Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference.

The TAM1 gene of Saccharomyces cerevisiae is expressed specifically during meiosis and encodes a protein that localizes to the ends of meiotic chromosomes. In a tam1 null mutant, there is an increase in the frequency of chromosomes that fail to recombine and an associated increase in homolog nondisjunction at meiosis I. The tam1 mutant also displays an increased frequency of precocious separation of sister chromatids and a reduced efficiency of distributive disjunction. The defect in distributive disjunction may be attributable to overloading of the distributive system by the increased number of nonrecombinant chromosomes. Recombination is not impaired in the tam1 mutant, but crossover interference is reduced substantially. In addition, chromosome synapsis is delayed in tam1 strains. The combination of a defect in synapsis and a reduction in interference is consistent with previous studies suggesting a role for the synaptonemal complex in regulating crossover distribution. tam1 is the only known yeast mutant in which the control of crossover distribution is impaired, but the frequency of crossing over is unaffected. We discuss here possibilities for how a telomere-associated protein might function in chromosome synapsis and crossover interference.

During meiosis, a single round of DNA duplication is followed by two successive nuclear divisions to generate four haploid progeny from a single diploid cell. The meiosis II division resembles mitotic chromosome segregation, but the meiosis I division is unique in that homologous chromosomes disjoin from each other. A complex series of events unique to prophase of meiosis I ensures that homologs segregate reductionally at the first division.
One important aspect of meiotic prophase is a high rate of recombination between homologous chromosomes. Crossing over establishes chiasmata, which are physical connections between homologous chromosomes that persist until metaphase. Chiasmata ensure the proper orientation of chromosomes on the meiosis I spindle and therefore promote reductional chromosome segregation (for review, see Carpenter 1994). The distribution of crossovers, and the chiasmata to which they give rise, is nonrandom in two respects. First, two crossovers rarely occur closely together--a phenomenon known as crossover interference. Second, every chromosome pair (no matter how small) almost always sustains at least one crossover--referred to as obligate chiasma. A number of studies suggest that crossover interference and obligate chiasma are different manifestations of the 1Corresponding author. E-MAIL: shirleen.roeder@yale.edu; FAX (203) 432-3263. same underlying mechanism (Jones 1967;Kaback et al. 1989Kaback et al. , 1992; King and Mortimer 1990;Sym and Roeder 1994;P. Ross-Macdonald, J. Novak, and G.S. Roeder, in prep.).
The pairing of homologous chromosomes during meiotic prophase culminates in the formation of an elaborate proteinaceous structure called the synaptonemal complex (SC), which holds homologs closely apposed along their entire lengths (von Wettstein et al. 1984). In yeast, meiotic recombination and chromosome synapsis occur concurrently. Double-strand breaks (the initiators of exchange) are formed just before the initiation of synapsis (Padmore et al. 1991). Joint molecules consisting of double Holliday junctions are present during pachytene, when chromosomes are synapsed fully Kleckner 1994, 1995). Mature recombinants are produced near the end of pachytene as chromosomes begin to desynapse (Padmore et al. 1991). A number of studies suggest that the SC has a role in regulating the distribution of meiotic recombination events (Arana et al. 1987;Parker 1987;Havekes et al. 1993;Sym and Roeder 1994;Egel 1995). Perhaps the SC provides a conduit for the transmission of an inhibitory signal from one crossover site to nearby potential sites of crossing over (Egel 1978;Maguire 1988;King and Mortimer 1990).
Little is known about the processes that mediate chromosome pairing before the initiation of chromosome synapsis. In some organisms, meiotic chromosomes form a bouquet, which results from the attachment of telomeres to the nuclear membrane followed by telomere movements that result in their clustering together on one side of the nuclear membrane (for review, see von Wettstein et al. 1984;Fussell 1987;Loidl 1990;Dernburg et al. 1995). It has been suggested that bouquet formation facilitates chromosome pairing by bringing homologs into approximate register with each other, thereby reducing a three-dimensional homology search to a twodimensional search (Fussell 1987;Dernburg et al. 1995). The timing of bouquet formation is consistent with a role for the bouquet in promoting the initiation of synapsis (Dawe et al. 1994;Scherthan et al. 1996;Bass et al. 1997).
In addition to the exchange-dependent pathway of chromosome segregation described above, there exists an exchange-independent pathway of reductional chromosome segregation called distributive disjunction (Dawson et al. 1986; for review, see Carpenter 1991;Hawley and Theurkauf 1993). This alternate pathway was first observed in Drosophila females, where the obligately nonexchange fourth chromosome almost always undergoes proper meiosis I disjunction (Grell 1963). In yeast, two nonexchange artificial chromosomes usually segregate away from each other at meiosis I, as do two nonhomologous authentic chromosomes that lack their normal pairing partners (Dawson et al. 1986;Guacci and Kaback 1991;Sears et al. 1992Sears et al. , 1994. A high rate of recombination and strong interference in yeast ensure that almost all homolog pairs are chiasmate. Therefore, distributive disjunction in wild-type yeast operates as a backup system for segregating rare achiasmate chromosomes (Dawson et al. 1986).
We have characterized a novel meiosis-specific yeast gene, TAMI, whose product localizes to the ends of meiotic chromosomes. The tam l mutant displays reduced spore viability resulting from chromosome missegregation at meiosis I. Recombination is not impaired in tamI strains, but there is a defect in crossover interference. In addition, chromosome synapsis and nuclear division are delayed in the tam l mutant. In this paper we consider models for how a telomere-associated protein might be involved in chromosome synapsis and crossover interference.

Isolation of the TAM1 gene
In a screen for transposon insertions that generate IacZ fusion genes in yeast, a strain (M45) carrying a fusion gene expressed specifically in meiotic cells was identified (Burns et al. 1994). The DNA immediately adjacent to iacZ in strain M45 was cloned (Burns et al. 1994) and used to probe a blot of ordered phage and cosmid clones of yeast genomic DNA (Olson et al. 1986). The cloned sequence localizes to cosmid c9348, which maps to the left arm of chromosome XV.
A diploid homozygous for the transposon insertion displays a decrease in spore viability relative to wild type (Burns et al. 1994). A 1.8-kb XhoI fragment ( Fig. 1)  phenotype. The gene present in the subclone has been sequenced and designated YOL104c in the course of yeast genome sequencing. Throughout the rest of this paper, YOL104c will be called TAM1 (for telomere-associated meiotic protein, see below). Analysis of the sequence reveals that the TAM1 open reading frame (ORF) encodes a protein of 352 amino acids with no significant sequence homology to other proteins in databases. A consensus URS1 regulatory element (TGGGCGGCTA), shown previously to be involved in the regulation of meiosis-specific genes (Buckingham et al. 1990), is located at bases -80 to -91.

