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.

最近在理解这一问题上取得了很大进展 在分子水平上调控真核细胞周期。一个 已有共识表明，单一蛋白质的激活 激酶，p34(Cdc2)，足以启动有丝分裂或减数分裂。 真核生物。然而，在尼杜拉曲霉中，有必要激活 P34(Cdc2)蛋白激酶和NIMA蛋白激酶(产物 NIMA基因)以启动有丝分裂(Osmani等人，1991a；1991b)。 为了充分了解有丝分裂的调控，因此有必要 了解NIMA蛋白激酶的作用。建议的工作是 目的是进一步明确NIMA在有丝分裂调控中的作用。 这项工作将提供知识，克隆和抗体，将是 用于理解与癌症相关的不受控制的细胞周期。 G2细胞中NIMA表达的调控主要是在转录后水平 控制力。缺失分析定义了一个潜在的翻译 NIMA mRNA5‘端的调控序列与NIMA开放阅读框有关。这 详细分析了区域及其调控机制 下定决心。在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 是NIMA活动所必需的，将通过突变来确定 NIMA的稳定性将被分析，因为作为细胞 有丝分裂NIMA活性下降的进展，我们将联系任何 有丝分裂时观察到的NIMA蛋白降解为降解 在有丝分裂期间破坏细胞周期蛋白的途径。有几个基因有 从酵母到老鼠等编码基因的物种中分离出来 与NIMA最相似的推定的蛋白激酶。这些将是 测试nimA5在根瘤拟青霉中的功能互补。我们有 从粗糙脉孢菌中分离到一个具有功能的NIMA同系物。使用 设计和使用可用的NIMA类序列聚合酶链式反应引物 从非洲爪哇克隆NIMA的功能同源物。抗体，以及 寡核苷酸直接去除非洲爪哇卵母细胞及其提取物中的NIMA 从它们衍生出来的将被用来将NIMA与成熟促进联系起来 因子(Mpf)和另外两个新分离的非p34基因(Cdc2) 包含强积金活动。最后，基因筛查将被采用 鉴定并分离曲霉的其他基因功能 参与NIMA细胞周期调控系统的尼杜兰。