The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

Meiosis is a dynamic process in which the behavior of the chromosomes and spindle is coordinated to allow the generation of haploid gametes from diploid precursor cells. This is especially evident in the dramatic movements of the chromosomes and spindle in anaphase I, when homologous chromosomes are drawn apart. Before this, during prophase, the chromosomes align and recombine with their homologous partners while an organized array of microtubules develops. The relationship of microtubule and chromosome activity in prophase is less clear; however, there is evidence to suggest that these are not independent processes. For example, in many organisms it has been shown that chemical agents or environmental conditions that disrupt meiotic spindle development have profound effects on chiasma formation or chromosome synapsis (for review see Loidl, 1990). The interdependence of microtubule function and chromosome behavior in early meiosis is also seen in certain oocytes in which the highly condensed meiotic chromatin serves as a foundation around which the microtubules are organized (Karsenti et al., 1984; Sawada and Schatten, 1988; Theurkauf and Hawley, 1992; Heald et al., 1996; for review see Rieder et al., 1993; McKim and Hawley, 1995).

Much of early meiosis (prophase) is dedicated to the establishment of a tight association among homologous chromosomes (for review see Bascom-Slack et al., 1997). In yeast, the initial steps of this process are thought to involve pairing interactions between homologous DNA sequences, eventually leading to homologous recombination (Kleckner and Weiner, 1993). The alignment process culminates in synapsis, the tight association of homologues along the proteinaceous structure known as the synaptonemal complex (SC).1 As these events occur, the early stages of spindle formation are in progress (Moens and Rapport 1971; Byers, 1981; Padmore et al., 1991). The spindle pole bodies (SPBs) duplicate during premeiotic DNA replication and an increasingly dense nuclear microtubule array emanates from the adjacent duplicated SPBs as prophase progresses (Moens and Rapport, 1971). An early landmark in meiotic prophase is the initiation of meiotic recombination by the generation of double-strand breaks (DSBs) in the DNA (Sun et al., 1989; Padmore et al., 1991). Recombination among homologues is important for chromosome alignment, as mutants that fail to initiate recombination do not achieve full SC formation (Engebrecht and Roeder, 1989; Giroux et al., 1989; Hollingsworth and Byers, 1989; Alani et al., 1990; Rockmill and Roeder, 1990; Roeder, 1990; Loidl et al., 1994). After SC assembly, and subsequent disassembly, the SPBs migrate apart to form a short bipolar spindle that rapidly lengthens at anaphase I, moving the homologues away from each other (Moens and Rapport, 1971; Byers, 1981; Padmore et al., 1991). In meiosis II the chromosomes undergo an equational division in which the sister chromatids separate away from one another.

Motor proteins have been implicated as playing critical roles in spindle assembly and function, and in chromosome segregation (for review see McIntosh and Pfarr, 1991; Sawin and Endow, 1993; Hoyt, 1994; Walczak and Mitchison, 1996). Whereas many of these studies have focused on mitotic functions, the analogous meiotic functions undoubtedly require motor proteins as well. Additionally, the spindle and chromosome behaviors that are unique to meiosis might require meiosis-specific motor protein activities. Among motor proteins with known meiosis-specific roles are two kinesin-like proteins (klps) from Drosophila (Endow et al., 1990; McDonald and Goldstein, 1990; Zhang et al., 1990). The nod protein has been proposed to push nonexchange chromosomes toward the center of the spindle prior to anaphase I (Afshar et al., 1995). The ncd protein has a very different function. In Drosophila oocytes, the microtubules normally assemble around a condensed meiotic chromosome mass and then become organized into a bipolar spindle. ncd mutant oocytes form wide or multipolar spindles in meiosis I (Kimble and Church, 1983; Hatsumi and Endow, 1992b). This observation, combined with the fact that the ncd protein bundles microtubules in vitro (McDonald et al., 1990), has led to the hypothesis that, in vivo, the ncd protein works to organize microtubule ends into spindle poles in meiosis I (Hatsumi and Endow, 1992a).

