ISOLATION AND ANALYSIS OF HUMAN DNA REPAIR GENES

着色性干皮病是一种常染色体隐性遗传 人类的一种疾病，特征是极易患上 皮肤癌和神经系统并发症。XP单元格 切除修补术的切开步骤不足，高度 辐射暴露的地区患皮肤癌的风险增加 身体都被归因于这一检测。XP在基因上是复杂的 已经确定了九个不同的互补小组。 补充XP细胞缺陷的DNA修复基因有 事实证明，用传统方法克隆是不容易的。最近 已开发的可复制的EB病毒(EBV)质粒 在人类细胞中的染色体外，有几个优点 现有的表达载体。它们在高密度下改变人类细胞 频率，以一致和高水平表达cDNAs插入， 以低频率重新排列，很容易从稳定的状态中解救出来 转化的细胞。分离DNA修复基因将是 以EB病毒为基础的cDNA表达文库的尝试 进入XP细胞并鉴定修复熟练者 通过紫外线照射进行表型选择的转化子。 抢救可能的DNA修复克隆将由Hirt执行 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 已执行。这些研究应该有助于长远发展 阐明人类DNA分子机制的目标 切除修复和XP的辐射敏感性。

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.