An mCherry-tagged Gemini Bac transgene provides a biosensor throughout D. melanogaster development and a tool for studying Geminin function. Robert C. Eisman, Samantha Young, Melissa A.S. Phelps, Amelia D. Tomlinson, Stacy L. Holtzman, Brian R. Calvi, Thomas C. Kaufman. Dept Biol, Jordan Hall A505, Indiana Univ, Bloomington, IN.

   Geminin is a conserved metazoan protein that prevents multiple rounds of DNA replication in a single cell cycle by binding Cdt1 and preventing the assembly of the DNA replication initiation complex. Additionally, normal Geminin function requires a cyclical increase in protein levels during the S and G2 phases of the cell cycle and subsequent degradation of the protein pool. In this study we have made two transgenic fly lines with gem Bac clones using the P[acman] system that express either an unmodified GEM or a recombineered GEM::mCherry fusion protein. Both transgenic fly lines survive when homozygous for the Bac in a WT genetic background and rescue gem mutant phenotypes, but four copies of gem reduce female fertility and perturb normal cleavage divisions in syncytial embryos. When GEM levels are high mitosis proceeds, but we find chromosome congression and alignment is aberrant, chromatin bridges are common at Anaphase, normal histone modifications are altered, and many nuclei fall out prior to cellularization. In addition to providing a biosensor throughout D. melanogaster development, these new D. melanogaster transgenic lines, in conjunction with publicly available gem mutant stocks, provide a cell cycle marker throughout development and a new tool for future investigations of GEM function and DNA replication in live animals and fixed tissues.