ROLE OF THE NIM-A GENE IN MITOTIC REGULATION

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Great progress has recently been made towards an understanding of the regulation of the eukaryotic cell cycle at the molecular level. A consensus has evolved indicating that activation of a single protein kinase, p34(cdc2), is Sufficient to initiation mitosis or meiosis in all eukaryotes. However, in Aspergillus nidulans it is necessary to activate both the p34(cdc2) protein kinase and the NIMA protein kinase (product of nimA gene) in order to initiate mitosis (Osmani et al., 1991a; 1991 b). To fully understand the regulation of mitosis it is therefore imperative to understand the role of the NIMA protein kinase. The work proposed is aimed at further defining the role of nimA in the regulation of mitosis. The work will provide knowledge, clones, and antibodies that will be of use in understanding the uncontrolled cell cycle associated with cancer. Regulation of NIMA expression in G2 is largely under post-transcriptional control. Deletion analysis has defined a potential translational regulatory sequence in nima mRNA 5' to the NIMA open reading frame. This region is to be analyzed in detail and the mechanism of its regulation determined. There appears to be a requirement for cyclin B during G2 for the increased expression of NIMA activity. The level at which cyclin B is required for nimA activity will be determined utilizing mutation of cyclin B. The stability of NIMA will be analyzed because as cells progress through mitosis NIMA activity drops and we will relate any degradation of NIMA protein observed at mitosis to the degradation pathway that destroys cyclin protein during mitosis. Several genes have been isolated from species ranging from yeast to mice that encode putative protein kinases with most similarity to nimA. These will be tested for functional complementation of nimA5 in A. nidulans. We have isolated a functional nimA homolog from Neurospora crassa. Using the available nimA like sequences PCR primers are to be designed and used to clone functional homologs of nimA from Xenopus. Antibodies, and oligonucleotide direct ablation of nimA in Xenopus oocytes and extracts derived from them will be used to relate nimA to maturation promotion factor (MPF) and also to two other newly isolated non-p34(cdc2) containing MPF activities. Finally, genetic screens are to be employed to identify, and then isolate, other gene functions of Aspergillus nidulans that are involved in the nimA cell cycle regulatory system.