NIM--A GENE AND MITOTIC REGULATION

最近，在理解 在分子水平上调节真核细胞周期。 一 一致性的发展表明，单个蛋白质的激活 激酶p34（cdc 2）足以启动所有细胞的有丝分裂或减数分裂。 真核生物 然而，在构巢曲霉中， p34（cdc 2）蛋白激酶和NIMA蛋白激酶（ nimA基因）以启动有丝分裂（Osmani等，1991年a; 1991年B）。 为了充分了解有丝分裂的调控， 了解NIMA蛋白激酶的作用。 拟议的工作是 旨在进一步确定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。 NIMA在G2细胞中的表达主要是在转录后水平调控的。 控制 缺失分析确定了一个潜在的翻译 nima mRNA中NIMA开放阅读框5'的调控序列。 这 区域进行了详细分析，其调节机制， 测定 G2期细胞周期蛋白B的表达似乎是 NIMA活性表达增加。 细胞周期蛋白B nimA活性所需的突变将利用 细胞周期蛋白B。将分析NIMA的稳定性，因为作为细胞， 通过有丝分裂NIMA活动下降的进展，我们将涉及任何 有丝分裂时观察到的NIMA蛋白降解 在有丝分裂过程中破坏细胞周期蛋白的途径。 一些基因 从酵母菌到老鼠的各种物种中分离出来， 与nimA最相似的推定蛋白激酶。 这些将是 在A. nidulans。 我们有 从粗糙脉孢菌中分离出功能性nimA同系物。使用 设计可用的nimA样序列PCR引物， 从非洲爪蟾克隆nimA的功能同源物。 抗体和 寡核苷酸直接切除非洲爪蟾卵母细胞和提取物中的nimA 从它们衍生的将被用于将nimA与成熟促进相关联 因子（MPF）和另外两种新分离的非p34（cdc 2） 强积金活动。 最后，将采用基因筛选 鉴定并分离曲霉菌的其他基因功能 nimA细胞周期调控系统中所涉及的nidulans。

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.