ROLE OF THE NIM-A GENE IN MITOTIC REGULATION

利用细胞周期特异性蛋白激酶(NIMA)的最新研究 已经导致了一种模型的制定 描述该物种有丝分裂的调节(Osmani et at.， 1988b)。该模型提出，细胞周期特异性表达 NIMA驱动有丝分裂周期。这项提议的目的是测试 以及扩展该模型以及在多大程度上建立 该模型也适用于其他系统。 我们将完成NIMA基因的分子表征 通过对尼玛野生型基因组座位的测序，获得了 该基因突变等位基因的序列。这些数据将是 为随后的基因操作和鉴定所需的 NIMA蛋白中与温度有关的重要残基 敏感度。我们打算进行体外诱变以修改 NIMA基因的催化蛋白激酶位点及其检测 通过将突变的基因重新引入到 曲霉菌，以便功能分析可以在 活着。这种方法将确定蛋白质的重要性 NIMA基因的激酶潜能。作为特定于细胞周期的 NIMA的表达被认为是驱动有丝分裂周期的因素 将决定细胞周期特异性表达在什么水平 尼玛已经被控制住了。这些实验还将确定 细胞周期特异性表达NIMA和NIMA的功能重要性 确定负性翻译控制是否由5‘ 在NIMA信使核糖核酸中发现了小的开放阅读框架。 针对NIMA蛋白的抗体将被诱导并亲和 纯化并用于揭示尼玛的保存程度 其他真核生物中的蛋白质。这些抗体还将被用于 建立蛋白质的亚细胞定位，以研究 NIMA蛋白丰度和蛋白的细胞周期特异性变异 激酶的催化能力和免疫沉淀蛋白质 与NIMA蛋白有关。我们打算提纯NIMA蛋白 来自过表达NIMA蛋白的曲霉菌株。这个 纯化的NIMA蛋白和NIMA特异性抗血清用于 建立NIMA与成熟促进的关系 在非洲爪哇卵母细胞中微量注射因子(MPF)。

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.