MITOSIS IN NORMAL AND NEOPLASTIC CELLS

正常细胞和肿瘤细胞的有丝分裂

• 批准号: 2633781
• 负责人: BILL R BRINKLEY
• 金额: $ 28.65万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2633781
• 关键词: DNA binding protein; aneuploidy; antinuclear autoantibody; antisense nucleic acid; cell cycle; centromere; chromosome movement; genetic manipulation; human genetic material tag; human tissue; laboratory mouse; laboratory rabbit; microinjections; neoplasm /cancer genetics; nucleic acid sequence; protein biosynthesis; protein structure function; yeast two hybrid system

DESCRIPTION: This is a first revision of a application for renewed support from a senior investigator, Dr. Bill Brinkley, to continue the investigation of kintechore assembly during mitosis in mammalian cells. Previous investigations have demonstrated that underlying mammalian centromeres are large blocks of repetitive DNA sequences, including one called satellite DNA. To dissect the complex DNA elements found in mammalian centromeres, the proposal will focus on the discovery in the previous granting period that centromeres can be fragmented by inducing cells with unreplicated genomes to enter mitosis (a process producing MUGS). With these, the present proposal will identify functional centromere sequences following chromosomal fragmentation by combining in situ hybridization and immunocytochemistry to follow specific DNA sequences and centromere proteins, particularly CENP-B and CENP-C. Three aims are now proposed. Using probes specific for alphoid DNA subfamilies known to localize within the centromere-kintechore complex of human chromosomes, the functional and molecular organization of these sequences will be followed using in situ hybridization. Second, to test the function of centromere protein B (CENP-B), the gene will be disrupted in embryonic stem cells to test whether elimination of this protein has a consequence in mice. Further, proteins that interact with CENP-B will be examined using the yeast two hybrid system and the properties of any proteins so identified will be further characterized. This will focus on using strategies to suppress protein function by overexpression of protein fragments, microinjection of antibodies, and suppression of protein expression using antisense oligonucleotides. Lastly, the properties of newly identified centromere protein-F (CENP-F) will be examined during kinetochore maturation as cells progress from interphase to mitosis, as well as using methods to interfere with CENP-F function to test its role in mitotic chromosome segregation.

描述：这是更新支持申请的第一次修订 高级调查员 Bill Brinkley 博士继续调查 哺乳动物细胞有丝分裂过程中动丝组装的过程。 以前的 研究表明，哺乳动物着丝粒的基本结构是 大块重复 DNA 序列，其中包括一个称为卫星的序列 脱氧核糖核酸。 为了剖析哺乳动物着丝粒中发现的复杂 DNA 元件， 该提案将重点关注上一个授权期间的发现 着丝粒可以通过诱导具有未复制的细胞而断裂 基因组进入有丝分裂（产生 MUGS 的过程）。 有了这些， 目前的提案将确定以下功能性着丝粒序列 结合原位杂交和染色体断裂 免疫细胞化学追踪特定 DNA 序列和着丝粒 蛋白质，特别是 CENP-B 和 CENP-C。 现在提出了三个目标。 使用已知定位于内的α类 DNA 亚家族特异的探针 人类染色体的着丝粒-动丝复合体，其功能和功能 这些序列的分子组织将使用原位跟踪 杂交。 二、检测着丝粒蛋白B的功能 (CENP-B)，该基因将在胚胎干细胞中被破坏，以测试是否 消除这种蛋白质会对小鼠产生影响。 此外，蛋白质 与 CENP-B 相互作用的物质将使用酵母两种杂交系统进行检查 并且如此鉴定的任何蛋白质的特性将被进一步 特点。 这将侧重于使用抑制蛋白质的策略 通过蛋白质片段的过度表达、显微注射 抗体，并使用反义抑制蛋白质表达 寡核苷酸。 最后，新鉴定的着丝粒的特性 蛋白质-F (CENP-F) 将在着丝粒成熟过程中作为细胞进行检查 从间期到有丝分裂的进展，以及使用干扰方法 具有CENP-F功能来测试其在有丝分裂染色体分离中的作用。