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.

最近已经取得了巨大的进步来理解 在分子水平上的真核细胞周期的调节。 一个 共识已经发展，表明单个蛋白的激活 激酶，p34（CDC2），足以在所有人中开始有丝分裂或减数分裂 真核生物。 但是，在Aspergillus nidulans中，有必要激活 p34（CDC2）蛋白激酶和NIMA蛋白激酶既有 Nima Gene）为了启动有丝分裂（Osmani等，1991a; 1991 b）。 为了完全了解有丝分裂的调节，因此必须 了解NIMA蛋白激酶的作用。 提出的工作是 旨在进一步定义NIMA在调节有丝分裂中的作用。 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中NIMA表达的调节主要在转录后下 控制。 删除分析定义了潜在的翻译 NIMA mRNA 5'的调节序列对NIMA开放阅读框架。 这 要详细分析区域及其调节机制 决定。 G2期间似乎需要Cyclin B的要求 NIMA活性的表达增加。 细胞周期蛋白B的水平 使用NIMA活动所必需的 Cyclin B.将分析NIMA的稳定性，因为作为细胞 通过有丝分裂的NIMA活性下降，我们将联系任何 在有丝分裂时观察到的NIMA蛋白的降解为降解 在有丝分裂过程中破坏细胞周期蛋白的途径。 几个基因有 从酵母到编码的小鼠的物种中分离出来 推定的蛋白激酶与NIMA最相似。 这些将是 测试了nima5在nidulans中的功能互补。 我们有 从Neurospora crassa分离出功能性的NIMA同源物。使用 可用的NIMA（例如序列PCR引物）将被设计和用于 Xenopus的NIMA的克隆功能同源物。 抗体和 NIMA在异爪蟾卵母细胞和提取物中的寡核苷酸直接消融 源自它们将用于将NIMA与成熟促进联系起来 因子（MPF），以及其他两个新隔离的非P34（CDC2） 包含MPF活动。 最后，要使用遗传筛选 识别曲霉的其他基因功能，然后隔离 涉及NIMA细胞周期调节系统的尼古拉人。