The yeast motor protein Kar3p shows structural and functional similarities to ncd protein. Unlike most klps, both Kar3p and ncd protein contain their conserved motor domains at the carboxy terminus and their nonconserved regions at the amino terminus. In addition, both proteins have been shown to move toward the minus ends of microtubules in vitro (McDonald et al., 1990; Walker et al., 1990; Endow et al., 1994). KAR3 was originally isolated in a screen for mutants defective for the fusion of gametic nuclei (karyogamy) in newly formed zygotes (Polaina and Conde, 1982). During karyogamy, Kar3p, in association with the protein Cik1, is found in the cytoplasm where it localizes to microtubules and SPBs (Meluh and Rose, 1990; Page et al., 1994). Kar3p may facilitate migration of the zygotic nuclei toward one another by cross-linking interdigitated cytoplasmic microtubules that radiate from SPBs within the two nuclei, and moving toward the minus end (the end at the SPB) of one microtubule while maintaining a hold on the other (Meluh and Rose, 1990). Additionally, Kar3p has the ability to depolymerize microtubules in vitro and may act at the SPB to shorten microtubules connecting the two nuclei (Endow et al., 1994). A characterization of spindle behavior in kar3 cells in mitosis suggests that depolymerization may be an important in vivo function as well (Saunders et al., 1997a).

KAR3 is important, but not essential, for mitosis. Mitotic cultures of kar3 mutants produce inviable large-budded cells containing short mitotic spindles (Meluh and Rose, 1990). Based on these phenotypes, it was proposed that KAR3 is involved in spindle development. This model is supported by the demonstration that certain kar3 alleles can suppress cin8-3 kip1Δ double mutants that have defects in mitotic spindle development (Saunders and Hoyt, 1992; Hoyt et al., 1993). A kar3Δ mutation in combination with a spindle checkpoint mutation (mad2, Li and Murray, 1991; bub, Hoyt et al., 1991) results in lethality, suggesting that in kar3 mutants, checkpoint genes must be used to delay entry into anaphase while a functional mitotic spindle is developed (Roof et al., 1992).

Here we demonstrate that KAR3 is essential for passage through meiosis I. kar3 mutants exhibit a novel meiotic phenotype that suggests Kar3p participates in meiotic events that culminate in chromosome synapsis.

Materials And Methods

Genetic Procedures

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 Canr Cyhr 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.

Plasmids and Strain Constructions

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.

Cytology

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).

Commitment to Heteroallelic Interhomologue Recombination

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.

Analysis of Double-Strand Breaks

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.

Results

Spindle Morphology of kar3 Mutants

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.

kar3 Null Mutants Fail to Produce Haploid Meiotic Products

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, Cyhr, Canr 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, Canr, Cyhr 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% Canr, Cyhr, Ade cells). No Ade, Canr, Cyhr 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.

kar3 Mutants Arrest Reversibly in Meiosis

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 to Heteroallelic Interhomologue Recombination in kar3 Mutants

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.

Double-Strand Break Formation in kar3 Mutants

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.

kar3 Mutants Fail to Form Mature SC

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.

Discussion

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.

Acknowledgments

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.

