9630618 Mandoli Dr. Mandoli's long-term goal is to understand how developmental age and/or size of an organism and blue light 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 systhesis, 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 3 specific aims in this proposal. AIM 1: Continue inbreeding morphologically distinct, near isogenic cell lines. She has inbred cell lines which are morphologically distinct and are more robust than their progenitors. She will determine which cell lines have the fastest growth rate, the highest numbers of progeny, and differ from each other in the greatest number of gross morphological phenotypes. She will continue inbreeding only those cell lines. AIM 2: Use cell grafting to study her developmentally arrested (da) cell lines. Developmentally arreseted cells grow normally at first but later arrest in vegatative growth. Based on morphology and behavior of cells in grafts and upon amputation, her 11 da cell lines comprise 4 classes. She has compensated for da defects in 3 ways: a wildtype nucleus can 'piggy-back' some mutants through reproduction in a birhizoidal heterokaryon; a wildtype apex, which always lacks a nucleus, permits some mutants to reproduce in a mutant::wildtype graft; some phenotypes reproduce if the defective apex is simply removed. Here, she will establish complementation groups and dominance using classical genetics for da cells that act like single gene traits and establish cell biological relationships between the da cell lines using mutant::mutant grafts between apices and rhizoids. AIM 3: Make transformants made via microparticle bombardment. In this funding period, she will optimize transformation frequency, examine the nature of the inserts, and determine inheritance, segregation, and stability of intruduced traits.
