Long-range restriction site mapping of a syntenic segment conserved between human chromosome 1 and mouse chromosome 3

A linkage map determined from segregation analysis of 338 meiotic events in an interspecific mouse cross was utilized to help investigate genomic organization of a linkage group conserved between human chromosome 1p and mouse chromosome 3. Using pulsed-field gel electrophoresis, the genes encoding the lymphocyte adhesion molecule human CD2/murine Ly-37, the alpha 1-subunit of Na, K-ATPase, the beta-subunit of thyrotropin, the beta-subunit of nerve growth factor, and muscle adenylate deaminase were similarly positioned on long-range restriction maps in both species. These studies indicate that the development of detailed genetic maps using interspecific Mus crosses facilitates rapid analysis of murine genomic organization and may enable physical mapping of syntenic regions within the human genome. Moreover, the data suggest profound conservation of genomic organization during mammalian evolution.     

Extreme heterogeneity in the molecular events leading to the establishment of chiasmata during meiosis i in human oocytes

In humans, ~50% of conceptuses are chromosomally aneuploid as a consequence of errors in meiosis, and most of these aneuploid conceptuses result in spontaneous miscarriage. Of these aneuploidy events, 70% originate during maternal meiosis, with the majority proposed to arise as a direct result of defective crossing over during meiotic recombination in prophase I. By contrast, <1%-2% of mouse germ cells exhibit prophase I-related nondisjunction events. This disparity among mammalian species is surprising, given the conservation of genes and events that regulate meiotic progression. To understand the mechanisms that might be responsible for the high error rates seen in human females, we sought to further elucidate the regulation of meiotic prophase I at the molecular cytogenetic level. Given that these events occur during embryonic development in females, samples were obtained during a defined period of gestation (17-24 weeks). Here, we demonstrate that human oocytes enter meiotic prophase I and progress through early recombination events in a similar temporal framework to mice. However, at pachynema, when chromosomes are fully paired, we find significant heterogeneity in the localization of the MutL homologs, MLH1 and MLH3, among human oocyte populations. MLH1 and MLH3 have been shown to mark late-meiotic nodules that correlate well with--and are thought to give rise to--the sites of reciprocal recombination between homologous chromosomes, which suggests a possible 10-fold variation in the processing of nascent recombination events. If such variability persists through development and into adulthood, these data would suggest that as many as 30% of human oocytes are predisposed to aneuploidy as a result of prophase I defects in MutL homolog-related events.     

Examination of sequence homology between human chromosome 20 and the mouse genome: intense conservation of many genomic elements

The conservation of genomic organization of mammalian species has been of interest for its usefulness in characterizing the genetics of traits and diseases and as one tool for examining evolution. The recent rough draft sequencing of the mouse and human genomes provides the opportunity for more detailed analyses. The current study examines the extent of homology between human chromosome 20 and the mouse genome by comparing putative coding and non-coding sequence to provide insight into organizational and sequence similarities between the species. The relative position of each of 460 putative coding orthologues was the same in both species, except for a single genomic segment rearrangement. The similarity extended to exon/intron structure, the size of introns, as well as strong evidence for the conservation of position of ancient LINE-1, LINE-2 and LTR repetitive sequence and the subtelomeric region of the long arm of human chromosome 20 and that of mouse chromosome 2. There was also evidence for conservation of a limited amount of non-coding single-copy sequence. Together these data provide additional insight into the extent of conservation of mammalian genomic organization and sequence.     

利用酵母人工染色体（YAC）减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。     

Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae

Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination. Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events. These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome. They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene. Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway. The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved. With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.     

Involvement of Sir2/4 in silencing of DNA breakage and recombination on mouse YACs during yeast meiosis

Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double-strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes. Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse major histocompatibility complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels. Moreover, segregation of these YACs during meiosis is seriously compromised. In meiotic yeast cells carrying the mutations sir2 or sir4, but not sir3, these YACs show DSBs, suggesting that a unique chromatin structure of the YACs, involving Sir2 and Sir4, protects the YACs from the meiotic recombination machinery. We speculate that the paucity of DSBs and recombination events on these YACs during yeast meiosis may reflect the refractory nature of the corresponding region in the mouse genome.     

Temporal expression of cell cycle-related proteins during spermatogenesis: establishing a timeline for onset of the meiotic divisions

During spermatogenesis, the complex events of the first meiotic prophase and division phase bring about dramatic changes in nuclear organization. One factor frustrating mechanistic dissection of these events is lack of knowledge about precisely what events occur, in what order they occur, and how they may be interrelated by temporal sequence; in other words, a precise timeline is lacking. This temporal ordering problem can be tackled by following expression and localization in mouse spermatocytes of proteins critical to events of the meiotic cell division process. These include ones that are primarily chromosomal and related to pairing and recombination, as well as kinases and substrates that mediate the cell cycle transition. Distinct and protein-specific patterns occur with respect to expression and localization throughout meiotic prophase and division and dramatic relocalization of proteins occurs as spermatocytes enter the meiotic division phase. This information provides a foundation for a meiotic timeline that can be augmented to provide, eventually, a complete catalog of meiotic events and their temporal sequence. Such a framework can clarify mechanisms of normal meiosis as well as mutant phenotypes and aberrations of the meiotic process that lead to aneuploidy.     