The Taml protein is meiosis-specific
Production of the Tam l protein was monitored in the original M45 strain heterozygous for a tam l ::lacZ translational fusion gene. As a control, an isogenic strain (M85; Burns et al. 1994) heterozygous for a zipl::lacZ translational fusion gene was used. ZIP1 has been shown previously to encode a meiosis-specific component of the SC (Sym et al. 1993). ~-Galactosidase assays were performed in mitosis and at various time points in meiosis. There is no detectable ~-galactosidase activity in vegetative cells, and both fusion proteins reach maximal abundance at 12-13 hr after the introduction into sporulation medium (Fig. 2). At this time, the majority of cells are in prophase, before meiosis I chromosome segregation (Sym et al. 1993). TAM1 therefore belongs to the early class of meiotic genes (Mitchell 1994). Compared with the Zipl fusion protein, the Taml-~-galactosidase fusion protein is produced at a very low level or is very unstable. At the time of maximal expression, there is 10 times more ~-galactosidase activity in the zipl::lacZ strain than in the taml::lacZ strain (Fig. 2).

The Taml protein localizes to discrete spots at the ends of meiotic chromosomes
Antibodies against the Tam l protein were generated (see Materials and Methods) and used to localize Taml on spread preparations of meiotic nuclei. At pachytene, when chromosomes are fully condensed and synapsed, the Tam l protein localizes to discrete spots at the ends of meiotic chromosomes (Fig. 3A), There are -25-35 Taml-staining foci per pachytene nucleus. No signal was observed when anti-Taml antibodies were used to stain spread meiotic chromosomes from a t a m 1 null mutant (strain PC664), indicating that the antibodies are specific to the Taml protein (data not shown). The number of chromosome ends associated with Taml spots was quantitated among individual well-defined chromosomes selected from spreads of pachytene chromosomes based on the pattern of staining with 4',6diamidino-2-phenyl-indole (DAPI) (Fig. 3B). This method of analysis eliminates chromosomes whose ends cannot be identified unambiguously. According to this analysis, 96% of chromosome ends (from a total of 56 selected chromosomes) are associated with Taml foci. No Taml foci were detected at interstitial sites on chromosomes. We cannot exclude the possiblity that Taml is located at interstital sites in amounts that are too low to be detected by immunofluorescence.
To confirm the telomeric localization of Taml, spread chromosomes from a strain carrying an epitope-tagged R A P 1 gene were stained with antibodies against Taml with DAPI (G) and anti-Zipl antibodies (H). Scale bars, 1 ~m. and the epitope. Previous studies have shown that Rapl is concentrated at the ends of chromosomes (Klein et al. 1992). There is extensive co-localization of Taml and Rap1 loci in pachytene nuclei ( Fig. 3C-E). In addition to a number of foci that stain intensely with antibodies to both Taml and Rapl, there are a number of sites that stain faintly with antibodies to Rap l, but contain no detectable Tam 1. These presumably correspond to interstitial Rap 1-binding sites (Shore and Nasmyth 198 7).
Nuclei judged to be in early prophase based on the pattern of anti-Zipl staining (Smith and Roeder 1997) also contain Taml spots, indicating that Taml localization occurs before synapsis is complete (data not shown). Tam l localization must be independent of chromosome synapsis and meiotic recombination, because Taml localizes to chromosomes in the spoll mutant (Fig. 3F), which does not initiate recombination or synapsis (Dresser and Giroux 1988;Cao et al. 1990).

A taml null mutation reduces spore viability
Null alleles of TAM1 were generated by deleting most of the coding region and replacing it with yeast selectable markers (Fig. 1). The taml null mutant is viable and does not display any obvious mitotic defects (e.g., taml strains exhibit wild-type resistance to the DNA-damaging agent, methylmethane sulfonate). When introduced into starvation medium, 86% of taml diploid ceils undergo sporulation to produce asci, compared with 96% in wild type (Table 1). Spore viability in the homozygous tam l null mutants is reduced significantly compared with wild type (Table 1). In one strain background, spore viability is 82% in taml compared with 96% in wild type. In another strain background, spore viability is 76% in the mutant compared to 94% in wild type. These differences are statistically significant (P < 0.0001). Additionally, the pattern of spore death in the taml mutant is significantly different from wild type (P < 0.0001). In tam l, 2-spore-viable and 0-spore-viable tetrads account for more than half of all tetrads containing inviable spores. In contrast, 3-spore-viable tetrads are the major source of inviable spores in wild type. taml mutants are proficient for recombination Meiotic gene conversion in tam 1 strains was assessed in two ways. Prototroph formation was measured at four different heteroallelic loci (Table 2A). tam 1 mutants display approximately wild-type levels of prototroph formation at all loci examined (0.8-to 1.6-fold decrease; differences not significant). Gene conversion was also measured by tetrad analysis (Table 2B). At five loci examined, the frequencies of 1:3 and 3:1 segregations in the tam l mutant are not significantly different from those in wild type (0.3-to 2.6-fold decrease).
Crossing over was measured in five intervals on chromosome III by tetrad analysis (Table 3). In four of the intervals measured (HML-HIS4, HIS4-CEN3, CEN3-MAT, and MAT-RAD18), the levels of crossing over in taml mutants are not significantly different from those in wild type. In the CEN3-MAT and RAD18-HMR intervals, crossing over in tam l is increased slightly compared to wild type.

