DESCRIPTION: This proposal focuses on the isolation of sex determination genes in the parasitic wasp Nasonia vitripennis. In Nasonia sex determination is by a haplo-diploidy mechanism. In these wasps, the males are haploid usually develop from unfertilized eggs and the females are diploid and develop from fertilized eggs. Some of the interest in Nasonia arises from the unusual ability of Nasonia females to control the sex ratio of their offspring by controlling fertilization of her eggs. It seems unlikely that Nasonia utilizes a simple heterozygosity/homozygosity principle for sex determination since long-term inbreeding studies have failed to produce biparental diploid males. The development of fertile diploid male progeny from unmated triploid females suggests that either fertilization itself or the presence of the male paternal genome is important in sex determination. The genetic mechanisms underlying sex determination are well understood for at least two organisms, Drosophila melanogaster and C. elegans. However, these two system share little direct homology except for the double-sex gene. In Drosophila the double sex gene produces two distinct transcripts, a male-specific transcript and a female-specific transcript which are the products of differential splicing of the same double sex primary transcript. This proposal asserts that existing mutagenesis systems in Nasonia are inadequate and so the first goal of the project is to try to optimize mutagenesis in Nasonia using either ENU (N-ethyl-N-nitrosourea) or DEB (Diepoxybutane). The goal of this phase of the project would be to identify a chemical mutagen that induces point mutations at an average frequency of 1 in 1000 to 1 in 3000 hits/genome. After optimizing the conditions for mutagenesis, males will be mutagenized and mated and the progeny of their potentially heterozygous mutant and unmated daughters will be scanned for sex ratio anomalies including high numbers of female progeny and/or intersexes. These females will then be mated to their sons in order to screen for female sex ratio anomalies. By appropriately marking the individuals in the original P generation, it should be possible to identify diploid males. The author estimates that screening 6000 genomes should yield at least one mutation in every locus, including all the sex determination loci. In addition to the general screen for mutations in sex determination, the author will be an effort to identify a Nasonia homologue of the Drosophila double-sex gene. The first approach to this problem will use PCR amplification of genomic DNA using degenerate primers generated from the sequence of the Drosophila doublesex gene. If a PCR amplification product is isolated it will be used to hybridize to Northern blots to determine if its expression is sex specific and to identify doublesex clones in a Nasonia cDNA library.
NIH ExPorter
NIH ExPorter