DNA REPAIR IN A HORMONE-RESPONSIVE GENE

这个提议的主要目标是了解分子的细节。 一种重要的防御机制，以抵御由 对哺乳动物细胞中DNA的侮辱。这种侮辱是由各种各样的原因造成的 环境因素，如紫外线(UV)辐射和化学品 致癌物质。因为这些侮辱中的大多数都是通过 同样的机理，紫外线辐射将被用作原型环境 这些研究的代理人。这项提案旨在审查 DNA修复效率、转录活性与基因表达的关系 两类不同哺乳动物细胞基因的染色质结构。这个 与RNA聚合酶II表达的关系将在一项 含单纯疱疹病毒胸苷的同基因小鼠L细胞系 小鼠乳腺肿瘤病毒长末端偶联的激酶(Tk)基因 重复(Ltr)。这个“LTL”结构的一份半拷贝是稳定的 整合到这些细胞的基因组和tk基因的表达 完全依赖于糖皮质激素的存在。我们会 同时使用Southern印迹和Northern印迹技术跟踪UV修复 LTL基因座的损伤具有以下特性：(1)不活跃和 被包装成大块染色质结构；(2)非活性并被包装成 “转录稳定”的染色质结构；以及(3)活跃的 转录。具有这些相同特征的RNA聚合酶I基因将是 在老鼠的朋友细胞中进行了研究。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，使用 核酸的限制性内切酶消化及两种形式的分离 制备凝胶上的rDNA。最后，我们将使用一个简单的酵母质粒， 含有诱导性基因和结构性表达基因的，如 酵母细胞染色质底物模型研究紫外线的特性 更详细地介绍活性基因的损伤和修复。这些研究将 包括间接末端标记和特定的质粒切割 DNA在紫外光下的产物由T4 Endo V. 因此，我们将研究基因表达和基因变化的影响。 局部染色质结构对去除DNA损伤的效率有影响。 因为这些损伤可能会改变所需的特定基因的表达 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 环境致癌物质的转化。

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.