taml strains undergo both homolog nondisjunction and precocious separation of sister chromatids
In a wild-type meiosis, homologous chromosomes segregate to opposite poles at meiosis I and sister chromatids segregate at meiosis II. This results in the production of four viable spores, each carrying one copy of each chromosome (Fig. 4A). During meiosis I nondisjunction, both homologs segregate to the same pole, resulting in the production of two viable sister spores that are disomic for the missegregating chromosome and two dead spores that are nullisomic (Fig. 4B). If there is precocious separation of sister chromatids (PSSC) at meiosis I, one chromosome and one chromatid from the homologous chromosome can segregate to the same pole, resulting in the The efficiency of sporulation (Spo. efficiency) and the distribution of 4-spore-viable (4-s.v.), 3-spore-viable (3-s.v.), 2-spore-viable (2-s.v.), 1-spore-viable (1-s.v.), and 0-spore-viable (0-s.v.) tetrads are shown for wild-type and the taml mutant in two different sets of isogenic strains. MY261 and PC873 are isogenic to each other, as are PC872 and PC871. The frequency of meiosis I nondisjunction (MI ndj.) for chromosome III is that of 2-spore-viable tetrads containing two disomic spores among total tetrads dissected (No. tetrads). The frequency of precocious separation of sister chromatids (PSSC) is that of 3-spore-viable and 2-spore-viable tetrads containing a single disomic spore among total tetrads. Trp § Ura + disomes in 1-spore-viable tetrads could have resulted either from PSSC, meiosis I nondisjunction of chromosome III accompanied by missegregation of other chromosomes, or random spore death. The total frequency of chromosome III meiosis I missegregation events (misseg.) includes events that could not be categorized in addition to meiosis I nondisjunction and PSSC events. The number of Eo tetrads refers to the number of 4-spore-viable tetrads in which chromosome III is nonrecombinant along its entire length (in strains PC872 and PC871). The number of Eo tetrads observed (obs.) was adjusted (see Materials and Methods) to account for the expected number of two-strand double crossovers to give the corrected Eo value (cor.).
Cold Spring Harbor Laboratory Press on July 19, 2018 -Published by genesdev.cshlp.org Downloaded from Data are from strains BR2625 (TAM1) and PC647 (tam 1). The rate of meiotic prototrophy was determined by subtracting the mitotic (premeiotic) frequency from the meiotic frequency (taken after 24 hr in sporulation medium) for each experiment and averaging the meiotic values. At least three cultures were analyzed for each strain. (B.) Absolute frequencies of gene conversion were measured by tetrad analysis at five different loci. Data are from strains PC872 (wild type) and PC871 (taml). The number of conversion events relative to the total number of 4-spore-viable tetrads scored is indicated in parentheses. The amount of gene conversion in the taml mutant is indicated as a percent of the wild-type level; the probability that the difference observed is statistically significant is indicated in parentheses.
production of one disomic spore, one nullisomic spore, and two haploid spores (Fig. 4C). The types of missegregation events occurring in taml strains, and the influence of recombination on chromosome disjunction, were determined using appropriately marked yeast strains (Fig. 5A). The centromere of chro-mosome III is marked with URA3 on one chromosome, and with TRP1 on the homolog; CEN3 is flanked by markers at HIS4 and MAT. A meiosis I nondisjunction event in which both chromosomes III segregate to the same pole (and all other chromosomes segregate correctly) produces a 2-spore-viable tetrad in which both Strains PC871 and PC872 are isogenic. Only 4-spore-viable tetrads that did not show gene conversion of the markers indicated were used to calculate map distances. Tetrads were scored as parental ditype (PD), tetratype (TT), or nonparental ditype (NPD). The probability of wild-type crossing over (Probability WT C.O.) refers to the likelihood that the observed differences in the frequency of crossing over between wild type and tamI are attributable to chance (see Materials and Methods). NPDs expected are the number of four-strand double crossovers expected in the absence of interference; the NPD ratio refers to the number of NPDs observed relative to the number of NPDs expected. The probability of wild-type interference (WT int.) indicates the likelihood that the difference in NPD ratio between wild type and taml is attributable to chance; the probability of no interference (no int.) is the likelihood that the difference between the number of NPDs observed and the number expected in the absence of interference is attributable to chance. Asterisks denote probability values in which the differences observed are statistically significant (P < 0.05).  viable spores are Trp § Ura § disomes. Meiosis I PSSC produces a 3-spore-viable tetrad in which only one of the spores is a Trp § Ura § disome. (Meiosis II PSSC events, which produce TRP1/TRP1 or URA3/URA3 disomes, are not detected.) Crossing over followed by missegregation produces disomes that are homozygous for one of the flanking markers and therefore either mating-competent (MATa/MATa or MATR/MATR) or histidine auxotrophs (his4/his4) (Sym and Roeder 1994). Chromosome segregation was assessed in two sets of isogenic strains (Table 1). There is an increase in total meiosis I missegregation events (meiosis I nondisjunction and PSSC) in taml mutants relative to the isogenic wild type in both sets of strains. In one set (MY261 and PC873), meiosis I nondisjunction is increased from 0% in wild type to 1.2% in the taml mutant, whereas PSSC is increased from 0.29% to 0.48%. In the other set (PC871 and PC872), meiosis I nondisjunction is increased from 0% in wild type to 0.3% in the taml mutant, whereas PSSC is increased from 0.24% to 0.82%. The total frequency of chromosome III missegregation events (meiosis I nondisjunction and PSSC) is increased from 0.24%-0.29% in wild type to 1.5%-1.9% in taml mutants. These differences between wild type and mutant are statistically significant (P ~ 0.005). Note that the relative distributions of chromosome missegregation events are different in the two different tam l strains studied. In one strain (PC873), meiosis I nondisjunction is 2.5-fold more frequent than PSSC. Conversely, in the other strain (PC871), PSSC is 2.7-fold more frequent than meiosis I nondisjunction. A possible explanation for these differences is presented in the Discussion Section.

A B C
A total of 14 pairs of disomes indicative of meiosis I nondisjunction for chromosome III was detected in tam 1 mutants. Based on the frequency of tetratype tetrads among 4-spore-viable tetrads (Table 3), 7 of the 14 pairs of disomes should be recombinant in at least one of two intervals monitored. However, none of the 14 pairs of disomes detected are recombinant, which is significantly different from the number of recombinants expected (P < 0.001). This result suggests that the failure to recombine is the cause of homolog nondisjunction in the tam 1 mutant. Of 15 PSSC events detected, three disomes contain chromosomes that are recombinant in one or both intervals, which is not significantly different from the five recombinant disomes expected based on the frequency of tetratype tetrads observed among 4-spore-viable tetrads (P > 0.2). This result suggests that PSSC occurs irrespective of recombination in taml mutants.