Abbreviations used in this paper

     
  • 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

References

References
Afshar
K
,
Barton
NR
,
Hawley
RS
,
Goldstein
LSB
DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein
Cell
1995
81
129
138
[PubMed]
Alani
E
,
Padmore
R
,
Kleckner
N
Analysis of wild-type and rad50mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination
Cell
1990
61
419
436
[PubMed]
Bascom-Slack, C.A., L.O. Ross, and D.S. Dawson. 1997. Chiasmata, crossovers, and meiotic chromosome segregation. In Advances in Genetics. Vol. 35. J. Hall and J. Dunlap, editors. Academic Press, Inc., San Diego, CA. 253–284.
Baudat
F
,
Nicolas
A
Clustering of meiotic double-strand breaks on yeast chromosome III
Proc Natl Acad Sci USA
1997
94
5213
5218
[PubMed]
Bishop
DK
,
Park
D
,
Xu
L
,
Kleckner
N
DMC1: a meiosis-specific yeast homolog of E. coli recArequired for recombination, synaptonemal complex formation, and cell cycle progression
Cell
1992
69
439
456
[PubMed]
Byers, B. 1981. Cytology of the yeast life cycle. In The Molecular Biology of the Yeast Saccharomyces cerevisiae. Vol. I. J.N. Strathern, E.W. Jones, and J.R. Broach, editors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 59–96.
de Massy
B
,
Baudat
F
,
Nicolas
A
Initiation of recombination in Saccharomyces cerevisiaehaploid meiosis
Proc Natl Acad Sci USA
1994
91
11929
11933
[PubMed]
Dernburg, A.F., J.W. Sedat, W.Z. Cande, and H.W. Bass. 1995. Cytology of telomeres. In Telomeres. E.H. Blackburn and C.W. Greider, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 295–338.
Dresser
ME
,
Giroux
CN
Meiotic chromosome behavior in spread preparations of yeast
J Cell Biol
1988
106
567
573
[PubMed]
Endow
SA
,
Henikoff
S
,
Soler-Niedziela
L
Mediation of meiotic and early mitotic chromosome segregation in Drosophilaby a protein related to kinesin
Nature (Lond)
1990
345
81
83
[PubMed]
Endow
SA
,
Kang
SJ
,
Satterwhite
LL
,
Rose
MD
,
Skeen
VP
,
Salmon
ED
Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends
EMBO (Eur Mol Biol Organ) J
1994
13
2708
2713
[PubMed]
Engebrecht
J
,
Roeder
GS
Yeast mer1mutants display reduced levels of meiotic recombination
Genetics
1989
121
237
247
[PubMed]
Esposito
MS
,
Esposito
RE
Genetic recombination and commitment to meiosis in Saccharomyces.
Proc Natl Acad Sci USA
1974
71
3172
3176
[PubMed]
Giroux
CN
,
Dresser
ME
,
Tiano
HF
Genetic control of chromosome synapsis in yeast meiosis
Genome
1989
31
88
94
[PubMed]
Goldman
ASH
,
Lichten
M
The efficiency of meiotic recombination between dispersed sequences in Saccharomyces cerevisiaedepends on their chromosomal location
Genetics
1996
144
43
55
[PubMed]
Goldway
M
,
Sherman
A
,
Zenvirth
D
,
Arbel
T
,
Simchen
G
A short chromosomal region with major roles in yeast chromosome IIImeiotic disjunction, recombination, and double strand breaks
Genetics
1993
133
159
169
[PubMed]
Hatsumi
M
,
Endow
SA
The Drosophilancd microtubule motor protein is spindle associated in meiotic and mitotic cells
J Cell Sci
1992a
103
1013
1020
[PubMed]
Hatsumi
M
,
Endow
SA
Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis
J Cell Sci
1992b
101
547
559
[PubMed]
Heald
R
,
Tournebize
R
,
Blank
T
,
Sandaltzopoulos
R
,
Becker
P
,
Hyman
A
,
Karsenti
E
Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts
Nature (Lond)
1996
382
420
425
[PubMed]
Hollingsworth
NM
,
Byers
B
HOP1: a yeast meiotic pairing gene
Genetics
1989
121
445
462
[PubMed]
Hoyt
MH
Cellular roles of kinesin and related proteins
Curr Opin Cell Biol
1994
6
63
68
[PubMed]
Hoyt
