抑制植物减数分裂重组的分子机理

减数分裂重组不仅保证了真核生物有性生殖过程中染色体数量的稳定,还通过父母亲本间遗传物质的互换在后代中产生遗传变异。因此,减数分裂重组是遗传多样性形成的重要途径,也是生物多样性和物种进化的主要动力。在绝大多数真核生物中,不管染色体数目的多少或基因组的大小,减数分裂重组的形成都受到严格的调控,但抑制减数分裂重组的分子机理目前仍不清楚。近年来,通过正向遗传学筛选鉴定出多个减数分裂重组抑制基因,揭示了抑制基因的功能和调控途径。本文基于拟南芥中减数分裂重组抑制基因的研究现状,综述了植物减数分裂重组抑制基因研究取得的突破性进展,并结合基因功能与其调控网络阐述了抑制植物减数分裂重组的分子机理。     

Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus.

In plant genomes as well as other eukaryotic genomes, meiotic recombination does not occur uniformly. At the level of the gene, high recombination frequencies are often observed within genetic loci in maize, but this feature of intragenic recombination is not seen at the csr1 locus in Arabidopsis. These observations suggest that meiotic recombination in plant genomes varies considerably among species. In the present study we investigated meiotic recombination at the wx locus in rice. The mutation sites of wx mutants induced by ethyl methanesulfonate (EMS) treatment or gamma-ray irradiation and a spontaneous wx mutant were physically characterized, and the genetic distances between those wx mutation sites were estimated by pollen analysis. Based on these results, the recombination frequency at the wx locus in rice was estimated as 27.3 kb/cM, which was about 10 times higher than the average for the genome, suggesting that there was a radically different rate of meiotic recombination for intra- and intergenic regions in the rice genome.     

The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination

Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.     

The double-stranded DNA helix in recombination at meiosis.

The nature of meiotic genetic recombination was resolved at the DNA level by the 1953 Watson and Crick model. What remains to be determined are the roles of the various recombination proteins and the distribution and localization of recombination events in the meiotic prophase nucleus.     

ReCombine: a suite of programs for detection and analysis of meiotic recombination in whole-genome datasets

In meiosis, the exchange of DNA between chromosomes by homologous recombination is a critical step that ensures proper chromosome segregation and increases genetic diversity. Products of recombination include reciprocal exchanges, known as crossovers, and non-reciprocal gene conversions or non-crossovers. The mechanisms underlying meiotic recombination remain elusive, largely because of the difficulty of analyzing large numbers of recombination events by traditional genetic methods. These traditional methods are increasingly being superseded by high-throughput techniques capable of surveying meiotic recombination on a genome-wide basis. Next-generation sequencing or microarray hybridization is used to genotype thousands of polymorphic markers in the progeny of hybrid yeast strains. New computational tools are needed to perform this genotyping and to find and analyze recombination events. We have developed a suite of programs, ReCombine, for using short sequence reads from next-generation sequencing experiments to genotype yeast meiotic progeny. Upon genotyping, the program CrossOver, a component of ReCombine, then detects recombination products and classifies them into categories based on the features found at each location and their distribution among the various chromatids. CrossOver is also capable of analyzing segregation data from microarray experiments or other sources. This package of programs is designed to allow even researchers without computational expertise to use high-throughput, whole-genome methods to study the molecular mechanisms of meiotic recombination.     

Biochemical analysis of genetic recombination in eukaryotes.

Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.     

Genetic control of chromosome synapsis in yeast meiosis

Both meiosis-specific and general recombination functions, recruited from the mitotic cell cycle, are required for elevated levels of recombination and for chromosome synapsis (assembly of the synaptonemal complex) during yeast meiosis. The meiosis-specific SPO11 gene (previously shown to be required for meiotic recombination) has been isolated and shown to be essential for synaptonemal complex formation but not for DNA metabolism during the vegetative cell cycle. In contrast, the RAD52 gene is required for mitotic and meiotic recombination but not for synaptonemal complex assembly. These data suggest that the synaptonemal complex may be necessary but is clearly not sufficient for meiotic recombination. Cytological analysis of spread meiotic nuclei demonstrates that chromosome behavior in yeast is comparable with that observed in larger eukaryotes. These spread preparations support the immunocytological localization of specific proteins in meiotic nuclei. This combination of genetic, molecular cloning, and cytological approaches in a single experimental system provides a means of addressing the role of specific gene products and nuclear structures in meiotic chromosome behavior.     

利用荧光标记高通量鉴定减数分裂重组抑制突变体

正向遗传学突变体筛选被广泛用于揭示减数分裂中涉及的遗传基因, 如调控减数分裂II型交叉形成途径的重组抑制基因。该研究利用拟南芥(Arabidopsis thaliana)花粉荧光标记系进行EMS突变体的正向遗传学筛选, 鉴定拟南芥野生型Col遗传背景下的重组抑制突变体, 共获得18个重组率显著提高3倍以上的重组抑制突变体, 其中包括显性和隐性遗传突变。研究表明, 基于荧光标记高通量鉴定重组抑制突变体是可行的, 可为植物减数分裂重组调控分子机制研究提供新方法和突变材料。     

Female site-specific transposase-induced recombination: a high-efficiency method for fine mapping mutations on the X chromosome in Drosophila

