Synaptonemal complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes.

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.     

Meiotic crossover hotspots contained in haplotype block boundaries of the mouse genome

Fertility requires successful chromosome segregation in meiosis, which in most sexual organisms depends on the formation of appropriately placed crossovers. The nonrandom genome-wide distributions of meiotic recombination events have been examined at the molecular level experimentally in yeast and by inference from linkage disequilibrium patterns in humans. Thus far, no method has existed for pinpointing sites of crossing-over on a genome-wide scale in an experimentally tractable animal whose genome size and complexity models that of humans. Here, we present a genomic approach to identify mouse crossover hotspots, based on targeting haplotype block boundaries. This represents a previously undescribed method potentially applicable to large-scale mouse hotspot identification. Using this method, we have successfully predicted the location of two previously uncharacterized crossover hotspots in male mice. As increasing amounts of single-nucleotide polymorphism data emerge, this approach will be useful for investigating the recombination landscape of the mouse genome.     

High rate of recombination and double crossovers in the mouse pseudoautosomal region during male meiosis.

The recombination rate in meiosis between the mouse X and Y chromosomes was analyzed. Mice heterozygous at two pseudoautosomal alleles, the steroid sulfatase gene and the Mov-15 provirus marker, were crossed. The provirus in the Mov-15 transgenic mouse strain had been previously shown to be carried in the pseudoautosomal region of the sex chromosomes. Recombination frequencies were shown to be 7-fold higher in this region in male meiosis than in female meiosis. Three-point crosses indicated the occurrence in male meiosis of double recombination events in the pseudoautosomal region, with little or no crossover interference, which is in marked contrast to observations made on the similar region of the human sex chromosomes. This result is contrary to a previous model, which predicted a single crossover event in male meiotic pairing of mammalian sex chromosomes.     

Meiotic recombination and segregation of human-derived artificial chromosomes in Saccharomyces cerevisiae.

We have developed a system that utilizes human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to study factors that contribute to the fidelity of meiotic chromosome transmission. Since aneuploidy for the YACs does not affect spore viability, different classes of meiotic missegregation can be scored accurately in four-viable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromatid loss, and meiosis II nondisjunction. Segregation of the homologous pair of 360-kilobase marker YACs was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment. By using this experimental system, a series of YAC deletion derivatives ranging in size from 50 to 225 kilobases was analyzed to directly assess the relationship between meiotic recombination and meiosis I disjunction in a genotypically wild-type background. The relationship between physical distance and recombination frequency within the human DNA segment was measured to be comparable to that of endogenous yeast chromosomal DNA--ranging from less than 2.0 to 7.7 kilobases/centimorgan. Physical analysis of recombinant chromosomes detected no unequal crossing-over at dispersed repetitive elements distributed along the YACs. Recombination between YACs containing unrelated DNA segments was not observed. Furthermore, the segregational data indicated that meioses in which YAC pairs failed to recombine exhibited dramatically increased levels of meiosis I missegregation, including both precocious sister chromatid separation and nondisjunction.     

Predicting crossover generation in DNA shuffling

We introduce a quantitative framework for assessing the generation of crossovers in DNA shuffling experiments. The approach uses free energy calculations and complete sequence information to model the annealing process. Statistics obtained for the annealing events then are combined with a reassembly algorithm to infer crossover allocation in the reassembled sequences. The fraction of reassembled sequences containing zero, one, two, or more crossovers and the probability that a given nucleotide position in a reassembled sequence is the site of a crossover event are estimated. Comparisons of the predictions against experimental data for five example systems demonstrate good agreement despite the fact that no adjustable parameters are used. An in silico case study of a set of 12 subtilases examines the effect of fragmentation length, annealing temperature, sequence identity and number of shuffled sequences on the number, type, and distribution of crossovers. A computational verification of crossover aggregation in regions of near-perfect sequence identity and the presence of synergistic reassembly in family DNA shuffling is obtained.     

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.     

Crossover and noncrossover recombination during meiosis: timing and pathway relationships.

During meiosis, crossovers occur at a high level, but the level of noncrossover recombinants is even higher. The biological rationale for the existence of the latter events is not known. It has been suggested that a noncrossover-specific pathway exists specifically to mediate chromosome pairing. Using a physical assay that monitors both crossovers and noncrossovers in cultures of yeast undergoing synchronous meiosis, we find that both types of products appear at essentially the same time, after chromosomes are fully synapsed at pachytene. We have also analyzed a situation in which commitment to meiotic recombination and formation of the synaptonemal complex are coordinately suppressed (mer1 versus mer1 MER2++). We find that suppression is due primarily to restoration of meiosis-specific double-strand breaks, a characteristic of the major meiotic recombination pathway. Taken together, the observations presented suggest that there probably is no noncrossover-specific pathway and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation. The biological significant of noncrossover recombination remains to be determined, however.     

Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe

In preparation for the unique segregation of homologs at the first meiotic division, chromosomes undergo dramatic changes. The meiosis-specific sister chromatid cohesins Rec8 and Rec11 of Schizosaccharomyces pombe are recruited around the time of premeiotic replication, and Rec10, a component of meiosis-specific linear elements, is subsequently added. Here we report that Rec10 is essential for meiosis-specific DNA breakage by Rec12 (Spo11 homolog) and for meiotic recombination. DNA breakage and recombination also depend on the Rec8 and Rec11 cohesins, strictly in some genomic intervals but less so in others. Thus, in addition to their previously recognized role in meiotic chromosome segregation, cohesins have a direct role, as do linear element components, in meiotic recombination by enabling double-strand DNA break formation by Rec12. Our results reveal a pathway, whose regulation is significantly different from that in the distantly related yeast Saccharomyces cerevisiae, for meiosis-specific chromosome differentiation and high-frequency recombination.     

Two levels of interference in mouse meiotic recombination

During meiosis, homologous chromosomes (homologs) undergo recombinational interactions, which can yield crossovers (COs) or noncrossovers. COs exhibit interference; they are more evenly spaced along the chromosomes than would be expected if they were placed randomly. The protein complexes involved in recombination can be visualized as immunofluorescent foci. We have analyzed the distribution of such foci along meiotic prophase chromosomes of the mouse to find out when interference is imposed and whether interference manifests itself at a constant level during meiosis. We observed strong interference among MLH1 foci, which mark CO positions in pachytene. Additionally, we detected substantial interference well before this point, in late zygotene, among MSH4 foci, and similarly, among replication protein A (RPA) foci. MSH4 foci and RPA foci both mark interhomolog recombinational interactions, most of which do not yield COs in the mouse. Furthermore, this zygotene interference did not depend on SYCP1, which is a transverse filament protein of mouse synaptonemal complexes. Interference is thus not specific to COs but may occur in other situations in which the spatial distribution of events has to be controlled. Differences between the distributions of MSH4/RPA foci and MLH1 foci along synaptonemal complexes might suggest that CO interference occurs in two successive steps.     

Diverse genetic mechanisms operate to generate atypical betaS haplotypes in the population of Guadeloupe

In a survey of the chromosomal background associated with the sickle cell gene in Guadeloupe, a French Caribbean island, we identified 37 unrelated patients with sickle cell disease (27 SS, nine SC, and one S-beta-thalassemia) of 477 unrelated sickle cell patients where the beta3 gene was linked to 20 different atypical haplotypes. These atypical chromosomes account for about 5% of the overall betaS chromosomes in this population. To investigate the origin of these atypical betaS haplotypes, we performed extensive typing of betaS and betaA chromosomes. Twenty-two different 5' subhaplotypes were identified among the betaS chromosomes. Fifteen of 20 different atypical haplotypes are likely to be the product of recombination by a single crossover around the 5' to the beta-globin gene, or between a major betaS haplotype and one of the betaS haplotypes present in the population. The remaining cases require genetic mechanisms (gene conversions, additional substitutions in a given haplotype) other than crossovers to generate these atypical haplotypes.     

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.     

The synaptonemal complex protein,Zip1, promotes the segregation of nonexchange chromosomes at meiosis I

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.     

HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events

In meiosis, gene conversions are accompanied by higher levels of crossing over than in mitotic cells. To determine whether the special properties of meiotic recombination can be attributed to the way in which Spo11p creates double-strand breaks (DSBs) at special hot spots in Saccharomyces cerevisiae, we expressed the site-specific HO endonuclease in meiotic cells. We could therefore compare HO-induced recombination in a well-defined region both in mitosis and meiosis, as well as compare Spo11p- and HO-induced meiotic events. HO-induced gene conversions in meiosis were accompanied by crossovers at the same high level (52%) as Spo11p-induced events. Moreover, HO-induced crossovers were reduced 3-fold by a msh4Delta mutation that similarly affects Spo11p-promoted events. In a spo11Delta diploid, where the only DSB is made by HO, crossing over was significantly higher (27%) than in mitotic cells (相似文献   

Mitotic recombination map of 13cen-13q14 derived from an investigation of loss of heterozygosity in retinoblastomas