The proportion of Eo tetrads is increased in taml mutants
The increased frequency of homolog nondisjunction at meiosis I in taml strains might be attributable to an increase in the number of tetrads in which one or more pairs of homologous chromosomes fail to receive a crossover (Eo tetrads). The frequency of Eo tetrads for chromosome III was determined in a pair of isogenic strains [PC872 (wild type) and PC871 (taml)] in which chromosome III has been marked along its entire length ( Fig. 5B; Table 1). Both the observed and corrected numbers of Eo tetrads (see Materials and Methods for determination of Eo tetrads) are at least an order of magnitude higher in tam 1 than in the isogenic wild type. The frequency of Eo tetrads among the total number of 4-spore-viable tetrads is 0.28% in wild type and 5.3% in the mutant, corresponding to a nearly 20-fold increase in the number of chromosome pairs that fail to cross over. The frequency of Eo tetrads is almost certainly underestimated, as these events are detected only in 4-spore-viable tetrads.

Crossover interference is impaired in tam l mutants
Crossover interference is the term used to describe the nonrandom distribution of crossovers, such that there is an inhibition of crossing over in regions that have already undergone exchange. One way to assess interference is to determine the NPD ratio (Snow 1979), which is the frequency of NPDs (nonparental ditype tetrads resulting from 4-strand double crossovers) observed divided by the frequency of NPDs expected in the absence of interference. The expected frequency is calculated based on the observed frequency of tetratype tetrads resulting from single crossovers. No interference results in an NPD ratio of 1.0 while absolute interference results in an NPD ratio of zero.
Crossover interference was measured in five different intervals on chromosome III in both wild type (PC872) and tarnl (PC871). Interference was found to be significantly weaker in the tam l mutant than in the isogenic wild type (Table 3). For wild type, the NPD ratios for all five intervals are significantly lower than the value of 1.0 expected in the absence of interference. In contrast, the tam l mutant shows a significant level of interference only in the HIS4-CEN3 interval. In each of the five intervals, the NPD ratio in the mutant is higher than in wild type (differences significant in every interval except Figure 5. Relevant configurations of markers in yeast strains used for genetic analysis. (A) Strains MY261 and PC873. The centromere of one chromosome III is marked with URA3 and the centromere of the homolog is marked with TRP1. Flanking markers at HIS4 and MAT allow crossing over to be monitored. (B) In strains PC871 and PC872, CEN3 is also marked with URA3 on one homolog and TRP1 on the other. Flanking markers on both arms of chromosome III allow crossing over to be monitored over almost the entire length of the chromosome. (C) Strains DS101 and PC874 contain nonhomologous YAC pairs, one of which is marked with URA3 and HIS3 and the other with LYS2 and TRP1. (D) PC875 and PC876 contain homologous YAC pairs, with the centromere marked by URA3 or TRP1 and flanked by LEU2 and HIS3. Crossing over can be detected on either YAC arm. RAD18-HMR), but less than 1.0. We conclude that crossover interference is impaired, but not completely abolished, in tam I mutants.
Another way to assess interference is to determine whether crossing over in one interval is inhibited by exchange in an adjacent interval. Such inhibition results in a reduced frequency of tetrads that sustain coincident crossovers in two adjacent intervals, compared with the frequency predicted if crossing over in the two intervals is independent. Table 4 shows the results of tetrad analyses for four pairs of adjacent intervals in wild-type (PC872) and tam l (PC871) strains. In wild type, the frequency of coincident crossovers is significantly lower than that predicted assuming no crossover interference in all pairs of intervals except for HIS4-CEN3-MAT. In contrast, the frequencies of coincident events in tam I are not significantly different from the predicted frequencies in all pairs of intervals analyzed. Therefore, crossovers in tam 1 strains are not strongly affected by crossovers nearby.

Distributive disjunction is impaired irl taml mutants
To examine the effect of tam l on the distributive disjunction system, the distributive disjunction of yeast artificial chromosomes (YACs) was measured in wild type and the taml mutant. Two sets of YACs, a nonhomologous pair (Fig. 5C) and a homologous pair (Fig. 5D), were examined.
In wild type, the nonhomologous pair of YACs segregates correctly to opposite poles at meiosis I in 76 % of all meioses and incorrectly to the same pole in 12% of meioses (Table 5). This is significantly different from the 1:1 ratio of correct segregations to meiosis I nondisjunctions expected if the YACs segregate randomly. In tam 1, proper segregation occurs 50% of the time and meiosis I  Distributive disjunction was measured for two different pairs of YACs. Strains DS 101 (TAM1) and PC874 (tam 1) were used to measure distributive disjunction of nonhomologous YACs. Strains PC875 (TAM1) and PC876 (taml) were used to measure distributive disjunction of homologous YACs; data shown are for nonrecombinant YACs. Probability wild type indicates the likelihood that the observed differences between wild type and tam 1 are attributable to chance alone. Probability random indicates the likelihood that the observed frequencies of disjunction follow a random (1:1) pattern. Only tetrads displaying correct disjunction or meiosis I nondisjunction were included in probability calculations. Asterisks denote probabilities for which the differences observed are statistically significant (P < 0.05).
nondisjunction occurs 26% of the time. This nondisjunction frequency is significantly higher than that observed in wild type, but it is still significantly lower than that expected for a random pattern of segregation. Meiosis I PSSC also occurs in both wild-type and taml strains, but is increased about twofold in the mutant. A similar result was obtained when homologous pairs of YACs were examined. In this case, only nonrecombinant YACs (-97% of meioses) were considered in calculations of distributive disjunction (Dawson et al. 1986). Nonrecombinant YACs disjoin from each other 92% of the time in wild type, but only 82% of the time in tarnl. Therefore, distributive disjunction is impaired, but not abolished, in tam l mutants.

