10.1101/SQB.1984.049.01.010Access the most recent version at doi: 1984 49: 67-76Cold Spring Harb Symp Quant Biol

R.H. Borts, M. Lichten, M. Hearn, et al. Saccharomyces cerevisiaePhysical Monitoring of Meiotic Recombination in

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Phys ica l Moni tor ing of Meiot ic R e c o m b i n a t i o n in Saccharomyces cerevisiae

R.H. BORTS, M. LICHTEN, M. HEARN, L.S. DAVIDOW,* AND J .E. HABER Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University,

Waltham, Massachusetts 02254

Genetic studies of meiotic recombination in Saccha- A romyces cerevisiae have provided a significant fraction of what we understand about the mechanism of recom- bination (Fogel et al. 1979; Esposito and Klapholz 1981; Szostak et al. 1983). A detailed genetic investi- gation of gene conversion events and associated recip- rocal exchange of flanking markers has provided a wealth of information indicating that such events are not uniformly distributed along the chromosome. These findings have led to the publication of several detailed molecular models of recombination, most no- tably the single-strand initiation model of Meselson and Radding (1975) and the double-strand-break model of Szostak et al. (1981). The recent development of recombinant DNA techniques to clone, modify, and replace genes in yeast has now made it possible to be- gin an investigation of meiotic recombination at the molecular level. In this paper we concern ourselves T js with three fundamental questions: (1) Are there se- quences that act as specific stimulators ("hot spots") of meiotic recombination? (2) When during meiosis does ~ ss reciprocal recombination occur, relative to other mei- otic events? (3) At what step relative to reciprocal re- D. combination do various meiotic-defective mutations block meiosis?

Construction of a Small Chromosomal Region to Study Meiotic Recombination

Our basic approach has been to examine recombi- nation in a small, well-defined region of the chromo- some that can be easily manipulated to accommodate in vitro modifications. The basic structure consists of a duplication of the 3.5-kb EcoRI-HindIII M A T (mat- ing-type) region that flanks pBR322 and a 1.2-kb HindllI fragment containing the yeast URA3 gene (Fig. IA). The duplication was created by the integra- tive transformation of a pBR322-MA T-URA3 plasmid (Rogers and Haber 1982). The mating-type (MA T) lo- cus may contain either a or a alleles, which are codom- inantly expressed; cells expressing both alleles are non- mating. Thus, a haploid strain containing the duplication MATa-URA3-pBR322-MATa will conju- gate with a haploid strain containing a MA Ta-URA3- pBR322-MATc~ region to yield a nonmating diploid. When such a diploid is placed under nitrogen-starva-

*Present address: Pfizer Central Research, Groton, Connecticut 06340.

9 kb =;

MATa URA3 MATo

Z T MAT~ MATer

i~ s %31kb ,U" Jl

i 14kb J'; - :

MATo M A T ~

MATe< MATa

~., 27 kb - ;

67

B

,To 43 I - ,To URA3 ] URA3

I Ikb ' '

LEU2

Figure 1. (A) Creation of two novel BgllI restriction frag- ments by meiotic recombination. Reciprocal exchange in the MA T-URA3-pBR322-MA Tinterval produces two nonmating segregants of the genotype MATa-URA3-pBR322-MATct and MA Ta-URA3-pBR322-MATa. Due to the two BglII recog- nition-site polymorphisms, these recombinants yield pBR322- containing BglIl fragments that can be distinguished from those produced by the two parents. (B) Location of sites where either the 1.2-kb URA3 fragment (Bach et al. 1979) or the 2.2-kb XhoI-Sall LEU2 fragment (Andreadis and Schimmel 1982) was inserted in the pBR322 backbone. (~) Direction of transcription. Restriction sites shown are BgllI (~7), HindIll (U), EcoRl (O), KpnI (•), Sail (e), SmaI (0), PvuII (0), and Aval (IlL The deletions described in Table 1 remove material between the Pvull site in pBR322 and the PvuII site in the right-hand MAT region (deletion 1), and between the Smal site in URA3 (inserted at the HindIII site) and the PvuII site in pBR322 (deletion H).

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68 BORTS ET AL.

tion conditions to induce meiosis and sporulation, asci containing four haploid meiotic products are pro- duced. Tetrads in which a recombination event has oc- cured within the MA T-URA3-pBR322-MA T interval can be recognized by the fact that two of the four meiotic products will give rise to spore colonies con- taining haploid nonmaters of genotype MA Ta-UR,43- pBR322-MATu and the reciprocal MATu-URA3- pBR322-MATa (Fig. 1A). The other two spores will give rise to an a-mating and an a-mating colony, with the parental configuration.