Loss of heterozygosity at tumor-suppressor loci is an important oncogenic mechanism first discovered in retinoblastomas. We explored this phenomenon by examining a set of matched retinoblastoma and leukocyte DNA samples from 158 patients informative for DNA polymorphisms. Loss of heterozygosity at the retinoblastoma locus (13q14) was observed in 101 cases, comprising 7 cases with a somatic deletion causing hemizygosity and 94 with homozygosity (isodisomy). Homozygosity was approximately equally frequent in tumors from male and female patients, among patients with a germ-line vs. somatic initial mutation, and among patients in whom the initial mutation occurred on the maternal vs. paternal allele. A set of 75 tumors exhibiting homozygosity was investigated with markers distributed in the interval 13cen-13q14. Forty-one tumors developed homozygosity at all informative marker loci, suggesting that homozygosity occurred through chromosomal nondisjunction. The remaining cases exhibited mitotic recombination. There was no statistically significant bias in apparent nondisjunction vs. mitotic recombination among male vs. female patients or among patients with germ-line vs. somatic initial mutations. We compared the positions of somatic recombination events in the analyzed interval with a previously reported meiotic recombination map. Although mitotic crossovers occurred throughout the assayed interval, they were more likely to occur proximally than a comparable number of meiotic crossovers. Finally, we observed four triple-crossover cases, suggesting negative interference for mitotic recombination, the opposite of what is usually observed for meiotic recombination.     

Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.     

Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice

Errors in meiotic chromosome segregation are the leading cause of spontaneous abortions and birth defects. Almost all such aneuploidy derives from meiotic errors in females, with increasing maternal age representing a major risk factor. It was recently reported that histones are globally deacetylated in mammalian oocytes during meiosis but not mitosis. In the present study, inhibition of meiotic histone deacetylation was found to induce aneuploidy in fertilized mouse oocytes, which resulted in embryonic death in utero at an early stage of development. In addition, a histone remained acetylated in the oocytes of older (10-month-old) female mice, suggesting that the function for histone deacetylation is decreased in the oocytes of such mice. Thus, histone deacetylation may be involved in the fair distribution of chromosomes during meiotic division. The high incidence of aneuploidy in the embryos of older females may be due to inadequate meiotic histone deacetylation.     

Loss of heterozygosity and mitotic linkage maps in the mouse.

Loss of heterozygosity is a significant oncogenetic mechanism and can involve a variety of mechanisms including chromosome loss, deletion, and homologous interchromosomal mitotic recombination. Analysis of H-2 antigen-loss variants from heterozygous murine cell lines provides an experimental system to estimate the relative contributions of different mechanisms for allele loss and to compare the chromosomal patterns of mitotic and meiotic recombination. Cytotoxic anti-H-2D antibodies and complement were used to isolate 161 independent target antigen-negative clones from H-2d/H-2b heterozygous cell lines; of these, 131 (84.5%) lost the allele encoding the target antigen. Allele-loss variants were typed and scored as either heterozygous or homozygous for six H-2D-proximal chromosome 17 markers and for one distal marker by restriction enzyme-site variations and Southern analysis. A single mitotic crossover could account for 50 clones (37%), with heterozygosity for at least one proximal marker and loss of heterozygosity for all markers distal to the putative recombination site. Eighty-two allele-loss variants (60%) were homozygous for all markers; the origin of these clones could be either chromosome loss or mitotic recombination between the centromere and the most proximal marker. Only 4 clones (3%) arose through more complex events such as multiple crossovers or deletion. A mitotic linkage map for mouse chromosome 17 was constructed, and the gene order deduced from somatic recombination was identical to that obtained by conventional transmission genetics. These results demonstrate that mitotic recombination is a common event leading to allele loss, in spite of the lack of evidence for frequent somatic pairing of homologous chromosomes. Mitotic mapping provides a defined system for comparison of mitotic and meiotic recombination and may lead to practical advances for elucidating somatic mechanisms of oncogenesis and for gene therapy in targeting mutations to specific sites through homologous recombination.     

