The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

Synthetic complete medium (CM) and rich yeast extract, peptone and dextrose medium (YPD) for growth of yeast strains have been described (Sherman et al., 1986). CM lacking one or more nutrient(s) is denoted by indicating the nutrient missing (e.g., CM-Arg is CM without arginine). Medium used to select for Can^r Cyh^r spores was CM-Arg-supplemented with cyclohexamide (10 μg/ml) and canavanine (60 μg/ml). For synchronous liquid sporulation, cells were grown at 30°C in presporulation medium (2% potassium acetate, 2% peptone, 1% yeast extract) to a concentration of ∼5 × 10⁷ cells/ml, washed in water, and incubated at the same density in 1% potassium acetate at 30°C with vigorous shaking (200 rpm). Presporulation medium and 1% potassium acetate were supplemented with nutrients for which the strain was deficient at one-fourth and one-half the concentrations recommended for CM, respectively.

Genotypes of all strains are as indicated in Table I. All strains used are S288C derivatives (Nicolas et al., 1989). MR820 (a gift from M. Rose, Princeton University, Princeton, NJ) contains a 3,461-bp PstI-BamHI KAR3 fragment in a YCp50 vector. To construct pD154.12, the 3,692-bp NarI-BamHI fragment from MR820 was inserted between the NarI and BamHI sites of the LEU2, and CEN3 vector, pUN105. pD153.9 was constructed by modifying MR820. A 1,528-bp BglII fragment from MR820 corresponding to the coding sequence for KAR3 (amino acids 12–571) was replaced by a BamHI fragment that carries the HIS3 gene.

To construct DJ1 + MR820, a and α haploids containing MR820 were transformed with the SspI-EcoRV kar3Δ::HIS3 fragment from pD153.9. His⁺ transformants were subjected to Southern blot analysis to confirm that the chromosomal KAR3 locus had been replaced by the kar3Δ::HIS3 fragment. These two haploid strains were mated to produce DJ1 + MR820. DJ1 was created by growing DJ1 + MR820 nonselectively at 25°C to allow isolation of derivatives that had lost MR820. DJ1 + pD154.12 was created by transforming DJ1 + MR820 with pD154.12 and then growing nonselectively to allow isolation of derivatives that had lost MR820. Dd519 + MR820 and Dd519 were constructed similarly to DJ1 + MR820 and DJ1. DC99-1, DC99-1C, DC152, and DC152B were constructed by standard genetic crosses.

Cells were induced to undergo synchronous sporulation in liquid medium as described in Genetic Procedures. At desired times after transfer to 1% KoAc, 7–10 ml of culture were removed and processed for immunofluorescence or EM. Preparation of DJ1 and DJ1 + pD154.12 cells for tubulin immunofluorescence was as described (Pringle et al., 1991). Monoclonal rat anti–tubulin antibody (Accurate Chemical and Scientific Corp., Westbury, NY) was used at a dilution of 1:200. Anti–rat FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at a dilution of 1:200. Mounting medium containing 25 μg/ml of 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was added and slides were viewed by fluorescence microscopy on a microscope using a 100× plan fluor lens (Axioscop; Carl Zeiss, Inc., Thornwood, NY).

Preparation of Dd519 and Dd519 + MR820 cultures for chromosome spreads was as previously described (Dresser and Giroux, 1988). The spreading procedure used was that described by (Loidl et al., 1991), except the slides were coated with poly-l-lysine before the addition of cells. Anti-Zip1 immunostaining was performed as in Sym et al. (1993), except that Zip1p antibody was used at a dilution of 1:100 in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 2% BSA).

For visualization of SCs by EM, 7–10 ml of cells were removed 14–16 h after cells were induced to sporulate, and were prepared for EM as in Loidl et al. (1991).

Interhomologue recombination was measured by methods previously described (Sherman and Roman, 1963; Esposito and Esposito, 1974). Isogenic KAR3 (Dd519 + MR820) and kar3Δ (Dd519) strains heteroallelic for mutations at arg4 were induced to undergo synchronous sporulation. Aliquots were removed at timed intervals following meiotic induction and plated onto YPD plates to measure the total cell viability and onto CM-Arg plates to measure the number of Arg⁺ recombinants. The frequency of interhomologue recombination was calculated by dividing the total number of Arg⁺ colony forming units (CFUs) by the total number of viable cells.