Nearly 16% (64/410) of the tetrads dissected from such diploids contained one a-mating, one a-mating, and two nonmating segregants characteristic of a re- ciprocal recombination event in the 9-kb MA T-URA3- pBR322-MAT interval. This level of exchange (0.9 cM/kb) is approximately 2.5 times the level of recom- bination seen along an average segment of chromo- some Il l (0.37 cM/kb) (Newlon et al. 1982). It should be noted that the region around MA T does not nor- mally exhibit high levels of meiotic recombination; in fact, the 21-kb interval between MA T and cryl is only about 2 cM long on a genetic map (Larkin and Wool- ford 1983). About 95% of the nonmating haploid spores are the products of reciprocal exchange in the interval between the flanking M A T alleles. A small number of nonmating haploid spores ( - 3 % ) are the products of events involving gene conversion of one M A T allele, whereas about 2% are produced by un- equal crossing-over between the flanking MA T regions.

Because 95% of the nonmating segregants are the products of reciprocal exchanges, it is possible to de- termine directly the amount of recombination in this region by measuring the amount of novel restriction fragments produced by recombination. We have taken advantage of the fact that the MA Ta sequence con- tains a BgllI site within the M A T locus that is absent in MATa (Astell et al. 1981). Consequently, when DNA from the parent diploid is digested with Bglll, two fragments that contain pBR322 are produced: a 9- kb fragment, derived from the MA Ta-URA3-pBR322- MA Ta region; and a 3 l-kb fragment, derived from the MATce-URA3-pBR322-MATa region (Fig. IA). The two reciprocal recombinants yield distinctly different restriction fragments containing pBR322. The MA Ta- URA3-pBR322-MA Ta region yields a 14-kb fragment, and the reciprocal MATa-URA3-pBR322-MATa re- gion yields a 27-kb BgllI fragment. Although the 27- kb fragment is difficult to distinguish from the paren- tal 31-kb band, the 14-kb (MATa-URA3-pBR322- MATct) fragment is clearly resolved from the parental bands.

The URA3 Region Contains a Stimulator of Meiotic Recombination

The MAT-URA3-pBR322-MAT dupl icat ion has provided an excellent opportunity to investigate the possibi l i ty that certain DNA sequences st imulate meiotic recombination. We have asked if any particu-

lar region of the MAT-URA3-pBR322-MA T interval was responsible for stimulating recombination. Using transplacement transformation (Rothstein 1983), we have replaced the original duplication with the series of deletions or substitutions shown in Figure lB. The results of these experiments are summarized in Table 1. Although deleting one half or the other of the pBR322 sequences in the region had only a moderate effect on recombination, removal of the 1.2-kb URA3 HindIIl fragment reduced reciprocal exchange from nearly 15.6%0 tetratypes (0.9 cM/kb) to 5.6% tetra- types (0.4 cM/kb) . Moreover, when URA3 was het- erozygous, the level of meiotic recombination was in- termediate (0.7 cM/kb). Thus, it appears that URA3 contains a semidominant stimulator of recombination and can act opposite a region of nonhomology.

The stimulating effect of URA3 is apparently inde- pendent of position. We have examined the effect of inserting URA3 in two other chromosomal locations (Table l). When URA3 was inserted at the Aval site of pBR322 (Fig. IB), virtually the same results were obtained as with URA3 inserted at the Hindlll site. Reciprocal recombination in the MA T-pBR322-MA T interval increased to I I % tetratypes (0.8 cM/kb) when URA3 was heterozygous and to 21% (1.3 cM/kb) when it was homozygous. In another series of experiments we examined the effect of URA3 in the adjacent cryl- M A T interval. The cryl-MA T interval is remarkably devoid of meiotic recombination, yielding only 2 cM in a 21-kb interval (Larkin and Woolford 1983). We inserted URA3 into a HindIII site approximately mid- way between the cry and M A T loci, using the gene re- placement method (Rothstein 1983). When URA3 was heterozygous, the frequency of recombination in- creased from approximately 2 cM to about 4 cM. When URA3 was homozygous, recombination in- creased to 9 cM (Table l).

The presence of a heterozygous stimulator of recom- bination has also enabled us to look at other aspects of meiotic recombination. For example, we have asked if the URA3 element stimulates exchange preferentially to one side of the element. Southern blot analysis can be used to demonstrate whether an a-URA3-a parental chromosome had recombined to yield an ot-URA3-a or an a-URA3-a segregant. In this way, the region in which an exchange event occurred can be determined. In 19 exchanges observed when URA3 was heterozy- gous at the HindIII locus and in 26 crossovers ob- served when URA3 was heterozygous at the AvaI site, exchanges occurred in rough proportionali ty to the size of the interval between URA3 and the flanking M A T loci. Thus, it appears that there is no striking polarity to crossovers stimulated by URA3 in this interval.

