MOLECULAR ANALYSIS OF THE CORTICAL GRANULE ENVELOPE

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DESCRIPTION (Adapted from the Applicant's Description): The cortical granule envelope (CGE) is a major post-fertilization coat that forms in the perivitelline space, where it has been hypothesized to block polyspermy and function in preimplantation development of eutherian mammals, including the human. The investigator's long term goal is to characterize the composition and function of the CGE. The CGE contains at least one protein, p75, which is the focus of this study. The major goals of this application are to isolate and sequence gene for p75 and to produce the molecular tools needed for subsequent functional studies of p75. The first Specific Aim is to determine the partial amino acid sequences of the NH2 terminus and several internal peptides for p75. These sequence data will be necessary to confirm that the correct gene has been isolated in Specific Aim #2. The second Specific Aim is to isolate and sequence the gene (cDNA and genomic DNA) for p75. A mouse ovarian cDNA expression library will be screened with an antibody to p75 to identify clones containing the p75 insert. The p75 insert will be sequenced and compared at the DNA and protein levels to data in Genbank and to the internal and NH2 terminus data obtained in Aim #1. Mouse p75 cDNA will then be used to screen the Stratagene 129/Sv mouse genomic library, and the entire gene sequence will be determined. The step is necessary for later construction of a targeting vector to produce knockout mice. The third Specific Aim is to make additional antibodies to recombinant p75. The gene for p75 will be expressed in E. Coli to obtain enough protein for antibody production in rabbits. This will yield a sufficient quantity of antibodies to use in subsequent functional experiments. Data obtained in this project will provide important basic information of p75 and the CGE, will help us understand the overall process of mammalian fertilization, and may lead to development of better ways to control reproduction in mammals, including humans. These Aims will also provide the tools needed to subsequently determine the function of p75 and the CGE using gene targeting and antibody inhibition experiments.