The meiosis I division is delayed in taml mutants
To monitor the timing of the meiotic nuclear divisions, cells from wild-type and taml strains were harvested at different time points after the induction of meiosis and stained with DAPI. Cells that have completed the meiosis I division contain two discrete nuclei, whereas cells that have completed both meiotic divisions contain four nuclei. The kinetics of nuclear division were examined in both sets of isogenic strains used to examine chromosome segregation (Table 1).
In tam 1 mutants, the generation of binucleate and tetranucleate cells is delayed by at least 2 hr compared with the isogenic wild type. In addition, formation of binucleate cells appears to be less synchronous in taml. Meiosis II is also delayed in tam l strains, presumably as a consequence of the delay in meiosis I.
The data also reveal that the two sets of strains examined differ with respect to the timing of the meiotic divisions. Strains MY261 and PC873 ( Fig. 6A-C) undergo meiosis faster than strains PC871 and PC872 (Fig. 6D-F). This difference may account for the different patterns of chromosome missegregation observed in tam l mutants (see above and Discussion Section).

C h r o m o s o m e synapsis is delayed in t a m l m u t a n t s
Spread meiotic chromosomes from wild type and a t a m 1 mutant were stained with silver nitrate and examined in the electron microscope. At the time of m a x i m u m synapsis, tam l nuclei form tripartite SCs that are morphologically similar to those seen in wild type (Fig. 7). Spread chromosomes from the tam l mutant were also stained with anti-Zipl antibodies. Zip l is a component of the central region of the SC; therefore, Zip l localization serves as a marker for chromosome synapsis. In the t a m l mutant, Zip l appears to localize along the length of every chromosome pair, indicating full synapsis (Fig.  3G,H).
To monitor the kinetics of SC assembly, chromosome spreads were prepared from sporulating cultures of tam 1 and wild-type strains at various times after the induction of meiosis. Anti-Zipl and anti-tubulin antibodies were used to visualize the SC and meiotic spindles, respectively. In the wild-type strain, the number of cells in pachytene reaches a m a x i m u m at 13 hr after the induction of meiosis (Fig. 8A). After this time point, nuclei that stain with anti-Zipl antibodies decrease in frequency, whereas nuclei with spindles increase. Compared with wild type, tam l mutants display a delay in chromosome synapsis (Fig. 8B). The number of nuclei in pachytene reaches a m a x i m u m after 17 hr of sporulation, -4 hr later than in wild type. Correspondingly, nuclei with spindles appear m u c h later in the t a m l mutant than they do in wild type.

D i s c u s s i o n t a m l is defective in meiotic c h r o m o s o m e segregation
Disruption of the TAM1 gene confers multiple defects in meiotic chromosome segregation. Compared with wild type, meiosis I nondisjunction and PSSC are increased significantly in the tam l mutant, and distributive disjunction is less efficient.
The increase in homolog nondisjunction in t a m l strains is the consequence of an increase in tetrads that contain nonrecombinant homologs. There are two possible explanations for this increase in Eo chromosomes. If a t a m l mutation confers a defect in homolog pairing, then homologs might fail to cross over because they fail  Rockmill et al. 1995). Open squares indicate nuclei in pachytene, as defined by Zip l localization along the entire lengths of most or all chromosome pairs. Nuclei with meiosis I spindles or meiosis II spindles are indicated by open and shaded triangles, respectively. The time course was performed in strains (PC712, wild type; PC664, taml) that sporulate very slowly compared with the strains examined in Fig. 6.
to find each other. Alternatively, or in addition, the increase in Eo chromosomes might result from a deregulation of crossover distribution. Crossover interference is thought to be important for distributing crossovers among chromosomes so that even small chromosomes undergo at least one crossover in every meiosis (King and Mortimer 1990;Kaback et al. 1992). Analysis of the z i p l mutant (Sym and Roeder 1994), in which crossover interference is abolished, is consistent with the hypothesis that interference is responsible for distributing crossovers among chromosomes. If a wild-type number of crossovers is distributed randomly among chromosomes (as a function of chromosome size), then chromosome III is expected to be nonrecombinant in 9.5% of meioses. Therefore, the frequency of Eo tetrads for chromosome III in taml (5.3%) could be accounted for by a deregulation of crossover distribution. The increase in Eo tetrads probably accounts for the reduced efficiency of distributive disjunction in the tam 1 mutant. Because the distributive disjunction pathway does not discriminate chromosomes on the basis of homology, an increase in the number of chromosomes entering this system might cause the YACs to disjoin from authentic chromosomes instead of from each other. In Drosophila, mutations that decrease crossing over cause missegregation of the fourth chromosome, which normally segregates exclusively via the distributive disjunction pathway (for review, see Baker et al. 1976). Even in wild-type flies, the fourth chromosome is more likely to nondisjoin in ova in which the X chromosome is nonrecombinant (for review, see Baker et al. 1976). It seems likely that the distributive disjunction pathway in yeast is also sensitive to the number of chromosomes it has to segregate. An alternative or additional explanation for the defect in distributive disjunction is that the Taml protein is directly involved in the pairing and/or segregation of chromosomes via the distributive system.
The increase in PSSC in the taml mutant might be attributable to the delay in meiotic prophase progression. If kinetochores continue to differentiate during this pause in the cell cycle, then sister chromatids may separate from each other before meiosis I, therefore leading to PSSC. A similar model was proposed by Carpenter (1994) to account for the increase in PSSC observed in strains containing nonrecombinant or dicentric chromosomes (e.g., Rockmill and Roeder 1994;Flatters et al. 1995). This model can explain the different relative frequencies of meiosis I nondisjunction versus PSSC observed in dif~ ferent tam 1 strains. A strain that spends a longer time in prophase would be expected to display more PSSC; a longer prophase might also decrease the probability of homolog nondisjunction by allowing time for nonrecombinant chromosomes to pair and segregate via the distributive system (Loidl et al. 1994). Of the two strains used to study chromosome segregation, the strain that has a higher frequency of PSSC relative to meiosis I nondisjunction spends a longer time in prophase than the strain in which the relative frequencies of homolog nondisjunction and PSSC events are reversed. tam l decreases interference without decreasing crossing over tam 1 mutants display approximately wild-type levels of crossing over in all intervals examined; however, crossover interference is reduced significantly. Therefore, the tam l mutation functionally separates the control of crossover distribution from the control of crossover frequency. In this respect, taml is unique among yeast mutants. In the two other yeast mutants known to be defective in interference, zipl (Sym and Roeder 1994) and rash4 P. Ross-Macdonald, J. Novak and G. S. Roeder, in prep.), crossing over is reduced about twofold compared with wild type. In other organisms as well, most genes that affect crossover distribution are also involved in determining recombination frequency. In Drosophila, the mei-41, mei-218, mei-251, mei-S282, mei-B, abo, and mei-68 L1 mutants are defective in recombination and display altered distributions of exchanges (for review, see Baker et al. 1976). In Caenorhabditis elegans, the him-l, him-5, and him-8 mutants affect both the frequency and distribution of crossovers on the X chromosome (Broverman and Meneely 1994).
The effect of taml in separating the control of crossover distribution from that of crossover frequency might be parallel to the effects of the mei-352 mutant in Drosophila (Baker and Carpenter 1972), the recl mutant in C. elegans (Zetka and Rose 1995), and/or an abnormal line of rye (Jones 1967). All of these mutants display approximately normal levels of recombination, but the distribution of crossovers along chromosomes is more random than in wild type. In mei-352 and the abnormal line of rye (but not recl), the absence of interference greatly increases the number of bivalents with no chiasmata, resulting in univalents at metaphase and consequent high rates of nondisjunction (Jones 1967;Baker and Carpenter 1972;Egel 1995). The molecular basis of the defect in crossover distribution is not understood for any of these mutants.
A recent model for crossover interference (Kleckner 1996;Storlazzi et al. 1996)postulates that recombination intermediates are resolved aberrantly when factors that normally regulate crossover distribution are absent. Under these conditions, it is proposed that pre-crossover intermediates, most of which are normally resolved as crossovers, are resolved randomly to generate approximately equal numbers of crossover and noncrossover products. The operation of such a default pathway could account for the observation that the zipl (Sym and Roeder 1994)and msh4 (P. Ross-Macdonald, J. Novak, and G.S. Roeder, in prep.) mutations both decrease crossing over approximately twofold and also reduce or eliminate crossover interference. However, the phenotype of the taml mutant demonstrates that a reduction in crossover interference need not be associated with a decrease in crossing over.

