ROLE OF THE NIM-A GENE IN MITOTIC REGULATION

NIM-A 基因在有丝分裂调节中的作用

• 批准号: 3301218
• 负责人: STEPHEN A OSMANI
• 金额: $ 15.32万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301218
• 关键词: Aspergillus; Xenopus; alleles; enzyme mechanism; eukaryote; gene expression; genetic manipulation; genetic promoter element; genetic regulation; genome; immunoprecipitation; microinjections; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid sequence; protein kinase; tissue /cell culture

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.

使用细胞周期特异性蛋白激酶（NIMA）的最新工作 曲霉的nidulans已导致模型的制定 描述该物种中有丝分裂的调节（Osmani et。 1988b）。 该模型提出了细胞周期的特定表达 NIMA驱动有丝分裂周期。 该建议旨在测试 并扩展该模型并确定在多大程度上 模型适用于其他系统。 我们将完成NIMA基因的分子表征 通过测序NIMA的野生型基因组基因座并获得 该基因突变等位基因的序列。 这些数据将是 随后对基因操纵的要求并确定 NIMA蛋白中的重要残留物负责温度 灵敏度。 我们打算进行体外诱变以修改 NIMA基因的催化蛋白激酶部位并测试 通过将突变基因引入重新引入功能意义 曲霉菌，使功能分析可以在 体内。 这种方法将确定蛋白质的重要​​性 NIMA基因的激酶潜力。 作为细胞周期特异性 已经提出了NIMA的表达来驱动有丝分裂周期 将在细胞周期的特定表达中确定 NIMA受到控制。 这些实验还将确定 细胞周期特异性表达NIMA和 确定阴性翻译控制是否由5'介导 在NIMA mRNA中发现的小开放式阅读框。 NIMA蛋白的抗体应引起并亲和力 纯化并用于揭示NIMA的保护程度 其他真核生物中的蛋白质。 抗体也将用于 建立蛋白质的亚细胞位置，以研究 NIMA蛋白丰度和蛋白质的细胞周期特异性变化 激酶催化能力和免疫沉淀蛋白 与NIMA蛋白有关。 我们打算纯化NIMA蛋白 从过表达NIMA蛋白的曲霉菌株中。 这 纯化的NIMA蛋白和NIMA特异性抗血清应用于 建立NIMA与成熟促进之间的关系 因素（MPF）通过爪蟾卵母细胞中的微注射实验。