Sites of recombination are local determinants of meiotic homolog pairing in Saccharomyces cerevisiae

Trans-acting factors involved in the early meiotic recombination pathway play a major role in promoting homolog pairing during meiosis in many plants, fungi, and mammals. Here we address whether or not allelic sites have higher levels of interaction when in cis to meiotic recombination events in the budding yeast Saccharomyces cerevisiae. We used Cre/loxP site-specific recombination to genetically measure the magnitude of physical interaction between loxP sites located at allelic positions on homologous chromosomes during meiosis. We observed nonrandom coincidence of Cre-mediated loxP recombination events and meiotic recombination events when the two occurred at linked positions. Further experiments showed that a subset of recombination events destined to become crossover products increased the frequency of nearby Cre-mediated loxP recombination. Our results support a simple physical model of homolog pairing in budding yeast, where recombination at numerous genomic positions generally serves to loosely coalign homologous chromosomes, while crossover-bound recombination intermediates locally stabilize interactions between allelic sites.     

DNA repeated sequences may be involved in synaptonemal complex formation

Synaptonemal complexes (SCs) are intranuclear structures that facilitate the reversible lateral synapsis of homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the attachment of chromatin to SC lateral elements. Considering the features of the dispersed repeated sequences (RSs), it is possible to assume that they participate in the structure and functional organization of the meiotic chromosomes. Using numerical analysis, we have investigated the relationship between the RS and the distribution of meiotic recombination events in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have examined the arrangement of different types of RSs relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localized predominantly in SCs regions. Based on the results, we proposed a model of meiotic chromosome organization. According to the model, RSs participate in the attachment of chromatin loops to SCs.     

Meiosis in mice without a synaptonemal complex

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1(-/-)Sycp3(-/-), here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups.     

DNA repeated sequences may be involved in the synaptonemal complexes formation

Synatonemal complexes (SCs) are the intranuclear structures which facilitate reversible lateral synapsis of the homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the chromatin attachment to the SC lateral elements. Considering the features of the dispersed repeated sequences (RS) it is worth to assume their participation in the structure functional organization of the meiotic chromosome. Using numerical analysis we have investigated the relationship between RS and the distribution of events of the meiotic recombination in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have demonstrated the arrangement of different types of RS relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localizating predominantly in the SCs regions. Our results allow us to suggest the model of the meiotic chromosome organization with the RS as the sequences, participating in the attachment of chromatin loops and SCs.     

Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.     

Genomic organization and chromosomal localization of the mouse IKBKAP gene.

The autosomal recessive disorder familial dysautonomia (FD) has recently been demonstrated to be caused by mutations in the IKBKAP gene, so named because an initial report suggested that it encoded an IkappaB kinase complex associated protein (IKAP). Two mutations in IKBKAP have been reported to cause FD. The major mutation is a T-->C transition in the donor splice site of intron 20 and the minor mutation is a missense mutation in exon 19 that disrupts a consensus serine/threonine kinase phosphorylation site. We have characterized the cDNA sequences of the mouse, rat and rabbit IKBKAP-encoded mRNAs and determined the genomic organization and chromosomal location of mouse IKBKAP. There is significant homology in the amino acid sequence of IKAP across species and the serine/threonine kinase phosphorylation site altered in the minor FD mutation of IKAP is conserved. The mouse and human IKBKAP genes exhibit significant conservation of their genomic organization and the intron 20 donor splice site sequence, altered in the major FD mutation, is conserved in the human and mouse genes. Mouse IKBKAP is located on the central portion of chromosome 4 and maps to a region in which there is conserved linkage homology between the human and mouse genomes. The homologies observed in the human and mouse sequences should allow, through the process of homologous recombination, for the generation of mice that bear the IKBKAP mutations present in individuals with FD. The characterization of such mice should provide significant information regarding the pathophysiology of FD.     