MA
,
Totis
L
,
Tibor
B
,
Roberts
S. cerevisiaegenes required for cell cycle arrest in response to loss of microtuble function
Cell
1991
66
507
517
[PubMed]
Hoyt
MA
,
He
L
,
Totis
L
,
Saunders
WS
Loss of function of Sacchromyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3motor domain mutations
Genetics
1993
135
35
44
[PubMed]
Karsenti
E
,
Newport
J
,
Hubble
R
,
Kirschner
M
Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into xenopus eggs
J Cell Biol
1984
98
1730
1745
[PubMed]
Kimble
M
,
Church
K
Meiosis and early cleavage in Drosophila melanogaster eggs: effect of the claret non-disjunctional mutation
J Cell Sci
1983
62
301
318
[PubMed]
Kleckner
N
,
Weiner
B
Potential advantages of unstable interactions for pairing of chromosomes in meiotic, somatic, and pre-meiotic cells
Cold Spring Harb Symp Quant Biol
1993
58
553
565
[PubMed]
Kurihara
LJ
,
Stewart
BG
,
Gammie
AE
,
Rose
MD
Kar4p, a karyogamy-specific component of the yeast pheromone response pathway
Mol Cell Biol
1996
16
3990
4002
[PubMed]
Li
R
,
Murray
AW
Feedback control of mitosis in budding yeast
Cell
1991
66
519
531
[PubMed]
Loidl
J
The initiation of meiotic chromosome pairing: the cytological view
Genome
1990
33
759
778
[PubMed]
Loidl
J
,
Nairz
K
,
Klein
F
Meiotic chromsome synapsis in a haploid yeast
Chromosoma (Berl)
1991
100
221
228
[PubMed]
Loidl
J
,
Klein
F
,
Scherthan
H
Homologous pairing is reduced but not abolished in asynaptic mutants of yeast
J Cell Biol
1994
125
1191
1200
[PubMed]
Lydall
D
,
Nikolsky
Y
,
Bishop
DK
,
Weinert
T
A meiotic recombination checkpoint controlled by mitotic checkpoint genes
Nature (Lond)
1996
383
840
843
[PubMed]
McDonald
H
,
Stewart
R
,
Goldstein
L
The kinesin-like ncd protein of Drosophilais a minus end-directed microtubule motor
Cell
1990
63
1159
1165
[PubMed]
McDonald
HB
,
Goldstein
LSB
Identification and characterization of a gene encoding a kinesin-like protein in Drosophila
Cell
1990
61
991
1000
[PubMed]
McIntosh
JR
,
Pfarr
CM
Mitotic motors
J Cell Biol
1991
115
577
585
[PubMed]
McKim
KS
,
Hawley
RS
Chromosomal control of meiotic cell division
Science (Wash DC)
1995
270
1595
1601
[PubMed]
Meluh
PB
,
Rose
MD
KAR3, a kinesin-related gene required for yeast nuclear fusion
Cell
1990
60
1029
1041
[PubMed]
Moens
PB
,
Rapport
E
Spindles, spindle plaques and meiosis in the yeast Saccharomyces cerevisiae(Hansen)
J Cell Biol
1971
50
344
361
[PubMed]
Nicolas
A
,
Treco
D
,
Schultes
NP
,
Szostak
JW
An initiation site for meiotic gene conversion in the yeast Saccharomyces cerevisiae.
Nature (Lond)
1989
338
35
39
[PubMed]
Padmore
R
,
Cao
L
,
Kleckner
N
Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae.
Cell
1991
66
1239
1256
[PubMed]
Page
BD
,
Satterwhite
LL
,
Rose
MD
,
Snyder
M
Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein
J Cell Biol
1994
124
507
519
[PubMed]
Polaina
J
,
Conde
J
Genes involved in the control of nuclear fusion during the sexual cycle of Saccharomyces cerevisiae.
Mol Gen Genet
1982
186
253
258
[PubMed]
Pringle
JR
,
Adams
AE
,
Drubin
DG
,
Haarer
BK
Immunofluorescence methods for yeast
Methods Enzymol
1991
194
565
602
[PubMed]
Rieder, C.L., J.G. Ault, U. Eichenlaub-Ritter, and G. Sluder. 1993. Morphogenesis of the mitotic and meiotic spindle: conclusions obtained from one system are not necessarily applicable to the other. In Chromosome Segregation and Aneuploidy. Vol. 72. B.K. Vig, editor. Springer-Verlag, Berlin, Germany. 183–197.
Rocco
V
,
de Massy
B
,
Nicholas
A
