ISOLATION AND ANALYSIS OF HUMAN DNA REPAIR GENES

人类DNA修复基因的分离与分析

• 批准号: 3197191
• 负责人: RANDY J LEGERSKI
• 金额: $ 14.85万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3197191
• 关键词: Baculoviridae; DNA repair; Epstein Barr virus; cell transformation; complementary DNA; cytogenetics; density gradient ultracentrifugation; fibroblasts; gene complementation; gene expression; gene mutation; genetic library; genetic mapping; human genetic material tag; microinjections; molecular cloning; nucleic acid sequence; protein purification; simian virus 40; synthetic peptide; transfection; transfection /expression vector; ultraviolet radiation; xeroderma pigmentosum

Xeroderma pigmentosus (XP) is an autosomal, recessive genetic disease of humans characterized by a high predisposition to cancers of the skin and neurological complications. XP cells are deficient in the incision step of excision repair and the highly elevated risk of skin cancer to radiation exposed areas of the body are attributed to this detect. XP is genetically complex in that nine different complementation groups have been identified. DNA repair genes that complement the deficiency in XP cells have proven refractory to cloning by traditional methods. Recently developed Epstein-Barr virus (EBV) plasmids which replicate extrachromosomally in human cells, have several advantages over existing expression vectors. They transform human cells at high frequency, express cDNA inserts at a consistent and high level, rearrange at low frequency and are easily rescued from stably transformed cells. Isolation of DNA repair genes will be attempted by transfection of EBV-based cDNA expression libraries into XP cells and identification of repair-proficient transformants by phenotypic selection with UV irradiation. Rescue of putative DNA repair clones will be performed by Hirt extraction and recovered into bacterial cells by electroporation. Structural and biochemical analyses of cloned genes will be performed. These studies should contribute to the long term goals of elucidating the molecular mechanisms of human, DNA excision repair and the radiation sensitivity of XP.

色素性干皮病 (XP) 是一种常染色体隐性遗传病 以高度易感性为特征的人类疾病 皮肤癌和神经系统并发症。 XP 细胞是 切除修复的切口步骤存在缺陷，且高度 暴露于辐射区域的皮肤癌风险增加 身体归因于此检测。 XP 的遗传复杂性 已经确定了九个不同的互补组。 补充 XP 细胞缺陷的 DNA 修复基因 已被证明难以通过传统方法进行克隆。 最近 开发了可复制的 Epstein-Barr 病毒 (EBV) 质粒 人类细胞染色体外，与 现有的表达载体。 他们以高水平转化人体细胞 频率，以一致和高水平表达 cDNA 插入片段， 低频重新排列，很容易从稳定状态中解救出来 转化的细胞。 DNA修复基因的分离 尝试转染基于 EBV 的 cDNA 表达文库 进入 XP 细胞并鉴定修复能力 通过紫外线照射的表型选择转化体。 Hirt 将负责拯救假定的 DNA 修复克隆 提取并通过电穿孔回收到细菌细胞中。 克隆基因的结构和生化分析将 执行。 这些研究应该有助于长期 阐明人类DNA的分子机制的目标 切除修复和 XP 的辐射敏感性。