. Genetics

KUPIEC, M., and G. SIMCHEN, 1984 Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae. Mol. Gen. Genet. 193:

LAMBIE, E. J., and G. S. ROEDER, 1988 A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences. Cell 52: 863-873.

MAGUIRE, M. P., 1978 Evidence for separate genetic control of crossing over and chiasma maintenance in maize. Chromosoma 65: 175-183.

MALONE, R. E., 1983 Multiple mutant analysis of recombination in yeast. Mol. Gen. Genet. 189: 405-412.

MALONE, R. E., and R. E. ESPOSITO, 1980 The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc. Natl. Acad. Sci. USA 77: 503-507.

MALONE, R. E., and R. E. ESPOSITO, 1981 Recombinationless meiosis in Saccharomyces cereuisiae. Mol. Cel. Biol. 1: 891-901. MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular

Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

MCCARTER, L. L., and M. SILVERMAN, 1987 Phosphate regulation of gene expression in Vibrio parahaemolyticus. J. Bacteriol. 169: 3441-3449.

1 1 0 187-216.

525-531.

MOENS, P. (Editor), 1987 Meiosis. Academic Press, New York. MORTIMER, R. K., and D. SCHILD, 1985 Genetic map of Saccha-

romyces cereuisiae, Ed. 9. Microbiol. Rev. 49: 18 1-2 12. PERKINS, D. D., 1949 Biochemical mutants in the smut fungus

Ustilago maydis. Genetics 34: 607-627.

ROCKMILL, B., and S. FOGEL, 1988 DISZ: a yeast gene required for proper meiotic chromosome disjunction. Genetics 119:

ROCKMILL, B., and G. S. ROEDER, 1988 R E D I : a yeast gene required for the segregation of chromosomes during the re- ductional division of meiosis. Proc. Natl. Acad. Sci. USA 85: 6057-6061.

ROSE, M . D., P. NOVICK, J. H. THOMAS, D. BOTSTEIN and G. R. FINK, 1987 A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 6 0

ROTHSTEIN, R., 1983 One-step gene disruption in yeast. Methods Enzymol. 101: 202-21 1.

SEIFERT, H. S., E. Y. CHEN, M. So and F. HEFFRON, 1986 Shuttle mutagenesis: a method of transposon mutagenesis for Saccha- romyces cerevisiae. Proc. Natl. Acad. Sci. USA 83: 735-739. SHERMAN, F., G. R. FINK and J. B. HICKS, 1986 Methods in Yeast

Genetics: Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

SMITH, P. A , , and R. C. KING, 1968 Genetic control of synapto- nemal complexes in Drosophila melanogaster. Genetics 60: 335- 351.

SOUTHERN, E. M., 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.

SYMINGTON, L. S., P. T. MORRISON and R. KOLODNER, 1984 Genetic recombination catalyzed by cell-free extracts of Saccha- romyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 4 9

VON WETTSTEIN, D., S. W. R A S M U S S E N ~ ~ ~ P. B. HOLM, 1984 The synaptonemal complex in genetic segregation. Annu. Rev. Ge- net. 18: 331-413.

261-272.

237-243.

805-8 14.

Figure

TABLE 1 Yeast  strains
TABLE 1-Continued
FIGURE 1 .-A, indicate transposon insertions (SEIFERT
FIGURE 2,“Patterns of chromosome marked with LYS2 alternative pattern of chromatid segregation gives rise to two CyhPP which papillate to CyhR, represented by  CyhPP
+4

References

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