CHROMOSOMAL COMPLEMENTATION OF BLOOM SYNDROME CELLS

布卢姆综合征细胞的染色体互补

• 批准号: 3196900
• 负责人: ROGER A. SCHULTZ
• 金额: $ 15.88万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3196900
• 关键词: Bloom syndrome; DNA; DNA repair; autosomal recessive trait; cell fusion; chromosome translocation; chromosomes; cytogenetics; endonuclease; fibroblasts; gene complementation; gene mutation; genetic crossing over; genetic mapping; genetic markers; glycosyltransferase; human genetic material tag; human tissue; hybrid cells; ligase; molecular cloning; monoclonal antibody; nucleic acid sequence; tissue /cell culture; transfection; uracil; western blottings

Bloom syndrome(BS) is an autosomal recessive, spontaneous chromosomal instability disorder demonstrating a clinical constellation which includes severe proportional dwarfing; sun-sensitive telangiectatic erythema; and a significant predisposition to the development of leukemia, with early onset of other neoplasias. Cytologically, the pathognomonic phenotype is a many- fold increase in the spontaneous rate of sister chromatid exchanges (SCE), elevated frequencies of chromosomal breakage and the appearance of quadriradial figures. Although BS cells exhibit normal responses for most DNA repair assays which have been studied, deficiencies and/or abnormalities have been found in the activities of both DNA ligase I and uracil DNA glycosylase. Taken collectively, enzymatic data would support a processing or regulation defect, rather than a primary mutation in either DNA ligase I or uracil DNA glycosylase. Whole-cell fusion experiments have demonstrated complementation of the elevated SCE rates in BS cells to normal frequencies. However, such investigative approaches are limited by lack of selective methods to specifically identify and stabilize hybrid cells resulting from those fusions, compromising efforts to accurately evaluate complementation. The present application proposes an alternative genetic approach to quantitatively characterize the correction of high SCE phenotype in BS fibroblasts and to chromosomally localize the complementing gene, leading to its molecular cloning. Individual normal human chromosomes will be transferred to BS cells by microcell mediated chromosome transfer (MMCT). The MMCT process will be facilitated by the use of an established collection of mouse/human hybrids bearing human chromosomes "tagged" with a dominant selectable marker (neo). Following MMCT and selection, each clone arising will be independently screened for correction of high SCE levels and the chromosome responsible for complementation will be identified with cytogenetic and molecular techniques. Complemented clones will be quantitatively characterized for BS associated cellular phenotypes, including rates of chromosome breakage, and the appearance of both DNA ligase I activity and the normal antigenic form of uracil DNA glycosylase. Cytogenetically detectable deletions and rearrangements of the complementing chromosome will be used to achieve finer mapping of the complementing locus. Quantitation of phenotypic complementation, fine mapping of the locus rendering correction, and the establishment of rodent/human hybrids bearing complementing and noncomplementing deviations of that single human chromosome, will provide the materials necessary for molecular cloning and characterization of the gene involved in BS complementation. The proposed study will provide valuable information leading to the ultimate identification of the specific protein defect associated with elevated SCE rates in BS cells and a better understanding of events which predispose to neoplasia in this syndrome.

Bloom综合征（BS）是一种常染色体隐性，自发性染色体 不稳定障碍，证明了一个临床星座，包括 严重的比例矮人；太阳敏感的远程直脉红斑；和 白血病的发育有明显的倾向，早期发作 其他肿瘤。 从细胞学上讲，病理学表型是多种多样的 姊妹染色单体交换（SCE）的自发率的折叠率增加， 染色体破裂的频率升高和出现 四边形数字。 尽管BS细胞对大多数人都表现出正常的反应 已研究的DNA维修分析，缺陷和/或 在DNA连接酶I和 尿嘧啶DNA糖基化酶。 集体采取的酶数据将支持 处理或调节缺陷，而不是任何一个主要突变 DNA连接酶I或尿嘧啶DNA糖基化酶。 全细胞融合实验已经证明了 BS细胞中的SCE速率升高到正常频率。 但是，这样 调查方法受到缺乏选择性方法的限制 明确识别并稳定由这些细胞产生的杂种细胞 融合，损害了准确评估互补的努力。 这 当前的应用提出了一种替代的遗传方法 定量地表征了BS中高SCE表型的校正 成纤维细胞并染色体将互补基因定位，领先 到其分子克隆。 个别正常的人类染色体将是 通过Microcell介导的染色体转移（MMCT）转移到BS细胞中。 MMCT过程将通过既定的 携带人类染色体的小鼠/人类杂种的收集用 主要可选标记（NEO）。 遵循MMCT和选择，每个克隆 将独立筛选出来以校正高SCE水平 并确定负责互补的染色体 细胞遗传学和分子技术。 互补的克隆将是 针对BS相关的细胞表型的定量表征， 包括染色体破裂的速率和两个DNA的出现 连接酶I活性和尿嘧啶DNA糖基化酶的正常抗原形式。 可检测到的细胞遗传学缺失和重排 补充染色体将用于实现更精细的映射 补充基因座。 表型互补的定量，细 基因座的绘制校正和建立 啮齿动物/人类杂种带有补充和不结合偏差 在那个单一的人类染色体中，将提供必要的材料 BS中涉及基因的分子克隆和表征 互补。 拟议的研究将提供有价值的信息 导致特异性蛋白缺陷的最终鉴定 与BS细胞中的SCE率升高和更好的理解有关 在该综合征中诱发肿瘤的事件。