We have also observed gene conversion events in- volving the stimulating element. If stimulation of re- combination occurred by the induction of a double- strand break within URA3, one would predict that be- cause of the absence of homology on the opposite chromatid those recombination events would be inev- itably accompanied by the loss of the URA3 insert

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PHYSICAL MONITORING OF MEIOTIC RECOMBINATION 69

Table 1. Effect of URA3 and LEU2 Inserts on Meiotic Recombination Random spore

Tetrad analysis recombinants Interval (tetratypes/total) (no./total) cM/kb

MA T-pBR322-MA T no insert 19/341 URA3 inserted heterozygous 20/209

at HindIII homozygous 64/410 URA3 inserted heterozygous 15/115

at A vaI homozygous 21/99 LEU2 inserted heterozygous 26/317

at SalI homozygous 30/247 URA3 inserted

at HindIII; pBR322 deletion I a homozygous 20/226

URA3 inserted at HindIII; pBR322 deletion//b homozygous 12/86

cry 1 -MAT no insert 17/465 URA3 inserted

9.5 kb distal heterozygous 17/233 to cryl homozygous

19/492 25/271

0.4 0.7 0.9 0.8 1.3 0.6 0.7

0.7

1.1

0.1

0.2 0.4

aDeletion I removes 2.5 kb of DNA between PvuII sites located in pBR322 and the distal MAT locus (see Fig. 1B).

bDeletion//is a SmaI-PvuII deletion removing the distal part of URA3 and 2 kb of pBR322 (see Fig. 1B). This deletion does not affect URA3 function.

(Szostak et al. 1983). We have examined gene conver- sion events in diploids heterozygous for the URA3 in- sert at either the HindIII site or the AvaI site. Among 73 tetrads containing a reciprocal exchange in the M A T-pBR322-MA T region, only 3 involved a conver- sion of the heterozygous URA3 insert (2 were 1 URA3:3 Ura- and 1 was 3 URA3:I Ura-) . Because the URA3 insert appears to be responsible for stimu- lating half of the exchange events in this region, it seems that URA3 is not frequently lost during ex- changes stimulated by the element. In addition, we have recovered a total of four gene conversion events that were not associated with exchange of the flanking M A T alleles. Two of these were of the type 3 URA3:1 Ura- and two were 1 URA3: 3 Ura- . These results are not consistent with the suggestion that stimulation of recombination by URA3 is the result of a double- strand break within URA3 sequences.

We have also carried out experiments in which the 2.2-kb XhoI-SalI yeast fragment containing LEU2 has been inserted into the SalI site of pBR322 in the M A T- pBR322-MA T region (Fig. 1B). LEU2 also appears to stimulate meiotic exchange in this interval, although not as strongly as URA3 (Table 1).

Physical Monitoring of Meiotic Recombination

The actual timing of recombination during meiosis has not been measured in any eukaryotic organism; rather, the time of exchange has been inferred from indirect methods. In the lily (Stern and Hotta 1977) or

Drosophila (Carpenter 1979, 1981), the time of recom- bination has been deduced from biochemical events or from the appearance of characteristic recombination nodules in electron micrographs. In yeast, it is possi- ble to measure the "time of commitment to meiotic re- combination," the time at which ce l l s - i f removed from conditions that promote meiosis and returned to mitotic g rowth-g ive rise to meiotic levels of recom- bination (Sherman and Roman 1963; Esposito and Es- posito 1974; Plotkin 1978). Yet even this method does not demonstrate exactly when exchanges are occur- ring. For example, it is possible that a cell becomes committed to a complex pathway of meiotic exchange events that culminate in exchange only several hours later. The ability to measure the appearance of novel restriction fragments produced by recombination in the M A T - U R A 3 - p B R 3 2 2 - M A T interval has made it pos- sible to determine when reciprocal recombination ac- tually occurs during meiosis.

We have constructed diploid strains containing the M A T - U R A 3 - p B R 3 2 2 - M A T region that are heterozy- gous for canl and are also heteroallelic at metl3 and lys2. All strains were predominantly of strain Y55 background. Cells were grown to stationary phase and then transferred to sporulation medium. At regular in- tervals, cells were removed for genetic and biochemical analysis. The time of commitment to intragenic re- combination was measured by the appearance of MET13 and L YS2 prototrophs upon return to mitotic growth on selective medium. The time of commitment to haploidization was measured by the appearance of canavanine-resistant colonies.

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70 BORTS ET AL.