For experiments using the rad50S strains DC99-1 and DC99-1C, cells were sporulated and, at each time point, 25 ml were removed, mixed with 25 ml of 95% ethanol and 1.25 ml of 0.5 M EDTA, and stored at −20°C overnight. Chromosomal DNA was extracted as described (Rocco et al., 1992). Approximately 5 μg of each sample were digested with BglII, subjected to electrophoresis, and then transferred to a nylon membrane (Boehringer Mannheim Corp., Indianapolis, IN) for Southern analysis (Southern, 1975). The ARG4 probe used was a 3.3-kb PstI fragment from pNPS558 (gift from N. Schultes and J. Szostak, Massachusetts General Hospital, Boston, MA). The THR4 probe used was a 4.9-kb PstI fragment from pMJ338 (gift from M. Lichten, National Institutes of Health, Bethesda, MD). Probe fragments were radioactively labeled using the Stratagene Prime-It system (La Jolla, CA).

DNA fragments were quantified using a Molecular Dynamics Phosphor Imager (Sunnyvale, CA). The DSB frequency was estimated by dividing the amount of radioactivity in the major DSB fragment by the total amount of radioactivity in that lane. The rates of DSB formation were calculated by determining the slope of the curves after normalizing for the maximal frequency of DSBs.

The approaches described above were also used to examine DSBs in the RAD50 strains Dd519 and Dd519 + MR820 immediately after transfer to sporulation medium and at intervals after transfer to sporulation medium.

It has been reported that kar3 null mutants fail to produce spores when assayed using bright field microscopy (Hoyt et al., 1993; Kurihara et al., 1996). To determine the stage of the meiotic block, homozygous kar3 mutant cells were analyzed by observing the spindle morphology and DNA staining patterns throughout meiosis (Fig. 1). At 5 h after the induction of meiosis, wild-type cells exhibit tubulin arrays typical of yeast prophase spindles (microtubules emanating from duplicated, unseparated SPBs). At the same time, most mutant cells possess similar tubulin arrays that appear slightly longer than those in wild-type cells. Additionally, the mutants often have elongated cytoplasmic microtubules that are not seen in wild-type cells (Fig. 1, 5 h), indicating that the kar3 mutation has an effect early in prophase. By 12 h most wild-type cells have spindles characteristic of metaphase I or later stages of meiosis, and at 24 h the wild-type cells have nearly all formed tetrads. In contrast, kar3 mutants continue to exhibit bush-like tubulin staining. At late time points (24 h and beyond, not shown), monopolar arrays with numerous and/or very long projections become more common (an example is shown in the 24-h time point, Fig. 1).

Staining of DNA with DAPI was also used to monitor progress through meiosis. Binucleate and tetranucleate cells were extremely rare in the mutant at late time points, indicating that these cells do not progress beyond anaphase I. To quantitatively demonstrate the lack of postprophase spindles in the mutant, we counted the number of recognizable postprophase cells compared to the total number of cells for both the wild type and mutant throughout meiosis. Only cells that exhibited unquestionable postprophase morphologies were classified as postprophase. Generally, anaphase spindles, some metaphase spindles, and binucleate and tetranucleate nuclei were easily distinguished. For wild-type cells, the fraction classified as postprophase was an underestimate as not all cells stained adequately for classification, and for others, critical features were not in the plane of focus in the photographs used for the quantification. The results shown in Fig. 2 indicate that by 12 h there is a significant difference in the number of postprophase cells for the mutant versus the wild type, and at 24 h no postprophase cells are observed in the kar3 mutants whereas 21.5% of the wild-type cells show unquestionable progression beyond prophase.

