Fertilization in Chlamydomonas reinhardtii, a novel model system for determining the protein(s) required for gamete fusion, may help define the basic requirements for sperm- egg fusion in more complex systems. Our long term goal is to understand the mechanism of fusion used by gametes. Mammalian fertilization requires so many interactions prior to fusion, that it is difficult to determine which genes/proteins are actually required for fusion of the sperm and egg membranes. While only a few proteins involved in gamete fusion have been identified, we have produced conditional and non-conditional (insertional) mutants of Chlamydomonas that carry out the early recognition, signaling and adhesion steps in mating but are blocked in the final stage of fertilization. In addition, this is the only available system in which gamete adhesion and fusion can be assayed independently and where mutations can therefore be defined as affecting plasma membrane adhesion or plasma membrane fusion. Using our insertional mutants, TAIL-PCR (Thermal asymmetric interlaced-PCR) has allowed us to identify 2 regions of the Chlamydomonas genome that may contain the gene that is defective in our insertional fusion-defective mutants. Transforming our fusion-defective mutants with bacterial artificial chromosomes (BACs) containing the wild type sequences from these identified regions of the genome, will allow us to determine if a gene in one of these regions can rescue fusion activity. Once we have identified the gene that is defective in our mutants, we will use our conditional mutants to map the functional domains of the identified protein. Fluorescence and electron microscopy as well as Northern blotting and RT-PCR will be used to study the regulation, production and localization of this protein and bioinformatics and molecular modeling will help us understand this protein's function. Future experiments may also involve site directed mutagenesis of the BAC sequences to allow further mapping of the protein's functional domains as well as real time RT-PCR, to learn more about the regulation of gene expression. Our hypothesis, supported by our genetic data, is that there is only a single gene controlling gamete fusion in Chlamydomonas. The power of Chlamydomonas genetics and molecular biology, the simplicity of the process of fertilization and the striking similarity of this system to fertilization in multicellular organisms makes it an important model system for studying the mechanism of gamete fusion. Understanding the molecular basis of gamete fusion in a simple model system may lead to new approaches for the treatment of human infertility (some types of infertility in mammals may be caused by the failure of sperm-egg fusion) as well as new types of contraceptives (molecules that impair fusion can exhibit contraceptive activity). ? ? ?