TELOMERASES AND TELOMERASE MOLECULAR BIOLOGY

端粒酶和端粒酶分子生物学

• 批准号: 6395754
• 负责人: BRAD E. WINDLE
• 金额: $ 0.71万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6395754
• 关键词: HeLa cells; RNA; cell line; cooperative study; drug discovery /isolation; enzyme inhibitors; enzyme reconstitution; gene expression; genetic mapping; hybrid cells; molecular cloning; nucleic acid repetitive sequence; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein structure function; site directed mutagenesis; telomerase; telomere

Telomeres have a G-rich repeated DNA sequence that maintains chromosome end stability. Telomerase is a reverse transcriptase-like ribonucleoprotein that maintains the telomere sequence. Telomerase is expressed in the majority of tumor cells but not in normal cells. Telomerase and telomeres are ideal tumor-specific targets for anti-cancer therapeutic strategies. Our preliminary studies show the discovery of the first telomerase inhibitors. Our studies also show that telomerase inhibitors induce telomere shortening, chromosome end fusion, and eventually cell death. Cells have shown the ability to adapt to telomerase inhibition. The goals of this proposal are to l) elucidate the molecular structure of telomerase and provide a more defined target for our anti-cancer strategies, and 2) determine the mechanism(s) of cellular adaptation to telomerase inhibitors. These goals will be accomplished by mapping and cloning the genes for the telomerase proteins and RNA template. Through our collaboration with Program l, purified telomerase will be used as a source of purified telomerase RNA for cloning. Somatic cell genetics will be used to map the telomerase activity. The new technique of Differential Display will be used to detect mRNAs expressed in posttelomerase-activated cells that are not expressed in pre-telomerase activated cells. The mapping will be coordinated with the isolation of candidate cDNA clones for the proteins and RNA to accelerate the identification of legitimate clones. The cDNA clones of the RNA will be screened for appropriate template sequence, secondary structure, and ability to direct in vitro and in vivo telomerase activity. The cDNAs for telomerase proteins will be expressed for production of antibodies. The antibodies and DNA clones will serve as probes for use in studies of telomerase expression in Program 3 and in tumor specimens from Program 4. The data from these studies will be used by Program 2 for structure modeling and specific drug design. Potential telomerase inhibitors, such as anti RNA oligonucleotides will be studied by Program l. The mechanism of adaptation to telomerase inhibitors will also be studied. Hypotheses concerning drug metabolism, telomerase up-regulation and telomerase specificity will be tested.

端粒具有富含G的重复DNA序列，可维持染色体 结束稳定性。端粒酶是一种逆转录酶 维持端粒序列的核糖核蛋白。端粒酶是 在大多数肿瘤细胞中表达，但在正常细胞中不表达。 端粒酶和端粒是抗癌的理想肿瘤特异性靶标 治疗策略。我们的初步研究表明了 第一个端粒酶抑制剂。 我们的研究还表明端粒酶 抑制剂诱导端粒缩短，染色体末端融合和 最终细胞死亡。细胞已经显示出适应端粒酶的能力 抑制。 该提议的目标是阐明 端粒酶并为我们的抗癌者提供更明确的目标 策略和2）确定细胞适应的机制 端粒酶抑制剂。这些目标将通过映射和 克隆端粒酶蛋白和RNA模板的基因。通过 我们与程序L合作的合作，纯化的端粒酶将被用作 纯化的端粒酶RNA的来源。体细胞遗传学将 用于绘制端粒酶活性。差异的新技术 显示将用于检测在telomerase激活后激活中表达的mRNA 未表达的细胞在前层酶激活的细胞中。这 映射将与候选cDNA克隆的分离协调 为了使蛋白质和RNA加速合法的识别 克隆。将筛选RNA的cDNA克隆以进行适当 模板序列，次要结构以及指导体外和 体内端粒酶活性。端粒酶蛋白的cDNA将是 表示生产抗体。 抗体和DNA克隆将用作研究 程序3中的端粒酶表达以及程序4的肿瘤标本中的表达。 这些研究的数据将由程序2用于结构 建模和特定的药物设计。 潜在的端粒酶抑制剂，这样 由于抗RNA寡核苷酸将通过程序L进行研究。机制 还将研究对端粒酶抑制剂的适应性。 假设 关于药物代谢，端粒酶上调和端粒酶 特异性将进行测试。