MOLECULAR ANALYSIS OF CHROMOSOME INSTABILITY SYNDROMES

染色体不稳定综合征是一组常染色体隐性遗传病， 与癌症风险较高相关的疾病。 对于其中一种疾病， 共济失调-毛细血管扩张症（A-T），杂合子携带者也在增加 相对风险，尤其是乳腺癌。 因此， 据估计，7%-8%患有乳腺癌的妇女是A-T 杂合子 来自A-T纯合子的细胞表现出异常 与DNA代谢缺陷和/或基因组蛋白质的维持相一致。 完整 A-T模型是一个重要的模型系统， 致癌作用、DNA修复和基因重组。 尽管进行了广泛的调查，但尚未分离出A-T基因，并且 潜在缺陷未知。 本提案的总体目标是 更好地理解A-T细胞对DNA损伤的反应 通过克隆和鉴定A-T基因以及研究 A-T表型的两个方面的病因学：辐射敏感性和 超重组 我们的方法概括为两个具体目标： a. 识别和表征导致A-T以及其他人类疾病的基因 通过以下方式补充A-T成纤维细胞的表型缺陷的cDNA： i）用包含在EBV-DNA中的cDNA文库转染A-T成纤维细胞， 基于附加型表达载体并选择 已变得具有诱变剂抗性的转染细胞; ii）筛选来源于这些存活集落的细胞， A-T表型的互补和从互补的表型中拯救cDNA 通过将核裂解物转染到E.大肠杆菌; iii）将回收的cDNA转染到新鲜的A-T成纤维细胞中， 通过候选cDNA与多个cDNA互补的能力来鉴定候选cDNA。 重复转染后A-T表型的方面; iv）通过定位候选cDNA并分析 正常人和受影响的候选基因的结构和表达 个人; v）通过分离和分析全基因组序列来表征A-T和相关基因， 长度cDNA和基因组序列和决定组织的模式 表达以及细胞周期中表达的变化， 在暴露于诱变剂之后。 B。 研究重组异常的病因， A-T的放射敏感性" i）确定p53和G1/S细胞的功能丧失的影响， 正常哺乳动物细胞中重组的周期检查点;以及 ii）研究细胞凋亡和p53功能在辐射中的作用， 诱变剂诱导的A-T成纤维细胞杀伤。 这项工作可能会进一步确定A-T的病因，确定新的基因， 参与细胞对DNA损伤的反应， 基因重组和癌症的遗传学。

The chromosome instability syndromes are a group of autosomal recessive diseases associated with higher cancer risks. For one of these diseases, ataxia-telangiectasia (A-T), heterozygotes carriers are also at increased relative risk, particularly for breast carcinoma. As a result, it has been estimated that 7-8% of women suffering from breast cancer are A-T heterozygotes. Cells from A-T homozygotes exhibit abnormalities consistent with a defect in DNA metabolism and/or maintenance of genomic integrity. A-T represents an important model system for the study of carcinogenesis, DNA repair and genetic recombination. Despite extensive investigation, no A-T gene has been isolated and the underlying defect is unknown. The overall goal of this proposal is to gain a better understanding of both A-T cellular responses to DNA damage by cloning and characterization A-T genes as well as studying the etiology of two aspects to the A-T phenotype: radiation sensitivity and hyper-recombination. Our approach is summarized in two specific aims: A. Identify and characterize genes that cause A-T as well as other human cDNAs that complement the phenotypic defects of A-T fibroblasts by: i) transfecting A-T fibroblasts with cDNA libraries contained in an EBV- based episomal expression vector and selecting for colonies of transfected cells that have become mutagen-resistant; ii) screening cells derived from these surviving colonies form complementation of the A-T phenotype and rescuing cDNAs from complemented fibroblasts by transfection of nuclear lysates into E. coli; iii) transfecting the recovered cDNAs into fresh A-T fibroblasts and identifying candidate cDNAs by their ability to complement multiple aspects of the A-T phenotypes upon repeated transfection; iv) identifying disease genes by mapping candidate cDNAs and analyzing the structure and expression of candidate genes in normal and affected individuals; v) characterizing A-T and related genes by isolating and analyzing full- length cDNA and genomic sequences and determining patterns of tissues expression as well as changes in expression during the cell cycle and after exposure to mutagens. B. Investigate the etiology of recombination abnormalities and radiosensitivity in A-T by" i) determining the effects of functional loss of p53 and the G1/S cell cycle checkpoint on recombination in normal mammalian cells; and ii) studying the role of apoptosis and p53 function in radiation- and mutagen-induced killing of A-T fibroblasts. This work may further define the etiology of A-T, identify new genes involved in cellular responses to DNA damage and further our knowledge of genetic recombination and the genetics of cancer.