TELOMERASES AND TELOMERASE MOLECULAR BIOLOGY

端粒具有富含G的重复DNA序列， 端稳定性。端粒酶是一种逆转录酶样酶， 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。 端粒酶和端粒是理想的肿瘤特异性抗癌靶点 治疗策略我们的初步研究表明， 首先是端粒酶抑制剂。 我们的研究还表明， 抑制剂诱导端粒缩短，染色体末端融合， 最终导致细胞死亡。细胞已经显示出适应端粒酶的能力 抑制作用 该提案的目标是：1）阐明 为我们的抗癌提供了一个更明确的靶点 策略，和2）确定细胞适应的机制， 端粒酶抑制剂。这些目标将通过绘制和 克隆端粒酶蛋白和RNA模板的基因。通过 我们与Program 1的合作，纯化的端粒酶将被用作 用于克隆的纯化的端粒酶RNA的来源。体细胞遗传学将 用于端粒酶活性图谱的绘制。微分新技术 显示将用于检测在端粒酶激活后的细胞中表达的mRNA。 在前端粒酶激活细胞中不表达的细胞。的 作图将与候选cDNA克隆的分离相协调 蛋白质和RNA加速识别合法的 克隆将筛选RNA的cDNA克隆，以进行适当的筛选。 模板序列、二级结构和体外定向能力， 体内端粒酶活性。端粒酶蛋白质的cDNA将被 表达以产生抗体。 抗体和DNA克隆将作为探针用于研究 程序3和程序4肿瘤标本中端粒酶表达。 这些研究的数据将用于程序2的结构 建模和特定药物设计。 潜在的端粒酶抑制剂，如 因为抗RNA寡核苷酸将由程序1研究。机制 对端粒酶抑制剂的适应性也将被研究。 假设 涉及药物代谢、端粒酶上调和端粒酶 将检测特异性。

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.