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 与较高癌症风险相关的疾病。对于其中一种疾病， 共济失调-毛细血管扩张(A-T)，杂合子携带者也在增加 相对风险，特别是对乳腺癌而言。因此，它已经 据估计，患有乳腺癌的女性中有7%-8%是A-T 杂合子。来自A-T纯合子的细胞出现异常 与DNA新陈代谢和/或基因组的维持缺陷一致 正直。A-T代表了一个重要的模型系统 癌症发生、DNA修复和基因重组。 尽管进行了广泛的调查，但尚未分离到A-T基因，并且 潜在的缺陷是未知的。这项提案的总体目标是 更好地了解A-T细胞对DNA损伤的反应 通过对A-T基因的克隆和鉴定，以及对A-T基因的研究 A-T表型的两个方面的病因：辐射敏感性和 超重组。我们的方法概括为两个具体目标： A.识别和表征导致房颤和其他人类疾病的基因 通过以下方式补充A-T成纤维细胞表型缺陷的cDNA： I)用EBV中包含的cDNA文库导入A-T成纤维细胞 基于异构体的表达载体及菌落筛选 对诱变剂产生抗药性的转基因细胞； Ii)筛选来自这些存活的克隆的细胞 A-T表型的互补性和从互补型中回收cDNA 将核裂解产物导入到大肠杆菌中的成纤维细胞； Iii)将回收的cDNA导入新鲜的A-T成纤维细胞，并 根据其互补多个基因的能力识别候选的cDNA 反复转染组A-T表型的变化； 四)通过绘制候选的cDNA并分析 正常和病变组候选基因的结构和表达 个人； V)通过分离和分析A-T及其相关基因的全长 长度c DNA和基因组序列与组织模式的确定 以及在细胞周期和细胞周期中表达的变化 在接触诱变剂之后。 B.调查重组异常的病因和 A-T的放射敏感性：“ I)确定P53和G1/S细胞功能丧失的影响 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功能在辐射损伤中的作用 诱变剂对A-T成纤维细胞的杀伤作用 这项工作可能会进一步确定房颤的病因，识别新的基因 参与细胞对DNA损伤的反应，并进一步了解 基因重组和癌症遗传学的研究。

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.