DNA was prepared from the same cells and digested with the restriction endonuclease BgllI. Southern blots of the separated fragments were probed with labeled pBR322 (Fig. 2A). The autoradiographs were exam- ined for the appearance of the recombined 14-kb MA Ta-pBR322-MA Tot BgllI restriction fragment (ab- breviated a---ot), which is the product of reciprocal re- combination between the a---a and ot---ot duplications (Fig. 1A). This 14-kb band, absent in vegetative cells and early during sporulation, was first visible in the sample taken after 7 hr in sporulation medium. Twelve hours after the initiation of meiosis, the 14-kb recom- binant band had increased to a maximum level corre- sponding to 3.5~ of all pBR322-containing DNA. This value is in accord with the genetic observation that nearly 16~ of the tetrads experienced recombination within the MA T-URA3-pBR322-MA T interval; con- sequently, as much as 4~ of the pBR322-containing DNA would be expected to appear in each of the re- combined BgllI restriction fragments. The timing of appearance of this recombinant DNA fragment, along with the timing of premeiotic DNA replication, the ap- pearance of MET13 and LYS2 prototrophs, and the appearance of canavanine-resistant colonies, is plotted in Figure 2B.

In this experiment, the time of appearance of the recombined restriction fragment was about l hr later than the time of appearance of MET13 prototrophs and was indistinguishable from the time of appearance of LYS2 prototrophs. In four different experiments, the time separating the half-maximum increase in MET13 prototroph formation from the half-maxi- mum level of recombined DNA ranged from signifi- cantly less than 1 hr to almost 2 hr. From these exper- iments we conclude that the appearance of physically recombined DNA occurs no more than 2 hr later than the time of commitment to intragenic recombination.

Although the 14-kb BglII a---ot fragment appeared only during meiosis, we were concerned that this band might not actually represent a bona fide recombina- tion event. The same fragment can also be generated by an incomplete digestion of DNA by the BglII re- striction endonuclease. Thus, the appearance of this band might represent some modification of DNA dur- ing meiosis rather than a recombination event. To eliminate this possibility we constructed another dip- loid whose parental haploids had the genotypes MA Ta- URA3-pBR322-MATot and MATot-LEU2-pBR322- MATa, abbreviated a---c~ and a---a. In this diploid, the two parental BgllI fragments homologous to

I00

90

80

70

6O

5O

4O

3O

2O

IO

2 4 6 8 I0 12 Hours

Figure 2. Time course of appearance of recombined MATa-URA3-pBR322-MATc~ BglII fragment. A diploid of genotype MATa-URA3-pBR322-MATct lys2-a met13-x can1 trp5 leu2 + MATa-URA3-pBR322-MATa lys2-b metl3-y + + leu2 his6

was sporulated, and samples were taken at intervals for DNA isolation and other measurements. (A) Autoradiograph of a Southern blot, probed with pBR322, showing appearance of the 14-kb recombined MATa-URA3-pBR322-MATc~ BglII frag- ment. Times (in hours) after initiation of sporulation are indicated. Unmarked lanes represent the intervening half hours. In the original autoradiograph, the 14-kb band could be detected at approximately 7 hr into sporulation. (B) Appearance of the 14-kb recombined BgllI fragment ([3) relative to premeiotic DNA replication (O), Lys + (0) prototroph formation, Met + (V) prototroph formation, and canavanine resistance (x). Values are expressed as percent of maximum value obtained. To deter- mine the relative level of the 14-kb recombined BglII fragment at each time point, the intensity of the 14-kb recombined band was determined by densitometry and normalized to the intensity of the 9-kb parental band determined in a lighter exposure.

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PHYSICAL MONITORING OF MEIOTIC RECOMBINATION 71

pBR322 are 14 kb and 28 kb, respectively. Reciprocal recombination in this interval would yield a 32-kb a---c~ and a 9-kb a---a band. The 9-kb BglII fragment, which is a product of recombination, cannot be gen- erated by partial digestion of the DNA. When this dip- loid was placed under sporulation conditions, the 9-kb a---a recombinant fragment appeared at 7 hr, approx- imately 3 hr after meiotic DNA replication (data not shown).

Heteroallelic Recombination within the MA T-pBR322-MA T Interval

In the experiments described above, the appearance of the recombiued BglII restriction fragment occurred within 2 hr of the time of appearance of L YS2 or MET13 prototrophs, which are measures of the time of commitment to meiotic intragenic recombination. However, these two genes are not located on the same chromosome as the MA T-pBR322-MA T region. It therefore remained possible that the time of commit- ment within the interval itself was distinctly different from the time of appearance of the physically recom- bined DNA. To address this issue in more detail, we constructed a set of diploid strains where the two ho- mologs contained different mutant alleles of the yeast LEU2 gene inserted in the SalI site of the pBR322 backbone of the duplication (Fig. 3). The two alleles (leu2-K and leu-R) were created by elimination of the KpnI or EcoRI restriction sites located in the coding region of the LEU2 gene (Andreadis and Schimmel 1982). This a-URA3-(leu2-K)-pBR322-a/a-URA3- (leu2-R)-pBR322-a diploid was also heteroallelic at lys2 and met13 and heterozygous for canl.

