Dr. Villeneuve is investigating the mechanisms underlying normal chromosome pairing, cross over, and segregation in C. elegans. She has identified at least 10 new loci defined by mutations causing meiotic chromosome non-disjunction. These mutations were isolated in screens for hermaphrodites that produced an abnormally high frequency of males as a result of non-disjunction of the sex chromosomes, and are denoted him mutations. Mutations in two loci only affect the segregation of the X chromosomes, whereas the others affect the segregation of all chromosomes. Dr. Villeneuve proposes that the non-disjunction in the him mutants could be due to a defect in pairing, recombination, chiasma maintenance, or the disjunctional process. The presence of chiasmata can be assessed cytologically, since C. elegans oocytes normally arrest at a late stage of meiotic prophase, with six pairs of homologues (bivalents) attached by chiasmata. Some of the him mutants exhibit up to 12 univalents, indicating defective or absent chiasmata. The majority of him mutants characterized result in a failure to form chiasmata. All seven him loci analyzed exhibit a reduction in global recombination frequencies. One of these genes, me14 is specifically defective in spermatocyte disjunction. Another mutation, me8, cis-acting defines a region on the X chromosome required for pairing; deletion of the locus results in a marked decrease in crossing over along the entire length of the X-chromosome. Dr. Villeneuve proposes to elucidate the function of the him genes and in particular will focus on him-14 and me9. The first specific aim will be to identify him mutants that exhibit pairing defects. Germ cell nuclei will be examined during early to mid meiotic prophase to detect anomalies in pairing. To detect pairing defects in the synaptonemal complex, EM specimens will be prepared and analyzed. In addition, fluorescence in situ hybridization (FISH) will be used to determine if tagged regions of homologous chromosomes are properly paired. Also, FISH will be used to determine the time point during prophase when pairing is first observed in normal germ cell nuclei. The second specific aim will be to follow-up on a preliminary finding that the me9 mutant exhibits reduced crossover interference. STS markers distributed over the X chromosome will be used to assess recombination between two strains differing for each of six markers to provide a more complete assessment of the reduced crossover interference. More alleles of me9 will be isolated to assist in the cloning of the me9 gene. Dr. Villeneuve proposes to clone the me9 and him-14 genes. The method of positional cloning and transformation rescue will be used to clone and confirm the identity of these genes. Once the genes are cloned, antibodies will be prepared against bacterially-expressed fusion proteins and the antisera will be used for immunolocalization of the him- 14 and me9 proteins. In addition HA-tagged him-14 and me9 genes will be constructed and transformed into worms to provide an additional method for immunolocalization. Different positions of the tag will be used to obtain tagged genes that are capable of complementation. The localization of these proteins will be studied in a variety of him mutants. The final specific aim is to identify other components that functionally interact with him-14. Him-14 mutants produce broods consisting mainly of inviable aneuploid embryos with only a few euploid survivors. Selection for EMS induced suppressors of him-14 will be performed. These suppressor mutants will be analyzed for defects in meiosis.
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