The Pan Gu Protein Kinase : Regulator of the Early Embryonic Cell Cycle in Drosophila

In Drosophila, fertilization is required for the embryo to undergo the first 13 rapid cell cycles, which consist of alternating DNA replication and mitosis without gap phases. In contrast, eggs from females mutant in the pan gu (png) locus no longer require fertilization to initiate DNA replication, and both fertilized embryos and unfertilized eggs from png mutant mothers have giant polyploid nuclei, indicating that S phase is not linked to mitosis and nuclear division in this mutant. png mutations fall into two classes: fertilized embryos from some alleles never undergo mitosis, while fertilized embryos from a second class of alleles are able to complete a few mitoses before the nuclei stop dividing and over-replicate. Four new mutations in the png gene were characterized, including new mutations of the multinuclear class, confirming that this phenotype results from loss-of-function of png and indicating that png functions during the first five divisions. To understand the function and mechanism by which the png gene product inhibits S phase, the gene was cloned. A screen for new alleles ofpng was completed, and a deficiency was isolated that delineated the gene to a 20 kb region. Smaller fragments that rescue png mutations were isolated, and the region was sequenced. All eight alleles of png have mutations in a single open reading frame, and a fragment containing only this transcription unit was capable of rescuing png mutants. The PNG protein shows strong homology to Ser/Thr protein kinases. The position and nature of the amino acid changes caused by the png mutations show that the severity of mutant phenotype correlates with the predicted degree of reduction of kinase activity. Phylogenetic analysis suggests that PNG represents a new family of protein kinases. Analysis of expression of png showed that the protein and mRNA are specifically expressed in mature oocytes and during early development, and ectopic expression of PNG later in development had no effect, indicating that PNG is a specific regulator of the early cell cycles. PNG protein co-immunoprecipitates with the PLUTONIUM protein, which also regulates the early embryonic cell cycles. Thesis Supervisor: Terry L. Orr-Weaver Title: Professor of Biology