To quantitatively measure whether the meiotic block in kar3 mutants was absolute, we designed a strain that would allow us to measure the efficiency with which a kar3 mutant produces viable haploid meiotic products compared to a KAR3 strain. Briefly, a homozygous kar3Δ::HIS3 diploid strain (TC12) was heterozygously marked for both canavanine and cyclohexamide resistances (CAN1/can1, CYH2/ cyh2). In both cases, the sensitive allele is dominant; therefore, with the exception of rare double recombinants, these diploids are unable to grow on medium containing canavanine and cyclohexamide. One quarter of the spores produced by such a diploid will inherit both the can1 and cyh2 genes and will grow in the presence of both compounds. In addition, the diploid strain is heterozygous for the ADE2 gene (ADE2/ade2) and produces white colonies, but haploid cells that have inherited only the recessive ade2 allele will produce red colonies. The haploidization frequency of this kar3 diploid was measured by inducing a culture to undergo synchronous meiosis and plating cells at timed intervals to measure the ratio of Ade⁻, Cyh^r, Can^r cells to the total number of viable cells (Fig. 3). An isogenic strain carrying a single copy number plasmid with the wild-type KAR3 gene (DJ1 + pD154.12) was used as the wild-type control. The wild-type culture began to produce Ade⁻, Can^r, Cyh^r cells at 8 h, and by 48 h 9.18% of the cells had this phenotype (100% sporulation would be expected to yield ∼12.5% Can^r, Cyh^r, Ade⁻ cells). No Ade⁻, Can^r, Cyh^r colonies were ever detected for the mutant even after 3 d in sporulation medium (Fig. 3), indicating that the kar3 defect causes a robust block of the meiotic process.

Whereas kar3 mutants appear to arrest during meiosis, they remain viable in sporulation medium. The total number of viable cells in the above experiments was determined by plating on rich nonselective medium that supports vegetative growth at each time point. Viability of the kar3 mutants remains high for the first 8 h of meiosis and then gradually decreases. In a series of experiments, ⩾50% of kar3 cells remain viable between 24–48 h of meiosis (data not shown). This demonstrates that although the kar3 cells are blocked in meiosis so that they cannot produce viable spores, the meiotic arrest is overcome when the cells return to mitotic growth.

Commitment of cells to meiotic levels of interhomologue recombination is a landmark event of meiosis that occurs in prophase I. To assay for interhomologue recombination, the frequency of prototroph formation at heteroallelic loci is measured at timed intervals in meiosis by returning meiotically-induced cells to vegetative growth (Sherman and Roman, 1963; Esposito and Esposito, 1974). Using a strain containing a pair of closely linked arg4 heteroallelic mutations, we were able to quantify the frequency of interchromosomal recombination events by selecting for Arg⁺ cells. The kar3 version of this strain was unable to commit to wild-type levels of heteroallelic recombination after 27 h, by which time the KAR3 control strain had reached maximal levels of recombination (Fig. 4). The mutant exhibited a gradual accumulation of Arg⁺ cells. By 27 h, the kar3 mutants showed ∼5% of wild-type levels of recombinants.

The reduced commitment to recombination could be an indication of a failure to either initiate or resolve recombination events. DSBs in the DNA are the initiating events in meiotic recombination and can be monitored using Southern analysis to detect the appearance of diagnostic restriction fragments. The ability of a kar3 mutant to form DSBs was measured by monitoring the accumulation of DSB products throughout meiosis in the region upstream of the ARG4 gene which contains hot spots for meiotic recombination (Sun et al., 1989). Identification of break products is facilitated by the presence of the rad50S mutation which allows DSBs to form but prevents their processing, resulting in the accumulation of what would otherwise be a transient product (Alani et al., 1990). The interhomologue recombinants measured in the commitment to recombination assay (above) are thought to arise from events initiated at DSBs. Restriction fragments diagnostic of the DSB products were apparent in both a KAR3 wild-type and kar3 mutant strain (Fig. 5,A). In the mutant, the breaks accumulated to 18.6% of wild-type levels (Fig. 5 C). The appearance of DSBs in the kar3 mutant demonstrates that, although this strain is unable to commit to wild-type levels of meiotic recombination, it is capable of initiating substantial levels of DSBs in a rad50S background. To verify that the differences in DSB accumulation are not unique to the ARG4 locus, we quantified DSBs upstream of the ARE1 gene on chromosome III, another hot spot for recombination (Goldway et al., 1993; Baudat and Nicolas, 1997). In a kar3 mutant, breaks accumulate to 33% of the level detected in an isogenic KAR3 strain (data not shown). For both the ARG4 and ARE1 loci, DSBs accumulate to a lesser amount in a kar3 mutant than in an isogenic KAR3 strain in the time span over which the breaks were assayed.

Certain mutants (e.g., rad50S and dmc1) able to form, but not properly process, DSBs accumulate broken DNA strands and exhibit a prophase arrest characterized by reduced levels of commitment to heteroallelic recombination (Alani et al., 1990; Bishop et al., 1992), phenotypes similar to those of kar3 mutants. To determine whether a kar3 mutation alone causes accumulation of DSBs, we tested for the appearance of DSBs in a strain that was wild-type at the RAD50 locus. Low levels of restriction fragments diagnostic of DSB formation appeared transiently in the kar3 mutants, concomitant with their appearance in the wild-type control (not shown). The transient nature of the diagnostic restriction fragments suggests the DSBs can be repaired or processed to some other intermediate in the kar3 mutants.