P-element transposons in the Drosophila germline mobilize only in the presence of the appropriate transposase enzyme. Sometimes, instead of mobilizing completely, P elements will undergo site-specific recombination with the homologous chromosome. Site-specific recombination is the basis for male recombination mapping, since the male germline does not normally undergo recombination. Site-specific recombination also takes place in females, but this has been difficult to study because of the obscuring effects of meiotic recombination. Using map functions, I demonstrate that it is possible to employ female site-specific transposase-induced recombination (FaSSTIR) to map loci on the X chromosome and predict that FaSSTIR mapping should be more efficient than meiotic mapping over short genetic intervals. Both FaSSTIR mapping and meiotic mapping were used to fine map the crossveinless locus on the X chromosome. Both techniques identified the same 10-kb interval as the probable location of the crossveinless mutation. Over short intervals (< approximately 7.6 cM), FaSSTIR produces more informative recombination events than does meiotic recombination. Over longer intervals, FaSSTIR is not always more efficient than meiotic mapping, but it produces the correct gene order. FaSSTIR matches the expectations suggested by the map functions and promises to be a useful technique, particularly for mapping X-linked loci.     

利用荧光标记高通量鉴定减数分裂重组抑制突变体

正向遗传学突变体筛选被广泛用于揭示减数分裂中涉及的遗传基因, 如调控减数分裂II型交叉形成途径的重组抑制基因。该研究利用拟南芥(Arabidopsis thaliana)花粉荧光标记系进行EMS突变体的正向遗传学筛选, 鉴定拟南芥野生型Col遗传背景下的重组抑制突变体, 共获得18个重组率显著提高3倍以上的重组抑制突变体, 其中包括显性和隐性遗传突变。研究表明, 基于荧光标记高通量鉴定重组抑制突变体是可行的, 可为植物减数分裂重组调控分子机制研究提供新方法和突变材料。     

Genetic Crossovers Are Predicted Accurately by the Computed Human Recombination Map

Hotspots of meiotic recombination can change rapidly over time. This instability and the reported high level of inter-individual variation in meiotic recombination puts in question the accuracy of the calculated hotspot map, which is based on the summation of past genetic crossovers. To estimate the accuracy of the computed recombination rate map, we have mapped genetic crossovers to a median resolution of 70 Kb in 10 CEPH pedigrees. We then compared the positions of crossovers with the hotspots computed from HapMap data and performed extensive computer simulations to compare the observed distributions of crossovers with the distributions expected from the calculated recombination rate maps. Here we show that a population-averaged hotspot map computed from linkage disequilibrium data predicts well present-day genetic crossovers. We find that computed hotspot maps accurately estimate both the strength and the position of meiotic hotspots. An in-depth examination of not-predicted crossovers shows that they are preferentially located in regions where hotspots are found in other populations. In summary, we find that by combining several computed population-specific maps we can capture the variation in individual hotspots to generate a hotspot map that can predict almost all present-day genetic crossovers.     

Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana

Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51‐DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three ‘non‐meiotic’ paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.     

Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions.     

Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recA-licBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.     

Maximal power tests for detecting defects in meiotic recombination

We have determined the marker separations (genetic distances) that maximize the probability, or power, of detecting meiotic recombination deficiency when only a limited number of meiotic progeny can be assayed. We find that the optimal marker separation is as large as 30-100 cM in many cases. Provided the appropriate marker separation is used, small reductions in recombination potential (as little as 50%) can be detected by assaying a single interval in as few as 100 progeny. If recombination is uniformly altered across the genomic region of interest, the same sensitivity can be obtained by assaying multiple independent intervals in correspondingly fewer progeny. A reduction or abolition of crossover interference, with or without a reduction of recombination proficiency, can be detected with similar sensitivity. We present a set of graphs that display the optimal marker separation and the number of meiotic progeny that must be assayed to detect a given recombination deficiency in the presence of various levels of crossover interference. These results will aid the optimal design of experiments to detect meiotic recombination deficiency in any organism.     

Exploring the pathways of homologous recombination.

There has been significant progress in elucidating the mechanisms by which meiotic and mitotic recombination occur. Double-strand breaks in particular have been the object of attention in studies on meiotic gene conversion, site-specific mitotic recombination, the repair of transposon excision and the transformation of cells with linearized DNA. A combination of genetic analysis and physical studies of molecular recombination intermediates have established that double-strand breaks can occur by two different mechanisms.     

Male mouse recombination maps for each autosome identified by chromosome painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.     

The Role of the SPO11 Gene in Meiotic Recombination in Yeast

Several complementary experimental approaches were used to demonstrate that the SPO11 gene is specifically required for meiotic recombination. First, sporulating cultures of spo11-1 mutant diploids were examined for landmark biochemical, cytological and genetic events of meiosis and ascosporogenesis. Cells entered sporulation with high efficiency and showed a near-doubling of DNA content. Synaptonemal complexes, hallmarks of intimate homologous pairing, and polycomplex structures appeared during meiotic prophase. Although spontaneous mitotic intra- and intergenic recombination occurred at normal levels, no meiotic recombination was observed. Whereas greater than 50% of cells completed both meiotic divisions, packaging of the four meiotic products into mature ascospores took place in only a small subset of asci. Haploidization occurred in less than 1% of viable colony-forming units. Second, the Rec- meiotic defect conferred by spo11-1 was confirmed by dyad analysis of spores derived from spo13-1 single-division meiosis in which recombination is not a requirement for viable ascospore production. Diploids homozygous for the spo13-1 mutation undergo meiotic levels of exchange followed by a single predominantly equational division and form asci containing two near-diploid spores. With the introduction of the spo11-1 mutation, high spore viability was retained, whereas intergenic recombination was reduced by more than 100-fold.     

Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination

The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles.     

Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.