Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites. This resulted in the high-resolution mapping of mei-W68 to a <18-kb region that contains a homolog of the S. cerevisiae spo11 gene. Molecular analysis of several mutants confirmed that mei-W68 encodes an spo11 homolog. Spo11 and MEI-W68 are members of a family of proteins similar to a novel type II topoisomerase. On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double-strand breaks needed to initiate meiotic recombination. This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks. Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects. In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.

Although in Saccharomyces cerevisiae the initiation of meiotic recombination, as indicated by double-strand break formation, appears to be functionally linked to the initiation of synapsis, meiotic chromosome synapsis in Drosophila females occurs in the absence of meiotic exchange. Electron microscopy of oocytes from females homozygous for either of two meiotic mutants (mei-W68 and mei-P22), which eliminate both meiotic crossing over and gene conversion, revealed normal synaptonemal complex formation. Thus, synapsis in Drosophila is independent of meiotic recombination, consistent with a model in which synapsis is required for the initiation of meiotic recombination. Furthermore, the basic processes of early meiosis may have different functional or temporal relations, or both, in yeast and Drosophila.In the classical view of meiosis, homologous chromosome synapsis, as indicated by the formation of an elaborate ribbonlike structure called the synaptonemal complex (SC), was thought to be the first and primary event of meiotic prophase, essential for the initiation of meiotic recombination (1). Studies in Saccharomyces cerevisiae, however, have created a different view of the meiotic process in which the initiation of recombination, as evidenced by a doublestrand break (DSB), precedes the initiation of synapsis (2, 3). Three lines of evidence support this view of early meiotic prophase in yeast. First, the initiating event of meiotic recombination, the formation of a DSB, appears before SC formation (4). Second, meiotic mutants that either fail to create DSBs or to process DSBs to make single-stranded tails prevent the formation of a mature SC (2). Third, some mutants allow high levels of meiotic recombination but prevent the production of a mature SC (5). These data are consistent with a model in which single-stranded DNA generated by a DSB carries out a homology search required for synapsis and SC formation. In contrast, synapsis is not an absolute prerequisite for either the initiation (6) or completion of meiotic recombination (7).To assess the relation between synapsis and the initiation of recombination in Drosophila oocytes, we examined both recombination and SC formation in oocytes homozygous for either of two null-recombination mutations. The mei-W68 and mei-P22 (8) mutants prevent the initiation of meiotic recombination as defined by four independent assays: (i) reduction or elimination of meiotic gene conversion; (ii) elimination of meiotic crossing over, as assayed by measuring either intragenic crossing over or the frequency of meiotic crossing over along entire chromosome arms; (iii) lack of doublestrand DNA breaks that persist into metaphase or anaphase I; and (iv) failure to produce either early or late recombination nodules (RNs).To assay the effects of the mei-W68 and mei-P22 mutations on meiotic crossing over, we examined intragenic recombination at the rosy locus (9). No gene conversion events or intragenic crossovers were observed among the progeny of mei-W68 or mei-P22 females (Table 1 and Fig...

Using an antibody against the phosphorylated form of His2Av (γ-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to γ-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to γ-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and γ-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All γ-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray–induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as γ-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of γ-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. Here, we test the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. We find that cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and that reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. We also find that cut expression is increased by a unique pds5 gene mutation that reduces the binding of cohesin to chromosomes. On the basis of these results, we posit that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B.

The D. melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes. Here we show that the predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins. There is also extensive functional overlap between mei-41 and ATM. Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability. We also demonstrate that mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells. Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene.