Meiosis is the fundamental process by which eukaryotic organisms produce gametes. The hallmarks of meiosis are homologous chromosome pairing, recombination and segregation. Defects in these processes lead to aneuploidy, which in humans results in birth defects and spontaneous abortions. A high correlation has been demonstrated between defects in meiosis and defects in the mitotic repair of ionizing radiation. The experiments proposed here will exploit this connection to study the genetic control of meiosis in Coprinus cinereus, a basidiomycete (mushroom) whose life cycle makes it an ideal model system for this work. The important features of meiosis in C. cinereus are that it is long and naturally highly synchronous. Each mushroom cap contain 107-108 meiotic cells; therefore sufficient material for biochemical analysis can be obtained at any stage of meiosis. Meiotic chromosomes can be studied using light and electron microscopy. In addition, both classical and molecular genetic techniques, including DNA-mediated cell transformation, are straightforward and well developed for this system. In previous work from this laboratory, radiation-sensitive mutants of C. cinereus were identified that have severe meiotic defects. These mutants, rad 3 and rad 9, will be analyzed further in the following experiments: 1. The effects of their mutations on DNA-mediated cell transformation will be determined. 2. The genes will be cloned and sequenced. The cloned genes will be used to study the expression of these genes during meiosis. It will also be determined whether the radiation sensitivity and meiotic dysfunction exhibited by rad 3 and rad 9 are pleiotropic effects of single gene mutations. In addition, null mutations of these genes will be created and analyzed. 3. The meiotic defects in these mutants will be further characterized by light microscopy. 4. Pseudorevertants of these mutants will be isolated, to identify other genes whose products function in meiosis and repair, and to reveal molecular interactions among these gene products.
