GENE PRODUCT RELATED TO XERODERMA PIGMENTOSUM GROUP

色素性干皮病组相关基因产品

• 批准号: 3186108
• 负责人: AUGUSTINUS R RINALDY
• 金额: $ 15.51万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186108
• 关键词: DNA repair; DNA replication; chemical carcinogen; chimeric proteins; cytogenetics; drug resistance; gene complementation; genetic library; genetic mapping; genotype; in situ hybridization; ionizing radiation; messenger RNA; molecular cloning; northern blottings; nucleic acid sequence; plasmids; radiation carcinogenesis; radiation genetics; radiation sensitivity; restriction fragment length polymorphism; transfection; ultraviolet radiation; western blottings; xeroderma pigmentosum

The human DNA repair mechanism is a complex system and an extremely important safeguard against DNA damage caused by both chemical and physical agents. A disturbance in the initial step of the excision repair pathway is involved in the human genetic disorder xeroderma pigmentosum complementation group A (XP-A). Phenotypically, this disease is associated with a very high incidence of skin cancer due to a failure of repair DNA damaged by ultraviolet irradiation as well as exposure to carcinogenic chemicals. Identification of the gene(s) responsible for DNA repair is not only important to elucidate the molecular defect of xeroderma pigmentosum, but is also crucial to understanding the mechanism of carcinogenesis in humans. A non-functional cDNA and its related functional genomic cosmid clone (partially complements the XP-A phenotype in transformed affected cells) have been isolated and characterized (Rinaldy et at., 1988, 1990; Mori et al., 1992; Kaur et al., 1992) . The non-functional CDNA and the functional cosmid genomic clone represent a novel gene that requires in- depth characterization in order to determine its role in DNA repair. A fragment of this gene was amplified by PCR and used as a marker for both the repair proficient cells and the partially complemented XP-A cells resulting from transformation with the cosmid clone. It has been demonstrated that this gene is strongly inducible with ultraviolet irradiation and exposure to benzpyrene diol epoxide (BPDE). It is therefore feasible to isolate a functional CDNA clone from the partially complemented XPA cells. The objective of the proposed experiments is to further investigate the gene product derived from the functional cDNA and its association with the defect in XPA. A cDNA library will be constructed from the BPDE induced XPA transformant and screened with the PCR amplified region of the non-functional cDNA. The cDNA will be completely sequenced and expressed using a shuttle vector in the XPA cell line in order to permanently complement the DNA repair defect. In-vitro transcription, followed by micro-injection of the transcript into the XPA cell line will be performed to determine the transient repair capability of the micro- injected cells. Additionally, cDNA will be expressed in E. coli and the gene product will be purified for antibody production. Western analyses will then be employed to detect the level of the gene product in repair deficient XPA cell lines compared to repair proficient cell lines. The translation of the cDNA will also provide valuable primary structural information about the XP complementation group A gene(s).

人类DNA修复机制是一个复杂的系统， 防止化学物质和化学物质引起DNA损伤的重要保障 物理代理 在切除的初始步骤中的干扰 修复途径与人类遗传性疾病干皮病有关 色素互补A组（XP-A）。 从表型来看，这种疾病 与皮肤癌的高发病率有关， 修复DNA损伤的紫外线照射以及暴露于 致癌化学物质。 鉴定负责以下疾病的基因 DNA修复不仅对阐明细胞的分子缺陷很重要， 着色性干皮病，但也是至关重要的了解机制， 致癌作用。 一个非功能cDNA及其相关功能基因组粘粒克隆 （部分补充转化受影响细胞中的XP-A表型） 已经被分离和表征（Rinaldy等人，1988年，1990年; Mori 例如，1992; Kaur等人，1992年）。 非功能性cDNA和 功能性粘粒基因组克隆代表了一种新的基因， 深入表征，以确定其在DNA修复中的作用。 一 通过PCR扩增该基因的片段，并用作两者的标记。 修复能力强的细胞和部分互补的XP-A细胞 这是用粘粒克隆转化得到的。 已经 表明该基因是强烈诱导与紫外线 辐照和暴露于苯并芘二醇环氧化物（BPDE）。是 因此，从所述部分cDNA克隆中分离功能性cDNA克隆是可行的。 补充XPA细胞。 所提出的实验的目的是进一步研究 来自功能cDNA的基因产物及其与 XPA的缺陷 将从BPDE构建cDNA文库 诱导XPA扩增，并用PCR扩增的 非功能性cDNA。 cDNA将被完全测序， 使用穿梭载体在XPA细胞系中表达， 永久地补充DNA修复缺陷。 体外转录， 随后将转录物显微注射到XPA细胞系中， 进行，以确定瞬态修复能力的微 注射细胞 此外，cDNA将在E.杆菌和 基因产物将被纯化用于抗体生产。 Western分析 将被用来检测修复中基因产物的水平 缺陷型XPA细胞系与修复熟练细胞系相比。 的 cDNA的翻译也将提供有价值的初级结构 关于XP互补组A基因的信息。