The defect in synapsis may account for the decrease in interference in the taml mutant
In addition to the defect in interference, tam1 mutants also display a delay in chromosome synapsis. Based on previous studies (summarized below) suggesting a role for the SC in the regulation of crossover distribution, it is attractive to speculate that the delay in synapsis is the cause of the defect in interference in the taml mutant. Many models of interference postulate the transmission of an inhibitory signal along the SC, starting at the site of a crossover and extending to nearby potential sites of crossing-over (Egel 1978;Maguire 1988;King and Mor-timer 1990). If commitment to crossing over occurs on time in tam 1 strains, or is delayed to a lesser extent than synapsis, then transmission of the inhibitory signal might be compromised.
A number of studies have implicated the SC in the control of crossover distribution. Most recombinationproficient organisms display interference and also make SC (for review, see Jones 1987). In contrast, two organisms that do not make SC, Schizosaccharomyces pombe (Olson et al. 1978) and Aspergillus nidulans (Egel-Mitani et al. 1982), do not display crossover interference. In yeast, analysis of the zipl mutant has provided evidence for a functional relationship between the SC and interference. Strains lacking the ZIP1 gene, which encodes a component of the central region of the SC ISym et al. 1993;Sym and Roeder 1995), undergo a substantial amount of recombination, but are completely defective in crossover interference (Sym and Roeder 1994). The asl and asb mutants of tomato also display defects in both synapsis and interference (Moens 1969;Havekes et al. 1994). In asb, analysis of partially synapsed chromosomes reveals that a recombination nodule (indicative of crossing over) is located at each synapsed segment (Havekes et al. 1993). A chromosome can contain as many as three nodules (which is rare for wild type) if it contains three independently synapsed segments, suggesting that interference does not extend beyond the boundaries of the synapsed segment. In addition, in plants and insects heterozygous for a chromosome rearrangement, interference does not operate across the interruption in the SC that results from rearrangement (Arana et al. 1987;Parker 1987).
Two different models have been proposed to account for the role of the SC in crossover interference. One model supposes that synapsis initiates at the sites of crossovers. Further exchange is inhibited in regions of the chromosomes that have synapsed, but crossovers can still be initiated in regions that have not yet synapsed (Egel 1978;Maguire 1988). Therefore, zippering up of the SC serves as the mechanical basis of interference. An alternate view is that interference acts after synapsis is complete and full-length SC acts as a substrate for the transmission of an inhibitory signal (King and Mortimer 1990;Sym and Roeder 1994). Signal transduction may involve a conformational change in proteins associated with the SC that starts at sites of crossing over and moves along the chromosome in a domino-like effect, or it may involve the polymerization of a protein along the SC starting at the sites of crossing over. According to both models, the signal that inhibits crossing over would not be transmitted on time if synapsis is delayed relative to the timing of commitment to crossing over.
A recent model for crossover interference suggests that interference is independent of the SC (Kleckner 1996;Storlazzi et al. 1996). This model is based on the observation that a zipl mutation reduces crossing over in a redl mutant and hinges on the assumption that there is no Zipl polymerization in the redl mutant. However, a recent study demonstrates that there is extensive polymerization of the Zipl protein in redl strains (Smith and Roeder 1997); therefore, Zipl need not participate in recombination in a manner that is independent of its role in SC formation. In addition, mutational analysis of the ZIP1 gene has failed to identify any mutations that separate the role of Zip l in chromosome synapsis from its role in crossover interference (K.-S. Tung and G.S. Roeder in prep.).

