Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly.Karyogamy is the process by which two nuclei fuse to produce a single nucleus. This process is critical in diploid organisms (e.g., mammals and plants) when haploid egg and sperm nuclei fuse to produce a diploid nucleus and zygote. In organisms with a haploid vegetative cycle (e.g., fungi and most algae), karyogamy is required to produce the diploid nucleus/cell, which will then enter meiosis. In both situations, karyogamy involves two steps: nuclear movement leading to nuclear juxtaposition (congression) and final fusion of the nuclear membranes. Cooperation of the cytoskeleton components is required for nuclear movement and correct positioning of the nuclei (e.g., ref. 1).In budding yeast, premeiotic karyogamy requires several genes (named Kar1 to Kar9) with their mutant defects corresponding to two steps: nuclear congression, which involves cytoskeleton components, motor proteins and the spindle pole body (SPB, mammal centrosome equivalent) and fusion of the two haploid nuclear envelopes (NEs), which involves either the endoplasmic reticulum (ER) per se or protein translocation into the perinuclear space (reviewed in refs. 2–4 and references therein). Kar homologs are present in fission yeast and Candida albicans (5, 6) and two fission-yeast proteins required for proper microtubule (MT) integrity, Mal3 and Mto1, are also required for karyogamy (7). Blast search of the basidiomycete Cryptococcus neoformans genome identified four of the known KAR genes; interestingly only the kar7 mutant showed a defect in vegetative nuclear movement and hyphae mating but not in premeiotic karyogamy (8), likely reflecting the different evolution of ascomycetes and basidiomycetes.Whereas karyogamy studies of fungi have provided significant insight into the steps and molecules involved, less is known concerning the outcome of the remaining nonfused haploid nuclei. Such “twin” meiosis has been analyzed in fission yeast in three cases: in the presence of the mating-type allele h⁹⁰ (9) or in absence of either the microtubule plus-end tracking protein Eb1/Mal3 (7, 10) or the type 1 membrane protein Tht1 (11), but without detailed analysis of the meiotic process per se.To further investigate these issues, we cloned and characterized the two Sad1p, UNC-84 (SUN)-domain genes of the fungus Sordaria macrospora. In most organisms investigated thus far, SUN-domain proteins are NE associated and are good candidates to play roles in karyogamy because they (i) transmit forces between the nucleus and cytoskeletal complexes; (ii) have conserved roles in meiotic chromosome movements, and (iii) in yeasts, are associated with the SPB (reviewed in ref. 12). One Sordaria gene is a member of the canonical SUN1/SUN2/MPS3/SAD1 family. Being essential for cell viability, it could not be analyzed for roles in the sexual cycle. The other gene is not essential for viability and is a member of the SLP1/OPT/SUCO/SUN3/4/5/SUNB mid–SUN-domain family (13–15). Like the other proteins of this family, Sordaria sun like protein 1 (Slp1) is localized in both the ER and NE. The precise role of those proteins remains unknown. Budding yeast Slp1 is implicated in folding of integral membrane proteins and is involved directly or indirectly in Sun1/Mps3 localization to the NE (15, 16). Dictyostellum discoideum SunB protein plays roles in both cell proliferation and differentiation (14). The mouse ortholog osteopotentia (Opt) protein is a key regulator of bone formation (17). The two splicing variants (CH1 and C1orf9) of the human SUCO gene were respectively found overexpressed in breast cancer cells or linked to the hereditary prostate cancer locus, HPC1 (18, 19).Interestingly, the Sordaria SLP1 gene is absolutely crucial for karyogamy but not for the overall meiotic program. In an slp1Δ null mutant, the two juxtaposed haploid nuclei progress synchronously and efficiently through the meiotic program, but the normal program of recombination-mediated interactions between homologous chromosomes (including synaptonemal complex formation and crossover interference) (20) now occurs regularly and efficiently between sister chromatids. These and other observations imply that the two haploid twin nuclei sense their change in status, either by detecting absence of karyogamy or by directly detecting absence of a homolog partner, and appropriately alter the program of effects to rescue a minimal level of gamete formation.     

Positional cloning and characterization of Mei1, a vertebrate-specific gene required for normal meiotic chromosome synapsis in mice

The mouse meiotic mutant Mei1 was isolated in a screen for infertile mice descended from chemically mutagenized embryonic stem cells. Homozygotes of both sexes are sterile due to meiotic arrest caused by defects in chromosome synapsis. Notably, RAD51 protein does not load onto Mei1 mutant meiotic chromosomes, suggesting that there is a defect in either recombinational repair or the production of double-strand breaks (DSBs) that require such repair. Here, we show that treatment of mutant males with cisplatin restores RAD51 loading, suggesting that mutant spermatocytes have intact recombinational repair mechanisms. Levels of histone H2AX phosphorylation (gammaH2AX) at leptonema are significantly reduced compared with wild-type controls but comparable to that seen in animals deficient for SPO11, the molecule required for catalyzing DSB formation during meiosis. These observations provide evidence that genetically programmed DSB induction is defective in Mei1 leptotene spermatocytes. We also report the positional cloning of Mei1, which encodes a product without significant homology to any known protein. Expressed almost exclusively in gonads, Mei1 has no apparent homologs in yeast, worms, or flies. However, Mei1 orthologs are present in the genomes of mammals, chickens, and zebrafish. Thus, Mei1 is required for vertebrate meiosis. To our knowledge, Mei1 is the first meiosis-specific mutation identified by forward genetic approaches in mammals.     

