GENE PRODUCT RELATED TO XERODERMA PIGMENTOSUM GROUP

色素性干皮病组相关基因产品

• 批准号: 2091251
• 负责人: AUGUSTINUS R RINALDY
• 金额: $ 13.93万
• 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/2091251
• 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，是一个极其复杂的系统 防止化学和化学物质引起的DNA损伤的重要保护措施 物理代理。在切除的初始步骤中的干扰 修复途径参与人类遗传性干皮病 色素互补组A(XP-A)。从表型来看，这种疾病 与由于失败而导致的皮肤癌的高发病率有关 修复因紫外线照射和接触辐射而受损的DNA 致癌化学物质。致病基因(S)的鉴定 DNA修复不仅对阐明细胞的分子缺陷具有重要意义 着色性干皮病，但也是了解其发病机制的关键 人类致癌的可能性。 一个非功能基因及其相关的功能基因组粘粒克隆 (部分补充了转化的受影响细胞中的XP-A表型) 被分离和鉴定(Rinaldy et at.，1988,1990；Mori 等人，1992；Kaur等人，1992)。非功能的CDNA和 功能粘粒基因组克隆代表了一种新的基因，需要在- 以确定其在DNA修复中的作用。一个 通过聚合酶链式反应扩增出该基因片段，并将其作为两个基因的标记 修复熟练细胞和部分互补的XP-A细胞 是由宇宙克隆变身而来的。一直以来 证明该基因对紫外线有很强的诱导性。 照射和暴露于苯并二醇环氧化物(BPDE)。它是 因此，从部分基因组中分离出具有功能的CDNA克隆是可行的 互补的XPA细胞。 拟议实验的目的是进一步研究 功能性cDNA衍生的基因产物及其与基因的关系 XPA的缺陷。我们将从BPDE中构建一个cDNA文库 XPA转化子的诱导及PCR扩增片段的筛选 非功能的cDNA.该基因将被完全测序，并 在XPA细胞系中使用穿梭载体表达，以便 永久性地补充DNA修复缺陷。体外转录， 然后将转录本显微注射到XPA细胞系Will 以确定微机械的瞬时修复能力。 注射细胞。此外，该基因还将在大肠杆菌中表达。 基因产物将被提纯以产生抗体。西方分析 将被用来检测修复中的基因产物的水平 缺陷的XPA细胞系与修复熟练的细胞系相比。这个 CDNA的翻译也将提供有价值的初级结构 关于XP互补A组基因的信息(S)。