A role for Taml in homologpairing?
How might a telomere-associated protein participate in chromosome synapsis and crossover interference? In higher eukaryotes, synapsis initiates at or near telomeres (yon Wettstein et al. 1984), raising the possibility that Tam 1 is required for the initiation of synapsis. In yeast, however, the evidence indicates that telomeres are not important for synaptic initiation. Loidl (1995) observed that synapsis can initiate interstitially by studying pairing partner switches in tetraploid yeast. Studies of the zipl mutant have identified sites of intimate association between paired but unsynapsed axial elements. These connections, which are presumed to be sites of synaptic initiation (Sym et al. 1993;Rockmill et al. 1995), are not concentrated at the ends of chromosomes. In addition, in zipl non-null mutants in which the rate of synapsis is decreased, regions near the ends of chromosomes are frequently unsynapsed even when most of the chromosome pair is engaged in SC formation (K.-S. Tung and G.S. Roeder, in prep.). Furthermore, when wild-type spreads are double-stained with anti-Zipl and anti-Taml antibodies, the Zipl and Taml foci do not overlap in nuclei presumed to be in zygotene (data not shown). Taken together, these observations argue against the possibility that synapsis initiates at telomeric regions in yeast.
It is possible that Tam l has a role in crossover interference that is independent of its role in synapsis. Kleckner and colleagues (Kleckner 1996;Storlazzi et al. 1996) have proposed that the attachment of meiotic chromatin to axial elements (the proteinaceous cores shared by sister chromatids) induces tension and that recombination intermediates are sensitive to this stress. When the level of tension reaches a certain threshold, a crossover event is initiated. The act of initiation relieves stress in the immediate vicinity of the crossover, which then reduces the probability that additional crossing over will be initiated nearby. Tam l might function in this context if attachment of chromosome ends to the nuclear membrane (or some other structure) is necessary to impose tension. In the absence of an attachment that confines the movement of a stressed chromosome, the tension required for crossover control might be more easily dissipated, resulting in reduced interference. This model does not easily account for the delay in synapsis in the tam 1 mutant.
We favor the view that the Taml protein has a role in homolog pairing, and that the delay in chromosome synapsis is the consequence of an earlier defect in chromosome alignment. The attachment of telomeres to the nuclear membrane and their clustering together during early meiotic prophase have been postulated to be im-portant for bouquet formation (for review, see von Wettstein et al. 1984;Fussell 1987;Loidl 1990;Dernburg et al. 1995). Formation of the bouquet may facilitate chromosome pairing by bringing homologous chromosomes into approximate alignment (Fussel 1987;Dernburg et al. 1995). Because the bouquet is a meiotic phenomenon, it is reasonable to suppose that a meiosis-specific protein like Taml is involved. Taml might be important for the early steps in chromosome pairing by promoting the attachment of chromosome ends to the nuclear membrane or by promoting their subsequent clustering.

Strain constructions and media
Yeast manipulations were performed and media were prepared according to Sherman et al. (1986). Substitutive and integrative transformations (Rothstein 1991) were carried out by the lithium acetate procedure (Ito et al. 1983). Genotypes of yeast strains are listed in Table 6. Strain BR2625 was obtained from Beth Rockmill (Yale University, New Haven, CT). Strain MY261 was obtained from Mary Sym (Sym and Roeder 1994). Strain DS101 was obtained from Phil Hieter (Sears et al. 1994). Dd99.1B andDd99.1C, containing YLp45 andYLp49, respectively (Dawson et al. 1986), were obtained from Dean Dawson. Strains PC872, PC871, PC875, and PC876 are congenic with SK1. To make PC875, the YACs present in Dd99.1B and Dd99.1C were introduced into an SKI strain background by nine consecutive backcrosses. The hmr::LYS2 locus, derived from AB 10-1D (obtained from Alan Rose, Princeton University, NJ), was introduced into an SK1 background by six consecutive backcrosses during the construction of PC871 and PC872. All transformations were verified by Southern blot analysis (Southern 1975). Escherichia coli strain XL1-Blue (Stratagene) was used for plasmid constructions and protein synthesis.

Plasmids
The cosmid clone c9348 (Olson et al. 1986) containing TAM1 was obtained from the American Type Culture Collection. A 1.8-kb XhoI fragment containing TAM1 was subcloned into pCB233, a pHSS6 (Seifert et al. 1986) derivative in which the EcoRI site in the polylinker has been destroyed, to generate pCB241. The Tyl sequences immediately upstream of TAM1 were then removed by cutting with BsaBI (which cuts between the Tyl element and the TAM1 promoter region) and HindIII (which cuts in the polylinker), filling in the overhanging ends with the Klenow fragment of DNA polymerase I, and religating to generate pCB243, pCB243 was used to make all tam l dele-  (Rockmill and Roeder 1990). Strain PC874 is isogenic to DS101 (Sears et al. 1994). Strains M45, M85, and VSG12 were derived from Y800 (Burns et al. 1994). taml:dacZ and zipl::lacZ fusions were created by mutagenesis with Tn3::lacZ::LEU2 (Burns et al. 1994), whereas the RAPI::HA fusion was created by mutagenesis with mTn3-3XHA/lacZ (Ross-Macdonald et al. 1997) followed by Cre-mediated excision of most of the transposon. Strains MY261 and PC873 were constructed in an SKI strain background (Kane and Roth 1974;Williamson et al. 1983). Strains PC872, PC871, PC875, and PC876 are congenic with SKI. The ADE2 gene was inserted upstream of RAD18 in strains PC872 and PC871. tion plasmids. To make the taml::LEU2, taml::LYS2, and tam l::ARG4 deletions, the 688-bp EcoRI fragment (comprising bases 68-756 of the TAM10RF1 was excised and replaced with LEU2, LYS2, or ARG4, resulting in plasmids pCB292, pCB295, and pCB435, respectively. The EcoRI ends of pCB243 were filled in with the Klenow fragment of DNA polymerase I and ligated to the following fragments: a 2.3-kb SrnaI-HpaI fragment containing LEU2 from YEp351 (Hill et al. 1986), a 5-kb XbaI-PvuII fragment (in which the XbaI overhangs had been filled in) containing LYS2 from pDP6 (Fleig et al. 1986), or a 2-kb HpaI fragment containing ARG4 (Beacham et al. 1984). The taml ::ADE2 deletion (pCB408) was made in two steps. First, a 3.6-kb BamHI fragment containing ADE2 from Yp3.6 ADE (Engelbrecht and Roeder 1990) was inserted into the BamHI site of pBR322 to generate pR940. Then, the 863-bp region between the EcoRI and EcoNI sites from pCB243 (comprising bases 68-931 of the TAM10RF) was replaced with the 4-kb EcoRI-EcoNI fragment containing ADE2 from pR940 to generate pCB408. To make the taml::URA3 deletion, the 1045-bp region between the EcoRI and DraI sites (comprising bases 68-1113 of the TAM1 gene) was replaced with a 1.6-kb EcoRI-HpaI fragment containing URA3 from YEp352 (Hill et al. 1986) to generate pCB249. All of these taml deletion plasmids were targeted for substitution by cutting with NotI. pJH455, containing the hrnl::LEU2 deletion, was a gift from Jim Haber (Brandeis University, Waltham, MA). The RAD18::ADE2 plasmid (pCB432) was made as follows. The 2.3-kb ClaI-XbaI fragment containing sequences in and upstream of RAD18 from pJH830 (Fabre et al. 1989) was subcloned into Bluescript SK(+) (Stratagene) to generate pCB430. A 2-kb BglII fragment containing ADE2 from pASZ11 (Stotz and Linder 1990) was inserted into the BglII sites of pSL301 (Invitrogen) to generate pCB429. Then, a 2-kb SmaI-SnaBI fragment containing ADE2 from pCB429 was inserted into the BsaBI site upstream of the RAD18 promoter region in pCB430. The resulting plasmid, pCB432, was targeted for substitution by cutting with ClaI and SalI. A TAM1 expression plasmid (pCB250) containing three-quarters of the TAM1 coding region, was made by ligating the 850-bp DraI fragment (bases 263-1113) into the filled-in EcoRI sites of the expression vector pGEX-KG (Guan and Dixon 1991).
Expression studies ~-Galactosidase assays were performed as described by Engebrecht and Roeder (1990). Y800 strains (Burns et al. 1994) heterozygous for tam I::lacZ and zipl::lacZ were assayed in duplicate, and the averages are shown in Figure 2.

