NIM--A GENE AND MITOTIC REGULATION

NIM--基因和有丝分裂调节

• 批准号: 2181486
• 负责人: STEPHEN A OSMANI
• 金额: $ 23.24万
• 依托单位: GEISINGER CLINIC
• 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 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181486
• 关键词: Aspergillus; Xenopus oocyte; alleles; cell cycle; cyclins; enzyme activity; gene expression; genetic promoter element; genetic regulation; high performance liquid chromatography; immunoprecipitation; microinjections; molecular cloning; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protein kinase; tissue /cell culture; western blottings

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.

最近在理解这一点方面取得了巨大进展 在分子水平上调节真核细胞周期。 一个 共识已经形成，表明单个蛋白质的激活 激酶 p34(cdc2) 足以启动所有细胞有丝分裂或减数分裂 真核生物。 然而，在构巢曲霉中，有必要激活 p34(cdc2) 蛋白激酶和 NIMA 蛋白激酶（ nimA 基因）以启动有丝分裂（Osmani 等人，1991a；1991b）。 因此，要充分了解有丝分裂的调节，就必须 了解 NIMA 蛋白激酶的作用。 提议的工作是 旨在进一步明确 nimA 在有丝分裂调节中的作用。 这项工作将提供知识、克隆和抗体 用于了解与癌症相关的不受控制的细胞周期。 G2 期 NIMA 表达的调节很大程度上受转录后调控 控制。 删除分析定义了潜在的转化 nima mRNA 中的调控序列位于 NIMA 开放阅读框的 5' 端。 这 有待详细分析区域及其调控机制 决定。 G2 期间似乎需要细胞周期蛋白 B NIMA 活性表达增加。 细胞周期蛋白B的水平 nimA 活性所需的值将利用突变来确定 cyclin B. NIMA 的稳定性将被分析，因为作为细胞 有丝分裂的进展 NIMA 活性下降，我们将联系任何 有丝分裂时观察到的 NIMA 蛋白降解 有丝分裂期间破坏细胞周期蛋白的途径。 有几个基因 从酵母到小鼠等编码的物种中分离出来 与 nimA 最相似的推定蛋白激酶。 这些将是 测试了构巢曲霉中 nimA5 的功能互补。 我们有 从粗糙脉孢菌中分离出功能性 nimA 同源物。使用 可用的 nimA 样序列 PCR 引物将被设计并用于 从非洲爪蟾克隆 nimA 的功能同源物。 抗体，和 爪蟾卵母细胞和提取物中 nimA 的寡核苷酸直接消融 从它们导出的将用于将 nimA 与成熟促进联系起来 因子 (MPF) 以及另外两个新分离的非 p34(cdc2) 包含强积金活动。 最后，将采用基因筛选 鉴定并分离曲霉属的其他基因功能 nidulans 参与 nimA 细胞周期调节系统。