UV irradiation of poplar cells of Drosophila melanogaster embryos. V. A study of the meiotic recombination in females with chromosomes of different structure

Polar cells of female embryos heterozygous for the genetic markers st in ri were UV irradiated. The effect of UV irradiation on the meiotic recombination within the st-ri segment was analysed. In individuals that contained this chromosome segment in its normal position, UV increased the rate of meiotic recombination. If this segment were separated from the centromeric heterochromatin and translocated on the left arm of chromosome II, UV did not influence meiotic recombination to a significant extent. On the other hand, in non-irradiated controls the individuals containing the translocated segment showed an increased rate of recombination within the segment compared with individuals having the segment in its normal position.These results can be explained by Suzuki's hypothesis according to which a heterocatalytic activity of heterochromatin shows an antagonism to meiotic recombination. UV seems to interfere with this antagonism indirectly via induction of physiological damage.     

A mouse c-ros genomic clone: identification of a highly conserved 22-amino acid segment in the juxta-membrane domain.

The mouse c-ros protooncogene genomic sequences have been cloned; an analysis of the partial genomic clone revealed a high conservation of the exons encoding the juxta-membrane (JM) and the 5' most protein tyrosine kinase domains. We have identified a segment of 22 amino acids conserved between the human and mouse JM domains; this segment may have a critical role in the function of the c-ros-encoded protein tyrosine kinase receptor.     

A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p.

The Saccharomyces cerevisiae Spo11 protein is thought to catalyze formation of the DNA double-strand breaks that initiate meiotic recombination. We have cloned cDNA and genomic DNA for a mouse gene encoding a protein with significant sequence similarity to conserved domains found in proteins of the Spo11p family. This putative mouse Spo11 gene maps to the distal region of chromosome 2 (homologous to human chromosome 20q13.2-q13.3) and comprises at least 12 exons, spanning approximately 15-18 kb. Strong expression of the Spo11 message is seen in juvenile and adult testis by RNA in situ hybridization, RT-PCR, and Northern blot, with much weaker expression in thymus and brain. In situ hybridization detects expression in oocytes of embryonic ovary, but not of adult ovary. RT-PCR and in situ hybridization analyses of a time course of juvenile testis development indicate that Spo11 expression begins in early meiotic Prophase I, prior to the pachytene stage, with increasing accumulation of mRNA through the pachytene stage. Taken together, these results strongly suggest that this gene encodes the functional homolog of yeast Spo11p, which in turn suggests that the mechanism of meiotic recombination initiation is conserved between yeast and mammals.     

Preaching about the converted: how meiotic gene conversion influences genomic diversity

Meiotic crossover (CO) recombination involves a reciprocal exchange between homologous chromosomes. COs are often associated with gene conversion at the exchange site where genetic information is unidirectionally transferred from one chromosome to the other. COs and independent assortment of homologous chromosomes contribute significantly to the promotion of genomic diversity. What has not been appreciated is the contribution of another product of meiotic recombination, noncrossovers (NCOs), which result in gene conversion without exchange of flanking markers. Here, we review our comprehensive analysis of recombination at a highly polymorphic mouse hotspot. We found that NCOs make up ～90% of recombination events. Preferential recombination initiation on one chromosome allowed us to estimate the contribution of CO and NCO gene conversion to transmission distortion, a deviation from Mendelian inheritance in the population. While NCO gene conversion tracts are shorter, and thus have a more punctate effect, their higher frequency translates into an approximately two-fold greater contribution than COs to gene conversion-based allelic shuffling and transmission distortion. We discuss the potential impact of mammalian NCO characteristics on evolution and genomic diversity.     

Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at,or Prior to,Establishment of Double-Strand Breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.     

Meiosis,recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants

Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes. However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early. Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans. Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.     

Divergent human and mouse orthologs of a novel gene (WBSCR15/Wbscr15) reside within the genomic interval commonly deleted in Williams syndrome

Williams syndrome (WS) is a contiguous gene deletion disorder resulting in complex and intriguing clinical features. Detailed molecular characterization studies of the genomic segment on human chromosome 7q11.23 commonly deleted in WS have uncovered numerous genes, each of which is being actively studied for its possible role in the etiology of the syndrome. Our efforts have focused on the comparative mapping and sequencing of the WS region in human and mouse. In previous studies, we uncovered important differences in the long-range organization of these human and mouse genomic regions; in particular, the notable absence of large duplicated blocks of DNA in mouse that are present in human. Aided by available genomic sequence data, we have used a combination of gene-prediction programs and cDNA isolation to identify the human and mouse orthologs of a novel gene (WBSCR15 and Wbscr15, respectively) residing within the genomic segment commonly deleted in WS. Unlike the flanking genes, which are closely related in human and mouse, WBSCR15 and Wbscr15 are strikingly different with respect to their cDNA and corresponding protein sequences as well as tissue-expression pattern. Neither the WBSCR15- nor Wbscr15-encoded amino acid sequence shows a statistically significant similarity to any characterized protein. These findings reveal another interesting evolutionary difference between the human and mouse WS regions and provide an additional candidate gene to evaluate with respect to its possible role in the pathogenesis of WS.