RNAi and heterochromatin repress centromeric meiotic recombination

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.     

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.     

Hybrid DNA formation during meiotic recombination.

G234 is a silent mutation located in the middle of gene b2, which controls spore pigmentation in Ascobolus immersus. Its effect on the aberrant segregation patterns of while spore mutants located in the same gene was investigated. When heterozygous, G234 decreases the frequency of aberrant segregations of the mutants located on its right, toward the low conversion end. It almost completely suppresses the aberrant 4:4 asci for mutants giving postmeiotic segregation and decreases the disparity between the 6 wild-type:2 mutant and 2 wild-type:6 mutant aberrant asci for mutants giving only these types of convertant asci. These effects are polar; G234 does not change the aberrant segregation pattern of the mutants located on its left, toward the high conversion end. This behavior suggests that G234 blocks the migration of the symmetric phase of hybrid DNA that diffuses from the high conversion end but does not prevent the formation of asymmetrical hybrid DNA. Taking into account previous observations, we conclude that the high conversion end corresponds to a region of asymmetric initiation of recombination rather than to a region of preferential ending of recombination. The asymmetric hybrid DNA first formed is further changed into a symmetric phase that extends via branch migration toward the low conversion end.     

Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis

Recombination, in the form of cross-overs (COs) and gene conversion (GC), is a highly conserved feature of meiosis from fungi to mammals. Recombination helps ensure chromosome segregation and promotes allelic diversity. Lesions in the recombination machinery are often catastrophic for meiosis, resulting in sterility. We have developed a visual assay capable of detecting Cos and GCs and measuring CO interference in Arabidopsis thaliana. This flexible assay utilizes transgene constructs encoding pollen-expressed fluorescent proteins of three different colors in the qrt1 mutant background. By observing the segregation of the fluorescent alleles in 92,489 pollen tetrads, we demonstrate (i) a correlation between developmental position and CO frequency, (ii) a temperature dependence for CO frequency, (iii) the ability to detect meiotic GC events, and (iv) the ability to rapidly assess CO interference.     

A meiotic recombination in a new isolated familial somatotropinoma kindred

We report here the genetic findings of a new isolated familial somatotropinoma (IFS) kindred in which the mother (subject I:2) and one daughter (subject II:2) are affected; their ages at diagnosis were 25 and 14 years respectively. Additionally, patient I:2 developed virilization due to an androgen-secreting adrenocortical mass, presenting clinical and molecular features of sporadic adrenal carcinoma. To genotype this family and to narrow down the candidate interval of the putative IFS gene at 11q13, we performed haplotyping on the DNA from all five members of the family and allelotyping of one available somatotropinoma using polymorphic microsatellite markers from chromosome region 11q12.1-11q13.5. Results indicated that the disease haplotype, between markers D11S956 and D11S527, was transmitted from subject I:2 only to subject II:2. A meiotic recombination event was detected in the fraternal twin sister of II:2 (subject II:1), but her disease status is unknown. Since she is only 18 years old this genetic event cannot yet narrow down the area involved in the pathogenesis of IFS. Allelotyping of the somatotropinoma from II:2 revealed loss of the chromosome carrying the wild-type copy of the putative IFS gene inherited from her father. These results support the involvement of a tumor suppressor gene at 11q13.1-q13.3 in the pathogenesis of IFS.     

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.     

Evidence for two pathways of meiotic intrachromosomal recombination in yeast.

This study shows that RAD50, a yeast DNA repair gene required for meiotic interchromosomal exchange between homologs, also is required for meiotic intrachromosomal recombination. However, only intrachromosomal events in nonribosomal DNA are dependent on RAD50; those in ribosomal DNA (rRNA-encoding DNA) occur in the absence of this gene. Furthermore, nonribosomal DNA sequences retain their RAD50-dependence even when inserted into the ribosomal DNA array. We argue that these data provide evidence for at least two pathways of meiotic intrachromosomal recombination whose activity depends on the specific sequences involved or their structural context in the chromosome. In contrast to its role in meiosis, RAD50 is not required for either inter- or intrachromosomal spontaneous mitotic recombination.     

Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope

Dynamic repositioning of telomeres is a unique feature of meiotic prophase I that is highly conserved among eukaryotes. At least in fission yeast it was shown to be required for proper alignment and recombination of homologous chromosomes. On entry into meiosis telomeres attach to the nuclear envelope and transiently cluster at a limited area to form a chromosomal bouquet. Telomere clustering is thought to promote chromosome recognition and stable pairing of the homologs. However, the molecular basis of telomere attachment and movement is largely unknown. Here we report that mammalian SUN-domain protein Sun2 specifically localizes to the nuclear envelope attachment sites of meiotic telomeres. Sun2-telomere association is maintained throughout the dynamic movement of telomeres. This association does not require the assembly of chromosomal axial elements or the presence of A-type lamins. Detailed EM analysis revealed that Sun2 is part of a membrane-spanning fibrillar complex that interconnects attached telomeres with cytoplasmic structures. Together with recent findings in fission yeast, our study indicates that the molecular mechanisms required for tethering meiotic telomeres and their dynamic movements during bouquet formation are conserved among eukaryotes.     

Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster.

Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing.     

A checkpoint control linking meiotic S phase and recombination initiation in fission yeast

During meiosis, high levels of recombination initiated by DNA double-strand breaks (DSBs) occur only after DNA replication. However, how DSB formation is coupled to DNA replication is unknown. We examined several DNA replication proteins for a role in this coupling in Schizosaccharomyces pombe, and we show that ribonucleotide reductase, the rate-limiting enzyme of deoxyribonucleotide synthesis and the target of the DNA synthesis inhibitor hydroxyurea (HU) is indirectly required for DSB formation linked to DNA replication. However, in cells in which the function of the DNA-replication-checkpoint proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, or Cds1p was compromised, DSB formation occurred at similar frequencies in the absence or presence of HU. The DSBs in the HU-treated mutant cells occurred at normal sites and were associated with recombination. In addition, Cdc2p is apparently not involved in this process. We propose that the sequence of meiotic S phase and initiation of recombination is coordinated by DNA-replication-checkpoint proteins.     

Initiation of meiotic recombination is independent of interhomologue interactions.

In yeast meiosis, crossing-over between homologues is dependent upon double-strand breaks. We demonstrate that the occurrence of these breaks is independent of pairing between homologues by showing that they occur with normal frequency, timing, and position in the absence of a homologue. This observation supports models that view double-strand breaks as initiating events and crossing-over as a consequence of repair of these breaks.     

Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae

Chromosome I is the smallest chromosome in Saccharomyces cerevisiae and contains a DNA molecule that is only 250 kilobases (kb). Approximately 75% of this DNA molecule has been cloned. A restriction map for the entire DNA molecule from chromosome I was determined and most of its genetically mapped genes were located on this physical map. Based on the average rate of recombination (centimorgans/kb) found for other S. cerevisiae chromosomes, the outermost markers on the genetic map of chromosome I were expected to be close to the ends of the DNA molecule. While the rightmost genetic marker was 3 kb from the end, the leftmost marker, CDC24, was located near the middle of the left arm, suggesting that the genetic map would be much longer. To extend the genetic map, a copy of the S. cerevisiae URA3 gene was integrated in the outermost cloned region located 32 kb centromere distal to CDC24, and the genetic map distance between these two genes was determined. The new marker substantially increased the genetic map length of chromosome I. In addition, we determined the relationship between physical and genetic map distance along most of the length of the chromosome. Consistent with the longer genetic map, the average rate of recombination between markers on chromosome I was greater than 50% higher than the average found on other yeast chromosomes. Owing to its small size, it had been estimated that approximately 5% of the chromosome I homologues failed to undergo meiotic recombination. New measurements of the zero-crossover class indicated that the enhanced rate of recombination ensures at least one genetic exchange between virtually every pair of chromosome I homologues.     

Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing

Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice, hotspots are largely defined by binding sites of the positive-regulatory domain zinc finger protein 9. To investigate the detailed recombination pattern in a flowering plant, we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination rates peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in positive-regulatory domain zinc finger protein 9–knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms.Meiotic recombination is a highly regulated process that enables pairing of homologous chromosomes and, by the formation of crossovers, ensures proper segregation (1). Along with mutation, drift, and selection, recombination is a critical factor in shaping genome-wide sequence variation. Recombination rates vary substantially across eukaryote genomes (2) in a manner that we are only beginning to understand. In humans and mice, the location of regions of strong recombination (“hotspots”) is largely determined by positive-regulatory domain zinc finger protein 9 (PRDM9) binding sites (3), whereas in yeast such regions are associated with nucleosome-depleted open chromatin often associated with gene promoters (4). When PRDM9 is disabled in mice, hotspots tend to relocalize to promoter regions (5). In flowering plants, at least one example of a promoter-associated hotspot has been reported (6), but it remains an open question whether this is a general tendency in plants.The positions of crossovers and the boundaries of gene conversion tracts resulting from meiotic recombination are often imprecisely known, because they can only be identified based on the location of nearby segregating markers. Within a species, genome-wide variation in recombination rates can be determined by following the inheritance of such genetic markers in crosses or pedigrees (7–10) or by examining patterns of linkage disequilibrium within a population (11–15). Population-based approaches have the advantage that in diverse populations, hundreds of thousands of historical recombination events can be sampled, compared with only hundreds in the largest pedigrees.The monkeyflower Mimulus guttatus has an exceptionally high nucleotide diversity, which makes it a particularly appealing system for characterizing the boundaries of recombination events. We observed an average pairwise nucleotide difference of π = 2.9% in a sample of 98 wild plants (196 haploid genomes) from four locations within a 16-km radius in the Sierra Nevada foothills in Northern California. At such high diversity, pairs of adjacent SNPs defining local haplotypes are often found on the same Illumina sequencing read (e.g., within 50 bases). Thus, short-range haplotypes can be determined cost-effectively by shotgun sequencing pooled samples rather than by sequencing each plant individually.For a pair of nearby segregating biallelic SNPs we expect to observe only three of the four possible haplotypes unless recombination and/or parallel mutation has occurred since the originating mutations. This is the essence of Hudson’s four-gamete test (16) and allows us to identify putative boundaries of historical crossovers and gene conversion tracts (Fig. 1) to within a fraction of a read length. If this information is combined with population genetic models, local recombination rates can then be inferred (17).Open in a separate windowFig. 1.Appearance of four haplotypes at a pair of SNP loci by recombination. From a single ancestral sequence (Top) a single mutation produces a second haplotype. A second mutation at a nearby site (Middle) generates a third haplotype In the population. Finally, a recombination boundary between the two SNP loci (Bottom) generates a fourth haplotype. Note that the recombination boundary can be due to a crossover event or a gene conversion tract. A fourth haplotype can also appear due to a parallel mutation (not shown), but this scenario can be distinguished from recombination because parallel mutation at a site should not depend on the distance to the nearest SNP.     

Ligand-activated site-specific recombination in mice.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.     

Relationships among chromatid interchanges, sister chromatid exchanges, and meiotic recombination in Drosophila melanogaster

Repair- and recombination-defective mutations at two loci (mei-9 and mei-41) of Drosophila melanogaster have been examined for their effects on the induction of chromosome aberrations by x-rays and the formation of sister chromatid exchanges (SCEs). Irradiation of larval neuroblast cells during the S phase with x-rays showed that mutants at both of these loci are about 10 times more sensitive than wild type to the induction of chromosome aberrations. The pattern of induced aberrations was characteristic for each mutant locus: in cells bearing mei-9 mutations most breaks were chromatid deletions, whereas in the presence of mei-41 mutations similar frequencies of chromatid and isochromatid deletions were observed. Furthermore, chromatid interchanges could not be induced in cells carrying mei-9 alleles; therefore these mutations define a step necessary for chromatid rejoining. mei-41 alleles also define a function involved in the formation of chromatid interchanges; total exchanges were less frequent than expected from nonmutant controls; and the proportion of exchanges arising by symmetrical rejoining was markedly reduced. These data indicate that chromatid and isochromatid deletions have different molecular steps in their formation, and that different molecular mechanisms are also involved in the symmetrical and unsymmetrical rejoining in chromatid interchanges. Neuroblast cells of larvae bearing mei-9 and mei-41 alleles were also treated for 13 hr with 5-bromodeoxyuridine at 9 μg/ml in order to differentiate sister chromatids for the scoring of SCEs. Whereas mei-41 had a normal level of SCEs, mei-9 exhibited a frequency of SCEs that was about 70% that of the control. Because both mei-9 and mei-41 mutations result in defective meiotic recombination, these data suggest that they define steps shared by symmetrical interchange formation and meiotic recombination that do not participate in the formation of most SCEs.     

Mnd1p: an evolutionarily conserved protein required for meiotic recombination

We used a functional genomics approach to identify a gene required for meiotic recombination, YGL183c or MND1. MND1 was spliced in meiotic cells, extending the annotated YGL183c ORF N terminus by 45 aa. Saccharomyces cerevisiae mnd1-1 mutants, in which the majority of the MND1 coding sequence was removed, arrested before the first meiotic division with a phenotype reminiscent of dmc1 mutants. Physical and genetic analysis showed that these cells initiated recombination, but did not form heteroduplex DNA or double Holliday junctions, suggesting that Mnd1p is involved in strand invasion. Orthologs of MND1 were identified in protists, several yeasts, plants, and mammals, suggesting that its function has been conserved throughout evolution.     

Tissue- and site-specific DNA recombination in transgenic mice.

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries.