Chromosome alignment and crossing over during meiosis is a complex and highly regulated process. This renewal application describes methods and experimental strategies that continue studies to identify the molecules that regulate and execute these functions in C. elegans. established to isolate genes required for chromosome synapsis and recombination. These experiments will screen collections of germline-expressed genes identified by microarray methods for RNAi knockout phenotypes consistent with roles in chromosome pairing and crossing over. True genetic mutations will be isolated for genes that score positive by RNAi and corresponding proteins will be localized using either antibodies or GFP-fusion transgenes. Experiments in SA2 and SA3 will examine cell biological and structural features of meiotic chromosomes during pairing/synapsis. Dr. Villeneuve's laboratory is skilled in the use these methods, having developed or improved many of them during the past five years. Chromosomal movement, nuclear reorganization, assembling synaptonemal complexes are among the processes that will be characterized in the course of these experiments. The features of these processes in wildtype animals will be examined for perturbations induced by mutants isolated in the course of other parts of this project. Previous work from Dr. Villeneuve's efforts has already been identified hal-1, sys-1 and sys-2 as candidates for pairing complex proteins. The synaptic and recombination behavior of chromosomes will be characterized in these mutants in order to determine more precisely what roles these proteins play during meiosis. These studies will also include efforts to clone hal-1, sys-1 and sys-2 using standard C. elegans techniques. Subsequent studies will include protein immunolocalization and biochemical immunoprecipitation experiments to characterize associated complexes. In SA4, Dr. Villeneuve will test the relationship between chromosome pairing and recombination. While chromosomes must align and become proximate to one another to crossover, it is not clear if such synapsis actively initiates recombination. To assay recombination initiation, Dr. Villeneuve plans to exploit the phenomenon of germ cell apoptosis that occurs as a consequence of unrepaired DSBs that remain from abortive recombination. If pairing mutants that affect synapsis maintenance permit recombination initiation, mutant germ cells will exhibit high apoptotic profiles. Subsequently, such apoptosis can be suppressed in spo-11 mutant backgrounds if recombination-dependent DSBs are the cause.