Recombination involving the leu2 alleles inserted into the MA T-pBR322-MA T region is similar to intra- genic recombination observed at other loci in S. cere- visiae. Approximately 0.307o of all asci contained a Leu + spore, as determined by random spore analysis. Of these LEU2 prototrophs, 267 were then selected for further analysis. The results are summarized in Table 2. Nearly half of the LEU2 recombinants that were linked to M A T had apparently occurred without ex- change of flanking markers, as they remained either a- mating or c~-mating type. A nearly equal number of segregants (53070) were nonmating haploids (either a-

K o !

MATe URA3 leu2 MATe

R - i

M A T a U R A 3 leu2 M A T a Ikb

Figure 3. Location of heteroallelic leu2-R and leu2-K muta- tions within the MA T-URA3-leu2-pBR322-MA T region.

LEU2-e~ or c~-LEU2-a), as determined by the fact that they were asporogenous. Thus, approximately half of the recombination events leading to LEU2 prototro- phy were accompanied by reciprocal recombination. An approximately 1:1 ratio of intragenic recombina- tion events with and without an accompanying recip- rocal recombination event has been observed at many other loci in S. cerevisiae (Fogel et al. 1979). In addi- tion, gene conversions of the leu2-K allele (to yield an a-LEU2-a segregant) occurred approximately three times as frequently as conversions of the leu2-R allele (to give ct-LEU2-a). This same difference was found in another diploid in which the two leu2 alleles were linked to the opposite mating types (Table 2). Differ- ences in the frequency of gene conversion of different alleles has also been observed at other loci (Fogel et al. 1979).

In addition to the 267 LEU2 prototrophs where Leu * was linked to MA T, we recovered five LEU2 proto- trophs, present as diploids, in which Leu § was not linked to MAT. These are presumably the products of recombination between the leu2 insert in the MA T- URA3-leu2-pBR322-MA T region and the leu2 gene at its normal location on the opposite arm of chromo- some III.

Timing of Commitment to Intragenic and Reciprocal Recombination in the MA T-pBR322-MA T Interval

Using the diploids described above, we have per- formed experiments that examined the timing of two different recombination events during meiosis. We measured the appearance of physically recombined BglII restriction fragments reflecting a reciprocal re- combination in the MA T-URA3-leu2-pBR322-MA T interval. In addition, we measured intragenic recom- bination events in the same region, which yielded LEU2 prototrophs. The timing of these two recombination events relative to meiotic DNA replication is shown in Figure 4. In this experiment, commitment to LEU2 prototroph formation occurred slightly later than the time of commitment to formation of Met + recombi- nants. Two independent Southern blots were meas- ured to determine the time of appearance of the re- combined DNA. In this experiment, the time of appearance of physically recombined DNA was ap- proximately 2 hr later than the time of LEU2 recom- bination within the same region of the chromosome (Fig. 4B). In a second experiment, the time of appear- ance of physically recombined DNA was indistinguish- able from the time of appearance of LEU2 proto- trophs. Thus, these experiments confirm the previous conclusion that physical exchange in meiosis occurs within 2 hr of the time that cells become irreversibly committed to carry out recombination.

DNA extracted from these diploids has also yielded evidence of unequal recombination between leu2 se- quences present in the MA T-URA3-leu2-pBR322-MA T region and leu2 sequences at their normal location on the opposite arm of chromosome III. In addition to

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72 BORTS ET AL.

Table 2. Analysis of Leu § Recombinant Segregants from leu2-K/leu2-R

Genotype Allele converted Number Percent

Experiment 1 a a--LEU2--a leu2-K 47 34.3 ct--LEU2--a leu2-R 19 13.9 a--LEU2--ot or

a--LEU2--a not determined 71 51.8 Experiment 2 b

a--LEU2--a leu2-R 15 11.6 t~--LEU2--oL leu2-K 45 34.6 a--LEU2--ot or

a--LEU2--a not determined 70 53.8 aA diploid of genotype

MA Ta-URA3-(Ieu2-K)-pBR322-MA Ta MA Ta- URA3-(leu2-R)-pBR322-MA Tot

was sporulated, and Leu + random spore colonies were analyzed. bThe diploid had the genotype

MA Ta-URA3-(Ieu2-R)-pBR322-MA Ta MA Ta-URA3-(Ieu2-K)-pBR322-MA Tot

the 16.2-kb recombinant M A Ta-URA3-1eu2-pBR322- M A T ~ band that appeared at 6 hr, several fainter bands appeared at approximately the same time (Fig. 4A). One of these bands was about 13 kb, and another was about 8 kb in length. These novel fragments were not evident in samples taken at early times in meiosis and persisted through times late in meiosis, at least 18 hr after initiation of sporulation. These novel frag- ments were also present in 12-hr DNA samples derived from diploids that were homozygous for either leu2-R or leu2-K. Restriction fragment mapping, using BglII, PstI, EcoRI, PvuII, and XbaI, has suggested that these fragments were the products of crossing-over between leu2 sequences present in the M A T-URA3-1eu2-MA T region and leu2 sequences at their normal location on chromosome III (data not shown).

