Recent work utilizing the cell cycle specific protein kinase (nimA) of Aspergillus nidulans has lead to the formulation of a model to describe the regulation of mitosis in this species (Osmani et at., 1988b). The model proposes that cell cycle specific expression of nimA drives the mitotic cycle. This proposal is aimed at testing and expanding this model and at establishing to what extent the model is applicable to other systems. We will complete the molecular characterization of the nimA gene by sequencing the wild type genomic locus of nimA and obtain the sequence of the mutant alleles of this gene. These data will be required for subsequent manipulations of the gene and identify important residues in the nimA protein responsible for temperature sensitivity. We intend to carry out in vitro mutagenesis to modify the catalytic protein kinase site of the nimA gene and test its functional significance by introducing the mutated gene back into Aspergillus such that a functional analysis can be carried out in vivo. This approach will establish the importance of the protein kinase potential of the nimA gene. As cell cycle specific expression of nimA has been proposed to drive the mitotic cycle we will determine at what level the cell cycle specific expression of nimA is under control. These experiments will also ascertain the functional importance of cell cycle specific expression of nimA and determine if negative translational control is mediated by the 5' small open reading frames found in the nimA mRNA. Antibodies to the nimA protein are to be elicited and affinity purified and used to reveal the degree of conservation of the nimA protein in other eukaryotes. The antibodies will also be used to establish the sub-cellular location of the protein, to study the cell cycle specific variation in nimA protein abundance and protein kinase catalytic capacity, and to immunoprecipitate proteins associated with nimA protein. We intend to purify nimA protein from strains of Aspergillus that overexpress the nimA protein. The purified nimA protein and nimA specific antisera are to be used to establish the relationship between nimA and maturation promotion factor (MPF) by micro injection experiments in Xenopus oocytes.
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