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.

利用细胞周期特异性蛋白激酶 (nimA) 的最新研究 构巢曲霉的研究导致了模型的制定 描述该物种有丝分裂的调节（Osmani 等人， 1988b）。 该模型提出细胞周期特异性表达 nimA 驱动有丝分裂周期。 该提案旨在测试 并扩展这个模型并建立到什么程度 模型适用于其他系统。 我们将完成nimA基因的分子表征 通过对 nimA 的野生型基因组位点进行测序并获得 该基因的突变等位基因的序列。 这些数据将 后续基因操作和鉴定所需的 nimA 蛋白中负责温度的重要残基 敏感性。 我们打算进行体外诱变来修饰 nimA 基因的催化蛋白激酶位点并测试其 通过将突变基因引入回体内来发挥功能意义 曲霉这样的功能分析可以在 体内。 这种方法将确定蛋白质的重要​​性 nimA 基因的激酶潜力。 由于细胞周期特异性 nimA 的表达被认为可以驱动有丝分裂周期 将确定细胞周期特异性表达的水平 nimA受到控制。 这些实验还将确定 nimA 和细胞周期特异性表达的功能重要性 确定负翻译控制是否由 5' 介导 nimA mRNA 中发现的小开放阅读框。 nimA 蛋白的抗体将被引出并进行亲和力 纯化并用于揭示 nimA 的保守程度 其他真核生物中的蛋白质。 这些抗体也将用于 确定蛋白质的亚细胞位置，以研究 nimA 蛋白丰度和蛋白的细胞周期特异性变化 激酶催化能力和免疫沉淀蛋白质 与 nimA 蛋白相关。 我们打算纯化nimA蛋白 来自过度表达 nimA 蛋白的曲霉菌株。 这 纯化的 nimA 蛋白和 nimA 特异性抗血清用于 建立 nimA 与成熟促进之间的关系 通过非洲爪蟾卵母细胞微量注射实验得到的因子（MPF）。