The Saccharomyces cerevisiae ARG4initiator of meiotic gene conversion and its associated double-strand DNA breaks can be inhibited by transcription interference
Proc Natl Acad Sci USA
1992
89
12068
12072
[PubMed]
Rocco
V
,
Nicholas
A
Sensing of DNA nonhomology lowers the initiation of meiotic recombination in yeast
Genes Cells
1996
1
645
661
[PubMed]
Rockmill
JB
,
Roeder
GS
Meiosis in asynaptic yeast
Genetics
1990
126
563
574
[PubMed]
Roeder
GS
Chromosome synapsis and genetic recombination: their roles in meiotic chromosome segregation
Trends Genet
1990
6
385
389
[PubMed]
Roof
D
,
Meluh
P
,
Rose
M
Multiple kinesin-related proteins in yeast mitosis
Cold Spring Harb Symp Quant Biol
1992
61
693
703
Saunders
W
,
Hoyt
MA
Kinesin-related proteins required for structural integrity of the mitotic spindle
Cell
1992
70
451
458
[PubMed]
Saunders
W
,
Hornack
D
,
Lengyel
V
,
Deng
C
The Saccharomyces cerevisiaekinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules
J Cell Biol
1997a
137
417
431
[PubMed]
Saunders
W
,
Lengyel
V
,
Hoyt
MA
Mitotic spindle function in Saccharomyces cerevisiaerequires a balance between different types of kinesin-related motors
Mol Biol Cell
1997b
8
1025
1033
[PubMed]
Sawada
TO
,
Schatten
G
Microtubules in Ascidianeggs during meiosis, fertilization and mitosis
Cell Motil Cytoskeleton
1988
9
219
230
[PubMed]
Sawin
KE
,
Endow
SA
Meiosis, mitosis, and microtubule motors
Bioessays
1993
15
399
407
[PubMed]
Sherman, F., G. Fink, and J. Hicks. 1986. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 163–169.
Sherman
F
,
Roman
H
Evidence for two types of allelic recombination in yeast
Genetics
1963
48
255
261
[PubMed]
Southern
EM
Detection of specific sequences among DNA fragments separated by gel electrophoresis
J Mol Biol
1975
98
503
517
[PubMed]
Sun
H
,
Treco
D
,
Schultes
NP
,
Szostak
JW
Double strand breaks at an initiation site for meiotic gene conversion
Nature (Lond)
1989
338
87
90
[PubMed]
Sym
M
,
Engebrecht
J
,
Roeder
GS
ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis
Cell
1993
72
365
378
[PubMed]
Sym
M
,
Roeder
GS
Zip1-induced changes in synaptonemal complex structure and polycomplex assembly
J Cell Biol
1995
128
455
466
[PubMed]
Theurkauf
WE
,
Hawley
RS
Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nodkinesin-like protein
J Cell Biol
1992
116
1167
1180
[PubMed]
Walczak
CE
,
Mitchison
TJ
Kinesin-related proteins at mitotic spindle poles: function and regulation
Cell
1996
85
943
946
[PubMed]
Walker
RA
,
Salmon
ED
,
Endow
SA
The Drosophilaclaret segregation protein is a minus-end directed motor molecule
Nature (Lond)
1990
347
780
782
[PubMed]
Weiner
BM
,
Kleckner
N
Chromosome pairing via multiple intersitial interactions before and during meiosis in yeast
Cell
1994
77
977
991
[PubMed]
Xu
L
,
Keeney
S
,
Kleckner
N
Sequence non-specific double strand breaks and interhomolog interactions prior to double strand break formation at a meiotic recombination hot spot in yeast
EMBO (Eur Mol Biol Organ) J
1995
14
5115
5128
[PubMed]
Zhang
P
,
Knowles
BA
,
Goldstein
LSB
,
Hawley
RS
A kinesin-like protein required for distributive chromosome segregation in Drosophila.
Cell
1990
62
1053
1062
[PubMed]

C.A. Bascom-Slack was supported in part by a Philanthropic Educational Organization Scholar Award and a National Institutes of Health predoctoral grant (T32 GM07310).

Address all correspondence to Dean S. Dawson, Tufts University, Department of Molecular Biology and Microbiology, 136 Harrison Avenue, Boston, MA 02111. Tel.: (617) 636-6750. Fax: (617) 636-0337. E-mail: ddawson@opal.tufts.edu