Effect of Meiot ic -defect ive Mutat ions on Recombina t ion

The ability to detect physically recombined DNA has also made it possible to examine the effects on recip- rocal recombination of several meiotic-defective mu- tations. To date, we have examined three -r-ray-sen- sitive mutations: rad50, rad52, and tad57. Other investigators have reported that diploids homozygous for these mutations sporulate poorly and yield vir- tually no intragenic or intergenic recombinants when cells are returned to selective growth media (Game et al. 1980; Prakash et al. 1980). However, the failure to recover recombinant spores or even recombinant veg- etative cells upon a shift from sporulation conditions back to growth medium does not demonstrate that re- combination per se has not occurred but only that vi- able recombinants are not produced.

To address this issue, we have constructed diploids that contain the MA T-URA3-pBR322-MA T region and are homozygous for one of these mutations. Diploids heterozygous for each mutation were used as controls. The diploids were also heteroallelic for lys2 and metl3

and heterozygous for canl. Cells were grown to sta- tionary phase and placed in sporulation medium, and samples were removed for biochemical and genetic analysis at various times. Because rad57-1 is a condi- tional allele that is 7-ray-sensitive and meiotic-defec- tive at or below 24~ but more normal at 34~ (Game et al. 1980), we examined homozygous rad57-1 dip- loids at the restrictive temperatures of 18~ and 24~ and at semipermissive temperatures of 30~ and 34~ In our hands, rad57-1 strains were more severely blocked in meiosis at 34~ than in sensitivity to 7-rays. The results of these experiments are summarized in Table 3.

In agreement with previous results, diploids homo- zygous for radSO, rad52, or rad57 (at its restrictive temperature) were severely blocked in the formation of prototrophs. Although none of these diploids suffered a large loss of viability during the course of these ex- periments, the few ascospores that were produced were inviable. The ability of these strains to complete meiotic recombination at the DNA level was deter- mined by monitoring the appearance of the 14-kb a- URA3-pBR322-a recombinant BglII fragment (Fig. 5). No recombinant band appeared in DNA isolated from either rad50 or rad52 homozygous diploids. In con- trast, the rad57-1 diploid clearly generated this recom- binant fragment, and the amount of this fragment pro- duced at the nonpermissive temperature was similar to that amount of recombinant fragment produced in the same diploid sporulated at the semipermissive temper- ature or in the heterozygous control.

DISCUSSION

A physical and genetic examination of a small inter- val of chromosome III has proven to be highly inform- ative about the initiation and timing of meiotic recom- bination in S. cerevisiae. For the first time it has been possible to determine when, during meiosis, physical

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PHYSICAL MONITORING OF MEIOTIC RECOMBINATION 73

L. #.

I10

I00

90

80

70

60

50

40

30

20

I0

2 4 6 8 I0 12 hr.

Figure4. Time course of appearance of recombined MA Ta-URA3-1eu2-pBR322-MA Ttx BglII fragment. Adiploidofgenotype MA Ta-URA3-(Ieu2-R)-pBR322-MA Tc~ lys2-c metl3-x canl trp5 leu2 + + MATa-URA3-(Ieu2K)-pBR322-MATa lys2-d metl3-y + + leu2 adel his6

was sporulated, and samples were taken at intervals for DNA isolation and other measurements. (A) Autoradiograph of a Southern blot, probed with pBR322, showing appearance of the 16.2-kb recombined MA Ta-URA3-1eu2-pBR322-MA T~ BglII fragment. Times (in hours) after initiation of sporulation are indicated. Unmarked lanes represent the intervening half hours. In addition to the two parental bands at 11.2 kb and 33 kb, one other band (18 kb) is seen throughout; this band represents the 18-kb BglII fragment proximal to the MATa locus and appears here because of the inclusion of a small amount of a second probe containing a portion of this region. The 16.2-kb recombined a---t~ band can be detected approximately 6 hr into sporu- lation. In addition, two other fragments of lower intensity ( - 13 kb and 8 kb) also appear. These two bands are apparently the product of unequal exchange between the leu2 region on the left arm of chromosome III and the leu2 portion of the MA T- URA3-leu2-pBR322-MA T region on the right arm of this chromosome. (B) Appearance of the recombined 16.2-kb BglII frag- ment (N), relative to premeiotic DNA synthesis (O) and "commitment to meiotic recombination," as measured by appearance of LEU2 prototrophs (A). Values are expressed as percent of the maximum values obtained. Levels of the 16.2-kb fragment were determined as described in the legend to Fig. 2.

recombination between homologous chromosomes oc- curs, relative to DNA replication and the time of com- mitment to intragenic recombination. The ability to detect restriction fragments containing physically re- combined regions has also enabled us to examine the effects of several meiotic-defective mutations that fail to yield viable recombinants but that might still allow recombination to occur. Finally, the ease with which yeast can be transformed has enabled us to begin a sys- tematic evaluation of the role of particular sequences in the stimulation of meiotic recombination.

Stimulation of Meiotic Recombination

In many fungal systems, including Saccharomyces, there is compelling evidence that recombination does not occur uniformly along a chromosome. Both the

polarity of gene conversion events within a gene and the existence of mutations that significantly increase recombination in specific intervals have been inter- preted as evidence of DNA sequences that stimulate meiotic recombination (Gutz 1971; Catcheside and Angel 1974; Fogel et al. 1979; MacDonald and White- house 1979). None of these stimulators have yet been characterized at the molecular level. Using defined DNA sequences, we have discovered that the 1.2-kb HindIII restriction fragment carrying URA3 can act as a semidominant stimulator of meiotic recombination when inserted at three different locations on chromo- some III. Our results to date indicate that URA3 does not promote exchanges primarily on one side of the element. Finally, there is no evidence that URA3 is preferentially lost during meiosis when it is heterozy- gons, as would be predicted by a model in which an

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74 BORTS ET AL.

Table 3. Meiotic Behavior of Diploids Homozygous for Different rad Mutations

Increase in Recombined Increase frequency of Increase in a---a

Diploid in DNA A s c i prototrophs frequency of Viability BglII band genotype (070) (r MET13 L YS2 Can r colonies (07o) (070) rad50 35 18 603 256 287 92 2.2 RAD50 rad50 60 0 0.7 1.4 2.4 95 < O. l radSO tad52 50 74 1937 299 862 100 3.0 RAD52 rad52 50 0 3.6 < 1 0.4 81 <O.l rad52 rad57 <24~ 50 18 346 25 108 82 3.6 RAD57 tad57 <24~ 73 0 4.2 1.9 1 53 2.3 rad5 7 rad57 >30~ 45 22 333 78 356 76 1.3 RAD5 7 rad57 >30~ 75 0 29 10 1.5 40 2.4 rad5 7

Sporulation of rad50 and rad52 strains was carried out at 30~ Values for prototroph formation, sporulation, viability, canavanine resistance (Cant), DNA replication, and amount of recombined BglII fragment were based on measurements at 24 hr. Diploids containing rad57 were sporulated at the restrictive temperatures of 24~ or 18~ and values for prototroph formation and other events were obtained after 48 hr in sporulation conditions. The results reported are averages of two experiments. In addition, the average values for the tad57 strains at the semipermissive temperatures of 30~ or 34~ are included. The increase in prototrophs and Can r colonies are expressed as the ratio of final frequencies to the frequencies obtained with stationary-phase cells plated just prior to initiating sporulation. Lcvels of the recombined a---~ BglII fragment are expressed as percent of total pBR322- containing DNA and were determined as described in the legend to Fig. 2.

enhancer of r ecombina t ion suffered a double-s t rand break (Szostak et al. 1983).

The 2.2-kb XhoI-Sa l I restr ict ion f ragment carrying the yeast L E U 2 gene also appears to st imulate meiotic exchange but not as s trongly as the URA3 f ragment . The fact that two t ranscr ibed yeast genes both st imu-

Figure 5. (Left) Effect of the rad52 mutation on recombina- tion. DNA was isolated at 3-hr intervals during sporulation from a tad52 homozygote and from a heterozygous RAD52/ rad52 control. The recombined 14-kb band, which is seen in the Rad + control, does not appear in the rad52 mutant. (Right) Effect of tad57 on recombination. DNA was isolated at 0, 12, 24, 48, and 72 hr after initiation of sporulation at the restrictive temperature of 24~ Both the rad57 homozy- gote and the heterozygous RAD57/rad57 control produced the 14-kb recombined band, even though no viable intragenic or intergenic recombinants were recovered from the tad57 homozygote (see Table 3).

late recombina t ion raises the possibili ty tha t any tran- scribed region may st imulate recombina t ion .

Timing of Meiotic Recombination

The use o f a pair o f restr ict ion endonucleasc recog- ni t ion site po lymorph i sms has enabled us to detect physically recombined c h r o m o s o m a l regions during meiosis. In the part icular system we have used, the re- str ict ion site dif ferences are associated with codomi- nantly expressed genetic markers , so tha t genetic and physical measurements o f r ecombina t ion are made in exactly the same region. In the MAT-URA3-1eu2- pBR322-MA T interval we have been able to measure both intragenic r ecombina t ion between leu2-K and leu2-R, as well as reciprocal exchange in the entire re- gion. Since, in our best exper iments , we can detect ap- proximately 0 .5% o f the total hybridizing DNA in a recombined band, it should also be possible to mea- sure the appearance of a r ecombined f ragment gener- ated by recombina t ion between the leu2-K and leu2-R alleles. Such exper iments are in progress .