Many mutations that prevent interhomologue recombination also prevent complete SC formation. To test the ability of kar3 mutants to form SC, we immunostained spread meiotic chromosomes harvested 14 h after induction of meiosis with antibody to Zip1, a component of the SC (Sym et al., 1993). Approximately equal fractions of mutant and wild-type nuclei showed Zip1 staining, indicating that the kar3 mutation does not block progression to this stage. In wild-type samples, most Zip1-stained nuclei (79%, n = 160) exhibited worm-like structures indicative of full-length SC. In many of these nuclei ∼16 synapsed pairs were clearly evident (Fig. 6,a). Mutant nuclei exhibited a range of Zip1 staining patterns, though none showed characteristic full-length SC staining. 46% (n = 129) of the kar3 Zip1-stained nuclei showed numerous dots or elongated signals indicative of partial or discontinuous SC formation (Fig. 6, b and c; the remaining 56% showed largely homogeneous nuclear staining). ∼25% of the kar3 cells (32 of 129 spreads) with Zip1 staining contained a single brightly stained structure (Fig. 6, b and c), which probably indicates the presence of a polycomplex. These structures were seen in <3% of the wild-type spreads (4 of 160) at the same time point. Polycomplexes are common in pachytene nuclei of mutants unable to assemble SC (Alani et al., 1990; Bishop et al., 1992; Loidl et al., 1994), or can result from overexpression of Zip1p (Sym and Roeder, 1995). The SC defect of the kar3 mutants does not improve over time. At a later time point, when >50% of the KAR3 cells have produced tetrads, the DNA in the kar3 mutants has decondensed and the Zip1 staining is barely visible (data not shown).

Consistent with the Zip1 staining, observation of silver-stained pachytene spreads with the electron microscope demonstrated a variety of SC phenotypes for the kar3 mutants, ranging from completely unstructured to nearly wild-type levels of SC formation (Fig. 7). Maximal SC structures in the wild-type and in the kar3 mutants appear simultaneously. Whether the SC present in the mutants represents small regions of synapsis among homologues or regions of illegitimate synapsis between nonhomologous chromosomes cannot be determined from this experiment. The electron microscopic observation of the mutants also revealed densely-stained bodies in many cells; these may represent polycomplexes. In many of the mutant and wild-type samples, duplicated but unseparated SPBs have been preserved and are visible in the mutant nuclei (Fig. 7, E and F). The timing of the appearance and subsequent disappearance of SC-related structures is similar in the wild-type and in the kar3 mutants, yet the mutants fail to assemble metaphase spindles. This indicates that the mutants can proceed through, but cannot exit from, prophase.

Kar3p is a versatile protein. It acts in the cytoplasm to mediate nuclear fusion when zygotes are formed (Meluh and Rose, 1990; Page et al., 1994), and in mitosis it localizes to the nucleus where it likely plays an important role in spindle development (Meluh and Rose, 1990; Roof et al., 1992; Page et al., 1994; Saunders et al., 1997a,b). Here we demonstrate that KAR3 is required for progression through meiosis I. Our data are consistent with a model in which Kar3p function is a prerequisite for homologous chromosome synapsis.

The difference between the mitotic and meiotic phenotypes of kar3 mutants raises the question of whether Kar3p performs the same role in these two types of chromosome segregation. In meiosis, kar3 cells arrest before the development of bipolar metaphase spindles, whereas the arrest exhibited by a fraction of cells in vegetative kar3 cultures features metaphase spindles (Meluh and Rose, 1990; Roof et al., 1992). In our strain background, pedigree analysis reveals that about one in three mitotic cell divisions produces a daughter that arrests, usually as a large-budded cell (Shanks, R., and D. Dawson, unpublished observations). If the kar3 mitotic and meiotic arrests are the same, then one would expect arrested meiotic cells to be inviable when returned to vegetative growth conditions. However, kar3 mutant cells arrested in meiosis can resume mitotic growth with nearly 100% efficiency. The differences between the mitotic and meiotic phenotypes of kar3 mutants could be attributable to KAR3 performing different functions in the two processes or, alternatively, to differences in the way the two processes are affected by a common defect. Regardless, it is clear from our results that KAR3 affects functions that are unique to meiosis and cause a prophase I arrest.

