CHROMOSOME SEGREGATION DURING FEMALE MEIOSIS

女性减数分裂期间的染色体分离

• 批准号: 6629127
• 负责人: PETER John DONOVAN
• 金额: $ 24.1万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-16 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629127
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; biological signal transduction; cell cycle proteins; chromosome movement; complementary DNA; confocal scanning microscopy; egg /ovum; enzyme activity; enzyme structure; female; gene expression; genetic library; green fluorescent proteins; immunoprecipitation; laboratory mouse; meiosis; mitogen activated protein kinase; mutant; oligonucleotides; phosphorylation; protein protein interaction; tissue /cell culture; western blottings; yeast two hybrid system

DESCRIPTION: (Adapted from the applicant's abstract) Formation of fertile oocytes requires orderly segregation of chromosomes during meiosis, which depends on proper assembly of a bipolar spindle and its actions. The PI has identified a novel kinase IAK1, one of very few components identified so far in the female meiotic spindle. The proposed study is to test the hypothesis that IAK1 is an important player of the meiotic chromosome segregation machinery. Specific Aim 1 is to localize the IAK1 protein and kinase activity in female germ cells throughout meiosis. In addition, IAK1 expression patterns and activity will be examined in mouse mutants defective in either checkpoint pathways or MPF/MAPK signal transduction pathways. Specific Aim 2 is to disrupt the function of IAK1 by injection of antisense oligonucleotides, kinase-negative mutant form of IAK1, and anti-IAK1 antibodies into oocytes. Spindle formation and phosphorylation as well as chromosome segregation will be assessed in the treated oocytes. Specific Aim 3 is to determine the functional domains of the IAK1 kinase by introducing wild-type and mutant forms of IAK1 into maturing oocytes. The mutated IAK1 proteins will be taqged with FLAG octapeptides or green fluorescent protein and localized in the injected oocytes by confocal microscopy. Simultaneously, the kinase activity will be examined in the oocyte extracts. Specific Aim 4 is to identify the IAK1-interacting proteins in meiotic germ cells. Immunoprecipitation and Western blotting will examine interaction of IAK1 with known IAK1-interacting proteins in other systems, such as Cdc37 and p100. Novel IAK1-interacting proteins will be identified in yeast two-hybrid screening system using a mouse oocyte/egg cDNA library.

描述：（改编自申请人的摘要）肥沃的形成 卵母细胞需要在减数分裂过程中有序分离染色体，这 取决于正确组装双极主轴及其作用。 Pi具有 鉴定出一种新型的激酶IAK1，这是迄今为止识别出的很少的成分之一 女性减数分裂纺锤体。拟议的研究是检验以下假设 IAK1是减数分裂染色体隔离机制的重要参与者。 具体目的1是在女性中定位IAK1蛋白和激酶活性 整个减数分裂的生殖细胞。另外，IAK1表达模式和 将在任何一个检查点中有缺陷的小鼠突变体中检查活动 途径或MPF/MAPK信号转导途径。 特定目标2是通过注射反义破坏IAK1的功能 寡核苷酸，激酶阴性突变体的IAK1和抗IAK1抗体 进入卵母细胞。主轴形成和磷酸化以及染色体 隔离将在处理的卵母细胞中进行评估。 特定目的3是通过确定IAK1激酶的功能域 将IAK1的野生型和突变形式引入成熟的卵母细胞。这 突变的IAK1蛋白将用旗八肽或绿色将其taq 荧光蛋白，并通过共焦点局部在注入的卵母细胞中 显微镜。同时，将在卵母细胞中检查激酶活性 提取物。 特定目的4是确定减数分裂细菌中的IAK1相互作用蛋白 细胞。免疫沉淀和蛋白质印迹将检查 IAK1在其他系统中具有已知的IAK1相互作用蛋白，例如Cdc37和 P100。新型的IAK1相互作用蛋白将在酵母两杂交中鉴定 使用小鼠卵母细胞/蛋cDNA库进行筛选系统。