ROLE OF FRAGILE SITES IN CHROMOSOME BREAKAGE AND CANCER

脆弱部位在染色体断裂和癌症中的作用

• 批准号: 3185312
• 负责人: THOMAS W GLOVER
• 金额: $ 2.94万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3185312
• 关键词: carcinogenesis; cell transformation; chromosome deletion; chromosome disorders; chromosome translocation; cytogenetics; developmental genetics; gel electrophoresis; gene expression; genetic disorder diagnosis; genetic mapping; genetic markers; genetic models; human population genetics; hybrid cells; in situ hybridization; leukemia; lung neoplasms; lymphoblast; lymphoma; neoplasm /cancer genetics; proline; small cell lung cancer; tissue /cell culture; transfection; transposon /insertion element

The overall aims of this proposal are to clone and characterize DNA sequences at the 3p14 fragile site (FRA3B). This fragile site will be studied because of its contribution to chromosome structure and due to its possible mechanistic involvement in chromosomal deletions seen in nearly all small cell lung cancers and many renal cell tumors. We have previously shown that this fragile site is a hot spot for chromosomal recombination as measured by sister chromatid exchange analysis and the induction of deletions and translocations at this site. As part of these experiments, we have utilized somatic cell hybrids containing human chromosome 3 to construct derivatives of this chromosome with deletions and translocations at FRA3B. The translocations have occurred with hamster chromosomes. We will use our translocation and deletion chromosomes as physical markers in complementary reverse genetics approaches to clone the fragile sites. We will attempt to identify the FRA3B fragile site translocation breakpoints with pulsed-field gel electrophoresis (PFGE) using currently available probes. We will also generate additional probes for this approach by preparing linking libraries from irradiation-reduced hybrids containing small segments of chromosome 3, including the fragile site, and physically map these clones with conventional hybrid panels that minimize the region around the fragile sites and by in situ hybridization. Once a probe is identified within close physical distance by PFGE analysis, protocols of chromosome jumping, cosmid walking and screening a general YAC library will be utilized to move close to, and ultimately clone, the fragile site sequences themselves. The irradiation-induced hybrids will be constructed in such a manner as to facilitate a complementary direct cloning strategy. Based on the observed high rate of chromosomal recombination at these sites, we will test the hypothesis that a selectable marker (the neor gene) can be inserted into the fragile sites and rescued together with flanking DNA sequences. Detection of the insertion event will be accomplished in part by co-segregation of the neor gene with DNA sequences close to FRA3B. Fragile site clones will be characterized at the DNA and RNA levels and tested in a series of biological experiments including testing for variation in normal individuals, transfection experiments, and in a direct test of the hypothesis that FRA3B plays a mechanistic role in chromosome breakage and deletions of chromosome 3 in human tumors including small cell carcinoma of the lung.

这项提案的总体目标是克隆和表征DNA 3p14脆性位点（FRA3B）。 这个脆弱的网站将是 研究是因为它对染色体结构的贡献， 可能的机制参与染色体缺失， 所有的小细胞肺癌和许多肾细胞肿瘤。 我们先前已经 表明这个脆性位点是染色体重组的热点， 通过姐妹染色单体交换分析和诱导 在这个位点上的缺失和易位。 作为这些实验的一部分， 我们已经利用含有人类3号染色体的体细胞杂交体， 构建带有缺失和易位的该染色体的衍生物 在FRA3B。 仓鼠的染色体发生了易位。 我们 将使用我们的易位和缺失染色体作为物理标记， 互补反向遗传学方法来克隆脆性位点。 我们 将尝试确定FRA3B脆性位点易位断裂点 脉冲场凝胶电泳（PFGE），使用目前可用的 probes. 我们还将为此方法生成其他探测器， 从辐射减少的杂交体制备连接文库，所述杂交体包含 3号染色体的小片段，包括脆性位点， 用传统的杂交板绘制这些克隆， 在脆性位点周围和通过原位杂交。 一旦探测器 通过PFGE分析在近物理距离内识别， 染色体跳跃、粘粒步移和筛选通用YAC文库将 被用来移动接近，并最终克隆，脆弱的网站， 序列本身。 将构建辐射诱导的杂交体 以便于辅助直接克隆策略。 根据在这些细胞中观察到的高染色体重组率， 位点，我们将测试的假设，一个选择标记（neor基因） 可以插入脆弱的网站和拯救一起侧翼 DNA序列 插入事件的检测将在 部分是由于neor基因与FRA3B附近的DNA序列共分离。 将在DNA和RNA水平表征脆性位点克隆， 在一系列生物实验中进行了测试，包括测试 在正常个体，转染实验中的变化，以及在直接 FRA3B在染色体中起机械作用的假设的检验 包括小细胞癌在内的人类肿瘤中3号染色体的断裂和缺失 肺癌