Though KAR3 encodes a motor protein, the kar3 meiotic arrest is most reminiscent of those exhibited by certain recombination mutants. kar3 mutants, like meiotic recombination mutants that are able to form but not properly process DSBs, show low commitment to heteroallelic recombination, failure to assemble a complete SC, and failure to complete the first meiotic division. Unlike these mutants, however, kar3 mutants do not cause DSBs to accumulate. In addition, spindle-associated defects in chromosome metabolism mutants such as rad50S and dmc1 have been reported to occur later than the aberrant tubulin arrays in kar3 mutants (Alani et al., 1990; Bishop et al., 1992).

DSBs are formed in kar3 mutants, but the level of DSBs was reduced from that of wild type. The reduction in DSB formation in kar3 strains seems likely to be an indirect result of the kar3 defect. One explanation stems from recent demonstrations that DSB levels are affected by homologous pairing interactions. Haploid chromosomes and loci without homologous pairing partners show reduced levels of DSB formation (de Massy et al., 1994; Xu et al., 1995; Rocco and Nicolas, 1996). For the two loci we have observed, our DSB results are consistent with the model that kar3 mutants are defective for meiotic homologous chromosome interactions that lead to full levels of DSB formation. The reduced frequency of DSBs alone does not explain the reduced level of heteroallelic recombination observed in the kar3 mutant (19% of wild-type DSB formation at ARG4, but 5% of wild-type heteroallelic recombination). Many explanations might account for this. First, this result would be obtained if higher levels of DSBs occur in a rad50S background (in which breaks were quantified) than in a wild-type background (in which commitment assays were performed). Second, the recombination machinery might be altered in kar3 mutants, such that heteroallelic recombinations are less likely to yield an Arg⁺ product. Finally, the disparity may reflect an inability of DSBs to be converted into interhomologue events in the kar3 background. This might be expected in a mutant defective in establishing homologous pairing interactions. In the absence of a homologous partner, breaks might be repaired from sister chromatid. Such repair events would go undetected in the commitment assay.

In addition to the recombination defects, kar3 mutants show a defect in SC formation as assayed by EM and immunofluorescence using antibodies against Zip1, a component of the SC. This indicates that KAR3 is required for events preceding chromosome synapsis. A relationship between recombination and SC formation has been well established in Saccharomyces cerevisiae. SC formation is likely dependent on recombination; mutants unable to initiate interhomologue recombination are also defective in SC formation (for review see Bascom-Slack et al., 1997). The meiotic phenotype of kar3 mutants is like that exhibited by a number of recombination mutants in that SC formation is incomplete. More SC is formed in kar3 mutants than in mutants that abolish recombination, such as rad50 and spo11 (Giroux et al., 1989; Alani et al., 1990). It has been proposed that synapsis is dependent on the initiation and maturation of recombination among homologous chromosomes (Roeder, 1990; Kleckner and Weiner, 1993). The amount of SC in a given kar3 nucleus might, therefore, be indicative of the number of successful interhomologue recombination events in that nucleus.

What is the basis of the terminal meiotic phenotype in kar3 mutants? In mitotic cells, Kar3p has been implicated in spindle development; spindle checkpoint genes are essential for viability of kar3 mutants. Is the spindle checkpoint mechanism responsible for the arrest? The kar3 mutants exhibit clear microtubule abnormalities, though it is possible this is partially due to, rather than a cause of, the arrest. Some pachytene-arrested recombination mutants develop bush-like microtubule arrays (Alani et al., 1990); kar3 mutants may share this tendency of arrested meiotic cells to accumulate aberrant microtubule arrays. The meiotic roles of the known mitotic spindle checkpoint genes have yet to be demonstrated (Hoyt et al., 1991; Li and Murray, 1991). If the mitotic checkpoint genes are responsible for the kar3 meiotic arrest, then the arrest point is different from their mitotic arrest point; kar3 mutants are blocked in prophase whereas mitotic spindle checkpoint blocks cell cycle progress at a later stage (onset of anaphase) (Hoyt et al., 1991; Li and Murray, 1991).

The recombination defects of the kar3 mutants suggest that the terminal phenotype might also be due to a recombination checkpoint mechanism. The meiotic prophase arrest of dmc1 mutants, which shows several parallels to the kar3 arrest, has been shown to be mediated by the RAD17, RAD24, and MEC1 genes that monitor DNA integrity in mitosis (Lydall et al., 1996).