Cytology
Spreads of meiotic chromosomes were prepared for electron microscopy according to Dresser and Giroux (1988), with modifications made by Engebrecht and Roeder (1990). Immunofluorescence procedures were performed according to Sym et al. (1993). A Leitz DMRB microscope equipped with fluorescence and a PL APO 100x objective was used to observe antibody-stained preparations. Images were captured using a Photometrics Imagepoint CCD camera. Meiotic cells were stained with DAPI as described by Thompson and Roeder (1989). Cells were grown and induced for meiosis as described by Sym and Roeder (1994) for SKI strains, and by Bhargava et al. (1991) for all other strains.

Antibodies
A glutathione S-transferase-Taml fusion protein was overproduced in bacteria using the plasmid pCB250 described above. The fusion protein was produced and purified according to Smith and Johnson (1988) and Guan and Dixon (1991). Rabbit antibodies against the fusion protein were generated by the Pocono Rabbit Farm and Laboratory ICanadensis, Pennsylvania) according to their fusion protein protocol. Anti-glutathione Stransferase-Taml antibodies were affinity purified according to Snyder (1989) and used at a 1:100 dilution. Cy3-or Texas redconjugated secondary antibodies against rabbit IgG (Jackson Laboratories) were used at a 1:200 dilution to detect anti-Taml antibodies. Mouse anti-HA antibodies [16B12; Babco (Richmond, CA)] were detected with fluorescein-conjugated secondary antibodies against mouse IgG (Jackson Laboratories). Anti-Zipl antibodies (Sym et al. 1993) were detected with fluorescein-conjugated secondary antibodies against rabbit IgG (Jackson Laboratories). Anti-tubulin antibodies [YOL1/34; (Kilmartin et al. 1982); Sera-lab, 1:100 final dilution of monoclonal supernatant] were detected with fluorescein-conjugated secondary antibodies against rat IgG (Jackson laboratories).

Genetic procedures and analysis
Recombination frequencies among disomes were calculated according to Ross-Macdonald and Roeder (1994). Interference was determined according to Sym and Roeder (1994). To determine the NPD ratio (NPDoB .... d/NPDexp .... d), the Papazian equation (Papazian 1952), NPD = 1/211 -TT -(1 -3TT/2)2/3], was used to calculate the proportion of NPDs expected, where TT is the proportion of tetratypes observed. To compare the number of NPDs observed with that expected in the sample, a X 2 test was performed for each interval. To compare the NPD ratios between wild type and taml, a contingency x 2 test was performed for each interval, using the observed and expected NPD values for wild type and tam I. To compare crossover frequencies between wild type and taml, a contingency • test was performed for each interval, using the number of nonrecombinant and recombinant tetrads (where the number of nonrecombinant tetrads = PD -NPD + 1/2[TT -2NPD] and the number of recombinant tetrads = 4NPD + 1/2ITT -2NPDJ). To compare the expected and observed frequency of coincident crossovers in adjacent intervals, a X 2 test was performed for each pair of intervals. X 2 tests and contingency X ~ tests were performed to compare spore viability and the pattern of spore death, respectively, between wild type and taml.
The observed frequency of Eo tetrads for chromosome III in strains PC872 and PC871 was determined as the number of 4-spore-viable tetrads in which all four spores retained the parental configuration of markers along the chromosome, divided by the total number of 4-spore-viable tetrads. The corrected Eo value was obtained by subtracting from the raw number of Eo tetrads the estimated number of tetrads within the Eo population that had sustained a two-strand double crossover in one or more intervals. Assuming no chromatid interference, the number of two-strand double crossovers in an interval is equal to the number of four-strand double crossovers detectable as NPDs. For each interval, (PD-NPD)/PD is the probability that an apparent PD is real. The sum of the probabilities of two-strand double crossovers in one, two or more intervals along the entire chromosome was subtracted from the observed number of Eo tetrads to generate the corrected frequency. To compare Eo frequencies between wild type and tam l, contingency X 2 tests were performed. plasmids and strains 9 We thank Janet Novak, Beth Rockmill, Pedro San Segundo, and Frank Stahl for comments on the manuscript. We are grateful to Junhyong Kim and Ed Louis for advice on statistical analysis. This work was supported by National Institutes of Health (NIH) grant GM28904 to G.S.R.
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Note added in proof
The TAM1 gene is identical to NDJI, which was recently described by Conrad et al. (1997) in Science 276: 1252-1255.