Relationship of DNA double-strand breaks to synapsis in Drosophila.

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV). Our results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation.

The Synaptonemal complex component C(2)M regulates meiotic crossing over in Drosophila.

BACKGROUND: The synaptonemal complex (SC) is a proteinaceous structure that forms between homologously paired meiotic chromosomes. Previous studies have suggested that the SC is required for meiotic crossing over in Drosophila. However, only one component of this structure, C(3)G, has been identified in Drosophila. RESULTS: Mutations in c(2)M cause a reduced frequency of meiotic crossing over due, in part, to how recombination events are resolved. Cytological evidence suggests that C(2)M is a component of the SC and is required for the assembly of C(3)G (a putative transverse filament of the SC) along the chromosomes. Additionally, C(2)M localizes along the chromosomes in the absence of C(3)G. Despite having a defect in C(3)G localization, c(2)M mutants unexpectedly affect crossing over less severely than a c(3)G mutant. There is virtually no crossing over in a c(3)G mutant, but c(2)M or c(2)M; c(3)G double mutants produce a substantial number of crossovers. The appearance of C(3)G-independent crossovers in c(2)M mutants suggests that C(2)M prevents recombination in the absence of complete SC formation. CONCLUSIONS: We have identified a new Drosophila SC component, C(2)M, that promotes the formation of crossovers. Furthermore, the appearance of C(3)G-independent crossovers in c(2)M mutants suggests a novel role in preventing recombination in the absence of complete SC.

Meiotic recombination and chromosome segregation in Drosophila females.

In this review, we describe the pathway for generating meiotic crossovers in Drosophila melanogaster females and how these events ensure the segregation of homologous chromosomes. As appears to be common to meiosis in most organisms, recombination is initiated with a double-strand break (DSB). The interesting differences between organisms appear to be associated with what chromosomal events are required for DSBs to form. In Drosophila females, the synaptonemal complex is required for most DSB formation. The repair of these breaks requires several DSB repair genes, some of which are meiosis-specific, and defects at this stage can have effects downstream on oocyte development. This has been suggested to result from a checkpoint-like signaling between the oocyte nucleus and gene products regulating oogenesis. Crossovers result from genetically controlled modifications to the DSB repair pathway. Finally, segregation of chromosomes joined by a chiasma requires a bipolar spindle. At least two kinesin motor proteins are required for the assembly of this bipolar spindle, and while the meiotic spindle lacks traditional centrosomes, some centrosome components are found at the spindle poles.

subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster.

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.

Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing-over in Drosophila.

During Drosophila oogenesis, the oocyte is formed within a 16-cell cyst immediately after four incomplete cell divisions. One of the primary events in oocyte development is meiotic recombination. Here, we report the intracellular localization of the MEI-218 protein that is specifically required for meiotic crossing-over. To understand the role of mei-218 in meiosis and to study the regulation of genes required for meiotic recombination, we characterized the expression pattern of its RNA and protein. Furthermore, we cloned and sequenced mei-218 from two other Drosophila species. The mei-218 RNA and protein have a similar expression pattern, appearing first in early meiotic prophase and then rapidly disappearing as prophase is completed. This pattern corresponds to a specific appearance of the mei-218 gene product in the region of the ovary where meiotic prophase occurs. Although mei-218 is required for 95% of all crossovers, the protein is found exclusively in the cytoplasm. Based on these results, we suggest that mei-218 does not have a direct role in recombination but rather regulates other factors required for the production of crossovers. We propose that mei-218 is a molecular link between oocyte differentiation and meiosis.