The demonstration that a microtubule-dependent motor is required to achieve normal meiotic recombination establishes the importance of microtubules for meiotic events that occur well before a bipolar spindle is assembled. Studies in a variety of organisms have provided cytological evidence that chromosome positioning early in meiosis might be important for achieving subsequent synapsis (for review see Loidl, 1990). The bouquet structure, in which the ends of the chromosomes are clustered along a small region of the nuclear envelope, is generally observed during meiotic prophase before full-length SC formation in a variety of organisms (for review see Dernburg et al., 1995). These data have led to the notion that the bouquet arrangement facilitates chromosome pairing by bringing the ends of chromosomes into close proximity. In Schizosaccharomyces pombe where meiosis immediately follows karyogamy (zygotic meiosis), the telomeres lead dramatic chromosome movements throughout karyogamy and early meiosis. These movements appear to result in a clustering of the telomeres near the SPBs. In S. cerevisiae, homologous chromosomes are roughly aligned in mitosis, lose this organization in premeiotic S phase, and become realigned in meiotic prophase. It is tempting to speculate that a protein required for both karyogamy and meiosis in S. cerevisiae may play a role in spindle and chromosome movements analogous to those observed during karyogamy and early meiosis in S. pombe.

Though no specific nuclear organization of meiotic chromosomes has been observed in S. cerevisiae, EM and fluorescence in situ hybridization (FISH) experiments have indicated that at some meiotic stages, nonhomologous telomeres are in closer proximity than nonhomologous interstitial regions (Dresser and Giroux, 1988; Weiner and Kleckner, 1994). Similarly, genetic experiments that tested the frequency with which pairs of homologous sequences recombined, when placed in assorted locations on homologous and heterologous pairs of chromosomes, provided evidence for chromosomal alignment before recombination (Goldman and Lichten, 1996). The low level of interhomologue recombination measured in the kar3 strains is similar to that observed between homologous sequences placed on heterologous chromosomes–in both cases nonalignment of the chromosomes can be used to explain the observed low recombination frequency.

We propose that Kar3p acts in meiotic prophase to mediate homologous chromosome interactions either indirectly, by promoting early microtubule organization, or directly, by positioning chromosomes in a microtubule-dependent manner in the early prophase nucleus. The favorable arrangement of chromosomes in the prophase nucleus might be critical for two key meiotic events. First, the alignment of homologous chromosomes might place homologous sequences in the same subregions, thereby reducing the effective volume that must be searched to achieve the homologous DNA–DNA contacts that lead to meiotic recombination. Second, rough alignment of homologues might be a precondition for efficient synapsis. The alignment of homologues would minimize the movement required for synapsis and would reduce the chance that heterologous chromosomes would be entangled between synapsed pairs.

We wish to thank N. Kleckner (Harvard University, Cambridge, MA), D. Bishop (University of Chicago, Chicago, IL), and B. McCarroll (University of Chicago) for critical comments on the manuscript. Additionally, we thank F. Slack (Harvard University), S. Rankin (Harvard University), R. Boumil (Tufts University, Boston, MA), R. Shanks (Tufts University), and B. Kemp (Tufts University) for their helpful discussions throughout the course of this work. Plasmids were generously provided by M. Rose (Princeton University, Princeton, NJ), N. Schultes (Massachusetts General Hospital), J. Szostak (Massachusetts General Hospital), and M. Lichten (National Institutes of Health). Zip1 antibody was provided by S. Roeder (Yale University, New Haven, CT). D. Bishop (Yale University), B. Rockmill (Yale University), J. Loidl (University of Vienna, Vienna, Austria), M. Dresser (Oklahoma Medical Research Foundation, Oklahoma City, OK), C. Giroux (Wayne State University, Detroit, MI), and M. Schuster (University of Pittsburgh, Pittsburgh, PA) contributed valuable advice for chromosome spreading and EM experiments. Finally we thank R. Isberg (Tufts University) and members of his laboratory for the use of their microscope.

CFU

colony forming unit

CM

complete medium

DAPI

4′, 6-diamidino-2-phenylindole dihydrochloride

DSB

double-strand break

SC

synaptonemal complex

SPB

spindle pole body

YPD

yeast extract, peptone and dextrose medium