The t ime of appearance o f physically recombined D N A during meiosis occurred approximate ly 3-5 hr af ter the increase in D N A synthesis. The fo rmat ion o f recombined D N A occurred within 2 hr o f the t ime o f commi tmen t to intragenic recombina t ion , as measured by the increase in L E U 2 pro to t rophs within the same D N A segment . We assume that measur ing L E U 2 pro- t o t r o p h f o r m a t i o n wi th in the M A T - U R A 3 - l e u 2 -

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P H Y S I C A L M O N I T O R I N G OF M E I O T I C R E C O M B I N A T I O N 75

p B R 3 2 2 - M A T interval is an accurate indication o f commi tment to other recombinat ion events occurring between the f lanking M A T alleles, a l though it has not yet been demonst ra ted that commitment to intragenic recombinat ion and commitment to reciprocal ex- changes necessarily occur at the same time. Genetic studies do indicate that half o f the L E U 2 protot rophs arising in this interval are associated with an exchange of flanking markers.

In previous studies, the t ime of genetic recombina- t ion has been inferred f rom the appearance of pachy- tene structures visible in the electron microscope (Byers and Goetsch 1982) or light microscope (Will iamson et al. 1983). Our direct measurement of the t ime of re- combina t ion is consistent with these previous obser- va t ions .

The basic approach we have used can be extended to investigate recombinat ion in other intervals of the yeast genome. In addit ion, we expect that this same systcm will be extremely useful in looking for intermediates of recombinat ion, such as the format ion o f heteroduplex DNA.

Unequal Crossing-over between leu2 Regions on Different Chromosome Arms

The D N A extracted f rom diploids that carry the M A T - U R A 3 - l e u 2 - p B R 3 2 2 - M A T region and are heter- oallelic for the leu2-K and leu2-R alleles has also yielded evidence o f unequal crossing-over between the leu2-3-113 locus on the left arm of chromosome III and the M A T - l e u 2 - M A T region on the right arm. It is somewhat surprising that these apparent unequal crossing-over events between regions sharing only 2.2 kb o f homology occur so frequently. The intensity of the 8-kb band resulting f rom exchange between the two different L E U 2 regions was approximate ly 10~ of the intensity of the recombined a---~ 16.2-kb band. Thus, given that recombinat ion ;in the M A T - U R A 3 - 1 e u 2 - pBR322-MA T region occurs in nearly 20O7o of all tet- rads, unequal exchanges involving leu2 appear to oc- cur in approximately 2O7o of the cells undergoing meiosis. A similar frequency of unequal crossing-over has been observed between the duplicated M A T re- gions in the M A T - p B R 3 2 2 - M A T interval (our data) and between two Tyl elements present at two locations on the left arm of this same chromosome (Roeder 1983).

Because crossing-over between the leu2 regions on opposi te sides of the ch romosome would lead to the format ion of a large acentric f ragment and either a de- ficiency ring chromosome or a dicentric chromosome, these products would most likely not have been ob- served among viable haploid cells. The physical mon- i toring of recombinat ion has made it possible to esti- mate their frequency.

Analysis of Meiotic-defective Mutations

We have examined three 7-ray-sensitive mutat ions that all block the format ion of ascospores and the ap-

pearance of meiotic levels of recombinat ion even in cells returned to growth medium. Al though rad50 and rad52 homozygotes do indeed fail to produce physi- cally recombined DNA, rad57-1 diploids must be de- fective at some other stage o f meiosis. At the restric- tive temperature of 24~ recombinat ion occurred within the M A T - p B R 3 2 2 - M A T interval at levels simi- lar to those found in wild-type controls, even though no viable spores containing intragenic or intergenic re- combinants were recovered. Thus, the rad57-1 defect allows at least some regions of the genome to undergo nearly normal levels o f recombinat ion. This approach should make it possible to make distinctions between other phenotypical ly identical mutat ions affecting meiosis and to identify mutat ions that might produce intermediates o f recombinat ion.

ACKNOWLEDGMENTS

Our experiments grew out of a prel iminary investi- gat ion of physical moni tor ing of meiot ic recombina- t ion carried out by L. Davidow, P. Shalit , B. Byers, and B. Hall. This work was supported by grant GM- 29736 from the Nat ional Institutes of Heal th . M.L. was supported by grant DRG-596 f rom the Waiter Winchel l -Damon Runyon Cancer Fund. R.H.B. was supported by grant PF-2313 f rom the Amer ican Can- cer Society.

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