ROLE OF FRAGILE SITES IN CHROMOSOME BREAKAGE AND CANCER

这项提议的总体目标是克隆和鉴定DNA 3p14脆性位点的序列(FRA3B)。这个脆弱的地方将是 因为它对染色体结构的贡献以及由于它的 染色体缺失可能的机械性参与 所有小细胞肺癌和许多肾细胞肿瘤。我们之前已经 表明这个脆弱的部位是染色体重组的热点，因为 姊妹染色单体互换分析和诱导 这个位点的缺失和易位。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 用缺失和易位构建该染色体的衍生品 在法兰克福机场3B。这种易位发生在仓鼠的染色体上。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克隆脆弱部位。我们 将尝试识别FRA3B脆性位点易位断点 使用现有的脉冲场凝胶电泳(PFGE) 探测器。我们还将通过以下方式为此方法生成额外的探测器 从含有以下物质的辐射减少的杂交体中制备连接库 3号染色体的小片段，包括脆弱部位，以及身体 用传统的混合电池板绘制这些克隆的地图，使区域最小化 在脆性部位周围和通过原位杂交。一旦探测器 通过PFGE分析在物理距离较近的范围内识别， 染色体跳跃、粘粒行走和筛选通用YAC文库将 被用来接近并最终克隆脆弱的部位 序列本身。将构建辐射诱导的杂交种 以这种方式促进互补的直接克隆战略。 根据观察到的高染色体重组率 ，我们将测试一个可选择标记(neor基因)的假设。 可以插入到脆弱的部位并与侧翼一起救援 DNA序列。插入事件的检测将在 部分是由于neor基因与接近FRA3B的DNA序列的共同分离。 脆性位点克隆将在DNA和RNA水平和 在一系列生物实验中进行了测试，包括测试 在正常个体中的变异，转基因实验，以及直接的 FRA3b在染色体中起机械性作用假说的检验 包括小细胞在内的人类肿瘤中3号染色体的断裂和缺失 肺癌。

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.