DNA REPAIR IN A HORMONE-RESPONSIVE GENE

这项提议的主要目标是了解分子细节 一个重要的防御机制，对表型变化诱导的 对哺乳动物细胞中DNA的损伤。 这种侮辱来自各种各样的 环境因素，如紫外线辐射和化学物质 致癌物质。 由于大多数这些损伤的修复是通过 同样的机制，紫外线辐射将被用作原型环境 代理这些研究。 这项建议旨在审查 DNA修复效率、转录活性和 两种不同类型的哺乳动物细胞基因的染色质结构。 的 与RNA聚合酶II表达的关系将在 含单纯疱疹病毒胸苷的同基因小鼠L细胞系 激酶（tk）基因与小鼠乳腺肿瘤病毒长末端偶联 重复（LTR）。 这种“LTL”结构的一个半拷贝稳定地 整合到这些细胞的基因组中并表达TK基因 完全依赖于糖皮质激素的存在。 我们将 使用Southern印迹和北方印迹技术来跟踪UV的修复 LTL基因座中的损伤具有以下性质：（1）失活， 包装成散装染色质结构;（2）无活性并包装成 “转录平衡”的染色质结构;和（3）主动 抄写 RNA聚合酶I基因具有这些相同的特征， 在小鼠Friend细胞中进行了研究。 这些细胞的核糖体RNA基因将 被分成转录活性和非活性形式， 限制酶消化细胞核并分离两种形式的 制备凝胶上的rDNA。 最后，我们将使用一个简单的酵母质粒， 含有诱导型基因和组成型表达基因， 在酵母细胞中模拟染色质底物以研究UV的特性 损伤和修复的活性基因更详细。 这些研究将 涉及间接末端标记和质粒的特异性切割 T4 endo V的UV光产物DNA。 因此，我们将研究基因表达和基因表达的变化对细胞的影响。 局部染色质结构对去除DNA损伤效率的影响。 由于这些病变可能会改变特定基因的表达， 建立肿瘤表型，这些研究应该提供 对细胞抵抗肿瘤的防御机制的有价值的见解 环境致癌物的转化。

The broad objective of this proposal is to understand the molecular details of an important defense mechanism against phenotypic changes induced by insults to DNA in mammalian cells. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of the majority of these insults occurs via the same mechanism, UV radiation will be used as the prototype environmental agent for these studies. This proposal is designed to examine the relationship between DNA repair efficiency, transcriptional activity and chromatin structure of two different classes of mammalian cell genes. The relationship to RNA polymerase II expression will be examined in an isogenic mouse L cell line containing the herpes simplex virus thymidine kinase (tk) gene coupled to the mouse mammary tumor virus long terminal repeat (LTR). One and a half copies of this "LTL" construction is stably integrated into the genome of these cells and the expression of the tk gene is totally dependent on the presence of glucocorticoid hormone. We will use both Southern blot and Northern blot techniques to follow repair of UV damage in the LTL locus having the following properties: (1) inactive and packaged into a bulk chromatin structure; (2) inactive and packaged into a "transcriptionally poised" chromatin structure; and (3) actively transcribing. RNA polymerase I genes, having these same features, will be studied in mouse Friend cells. The ribosomal RNA genes of these cells will be fractionated into transcriptionally active and inactive forms using restriction enzyme digestion of nuclei and separation of the two forms of rDNA on preparative gels. Finally, we will use a simple yeast plasmid, containing an inducible gene and a constitutively expressed gene, as a model chromatin substrate in yeast cells to study specific feature of UV damage and repair of active genes in more detail. These studies will involve indirect end-labeling coupled with specific cleavage of the plasmid DNA at UV photoproducts by T4 endo V. Thus, we will examine the effects of both gene expression and changes in local chromatin structure on the efficiency of removal of DNA lesions. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.