Interference-mediated synaptonemal complex formation with embedded crossover designation

Biological systems exhibit complex patterns at length scales ranging from the molecular to the organismic. Along chromosomes, events often occur stochastically at different positions in different nuclei but nonetheless tend to be relatively evenly spaced. Examples include replication origin firings, formation of chromatin loops along chromosome axes and, during meiosis, localization of crossover recombination sites (“crossover interference”). We present evidence in the fungus Sordaria macrospora that crossover interference is part of a broader pattern that includes synaptonemal complex (SC) nucleation. This pattern comprises relatively evenly spaced SC nucleation sites, among which a subset are crossover sites that show a classical interference distribution. This pattern ensures that SC forms regularly along the entire length of the chromosome as required for the maintenance of homolog pairing while concomitantly having crossover interactions locally embedded within the SC structure as required for both DNA recombination and structural events of chiasma formation. This pattern can be explained by a threshold-based designation and spreading interference process. This model can be generalized to give diverse types of related and/or partially overlapping patterns, in two or more dimensions, for any type of object.Meiosis is the specialized cellular cycle that yields haploid gametes for sexual reproduction. A central feature of the meiotic program is recombination (1, 2). DNA/DNA recombination interactions, initiated by programmed double-strand breaks (DSBs), mediate the recognition and juxtaposition (pairing) of homologous chromosomes. A minority subset of these interactions matures into reciprocal crossover recombination products (COs); the remaining majority matures primarily into interhomolog non–crossover products (NCOs). COs promote genetic diversity but also are required for the segregation of homologous chromosomes (homologs) via their role in creating chiasmata (3).A nearly universal feature of meiosis is that COs occur along a particular chromosome at different positions in different meiotic nuclei. Nonetheless, along any given chromosome, COs tend to be evenly spaced. This pattern results from the reduced possibility that a second cross-over will occur if a crossover has occurred nearby. The existence of such a pattern was identified more than a century ago as the genetic phenomenon of CO interference (4, 5). In this phenomenon, occurrence of a CO in one genetic interval is accompanied by a reduced probability that another CO will occur along the same chromosome in a nearby interval. This effect implies the existence of communication along chromosomes, with an event at one position triggering occurrence of an “interference signal” that spreads outward, inhibiting occurrence of subsequent events nearby.A second central feature of the meiotic program is the synaptonemal complex (SC). This prominent structure links the axes of paired homologs along the lengths of the chromosome at midprophase, the “pachytene” stage (3, 6, 7). In the canonical meiotic program, as shown in several organisms, CO patterns and SC formation arise concomitantly at zygotene (Discussion and ref. 8). Also, in budding yeast, the SC is not required for CO patterning (9, 10). Intriguingly, however, in two organisms, Schizosaccharomyces pombe and Aspergillus nidulans, SC and CO interference are concomitantly absent (3), indicating some type of relationship between the two processes.The present study began by investigating the possibility that interference is not confined specifically to COs but instead pertains more broadly to include SC nucleations. Such integration could concomitantly ensure regular SC formation along the chromosomes and the embedding of CO recombinational interactions in a specialized local relationship to the SC.Here we analyze chromosomal events in the filamentous fungus Sordaria macrospora. Sordaria exhibits the canonical meiotic program and provides uniquely detailed readouts for recombination and SC formation including both ultrastructural data from 3D serial section reconstructions and whole-cell analysis of fluorescent signals for recombination complexes and the SC as they evolve through prophase (8, 11–13). Our findings support the existence of regularly-spaced SC nucleation sites, a subset of which comprise CO sites that exhibit classical interference.We next investigated the possible scenarios by which such a pattern might arise. The entire pattern, with all its component features, appears to emerge during a single stage, zygotene. Correspondingly, the notion that the entire array arises in a single patterning process is attractive. Detailed analysis supports a scenario in which SC nucleations, with embedded CO designation, emerge via a single interference-mediated process, i.e., a process that involves a spreading interference signal. The basic principles that emerge could generate diverse complex interrelated patterns.     

Long-range organization of tandem arrays of alpha satellite DNA at the centromeres of human chromosomes: high-frequency array-length polymorphism and meiotic stability.

The long-range organization of arrays of alpha satellite DNA at the centromeres of human chromosomes was investigated by pulsed-field gel electrophoresis techniques. Both restriction-site and array-length polymorphisms were detected in multiple individuals and their meiotic segregation was observed in three-generation families. Such variation was detected in all of the alpha satellite arrays examined (chromosomes 1, 3, 7, 10, 11, 16, 17, X, and Y) and thus appears to be a general feature of human centromeric DNA. The length of individual centromeric arrays was found to range from an average of approximately 680 kilobases (kb) for the Y chromosome to approximately 3000 kb for chromosome 11. Furthermore, individual arrays appear to be meiotically stable, since no changes in fragment lengths were observed. In total, we analyzed 84 meiotic events involving approximately 191,000 kb of alpha satellite DNA from six autosomal centromeres without any evidence for recombination within an array. High-frequency array length variation and the potential to detect meiotic recombination within them allow direct comparisons of genetic and physical distances in the region of the centromeres of human chromosomes. The generation of primary consensus physical maps of alpha satellite arrays is a first step in the characterization of the centromeric DNA of human chromosomes.