9305473 Mandoli Dr.Mandoli's long term goal is to understand how developmental age and/or size of an organism and environmental signals co-regulate initiation of reproductive development. A genetic dissection of the initiation of reproduction in Acetabularia acetabulum will provide unique insights into development at the level of the single cell. Initiation of reproduction in A. acetabulum, requires de novo protein synthesis, and is regulated at the translational level by a) developmental age/size of the cell, b) blue light and c) a putative cytosolic, nuclear encoded inhibitor. There are three specific aims in this proposal: 1) To breed an isogeneic cell line. Isogeneic cells will provide a homogeneous background for mutants. The cell line also will be screened for increased fecundity and DNA content using four parameters which correlate with these phenotypes; 2) To screen for two types of developmental mutants; those which are developmentally arrested at specific points during vegetative growth (da mutants) and those which initiate reproduction or 'cap' prematurely (early mutants) which may be defective in, or null for, an inhibitor. Mitotically active haploid nuclei will be mutagenized with EMS prior to gametogenesis at a dose known to induce 50% kill of the subsequent generation. Treatment of mitotically active haploid nuclei will propagate populations of mutant gametes inside gametangia. Selfs and backcrosses of gametes will recover recessives; 3) To characterize these mutants using nuclear and cytoplasmic complementation in trans (cell grafting and nuclear transplantation) and classical genetic techniques. Dr. Mandoli can rescue da mutants in three ways. First, implanting a wild type nucleus will 'piggy-back' the mutant nucleus through reproduction. Second, grafting a wild type cytoplasm may complement the mutant in trans. Third, repetition of development post amputation will determine if regrowth and normal development are the same. Classical genetics of early and rescued da mutants will determine dominance, numbers of alleles and complementation groups. ***
