MECHANISMS REGULATING EXPRESSION OF TGF- MRNA

转化生长因子α是一种由50个氨基酸组成的促有丝分裂多肽。 这与EGF在结构和功能上具有同源性。它是生产出来的 由许多转化的细胞以及发育中的和正常的哺乳动物 组织，表明在自然界中有广泛的作用。然而，人们对此知之甚少， 关于转化生长因子-αmRNA表达的调控机制。这个 化学来源的克隆肿瘤细胞系的存在 大鼠肝上皮细胞的转化提供了一种模型 以此来探索这些机制。使用来自此的某些细胞系 血统，这是这项建议的长期目标是澄清 转化生长因子-α基因的调控机制及转化生长因子-αmRNA的鉴定 表达式在其不同的上下文中被更改。因此，我们将：(1) 进行核连续分析，以及甲基化和DNA酶I敏感性 用于确定监管控制是否发生在 转录并受基因结构变化的影响；(2) 进一步定义那些表示正和负的序列 野生型CAT法检测转化生长因子-α启动子的调控元件 和含有假定调控序列的诱变构建体； (3)识别与已识别的细胞提取液结合的活性 通过进行凝胶移位和DNA足迹分析来调节序列； (4)检测3‘端非翻译序列在转化生长因子-α基因中的作用 稳定性，因此转化生长因子-αmRNA积累。这将是 通过制备包含不同量的嵌合结构来完成 来自转化生长因子-α基因的3‘未翻译序列和一个简单的 确定了报告基因。这些将被用于转基因实验。 并用S1核酸酶评价转录本的稳定性 转染体处理后的酶切分析 放线菌素D

Transforming growth factor alpha is a 50 amino acid mitogenic peptide that shares structural and functional homology with EGF. It is produced by many transformed cells, as well as developing and normal mammalian tissues, indicating a broad role in nature. Very little is known, however, about the regulatory mechanisms involved in TGF-alpha mRNA expression. The existence of cloned neoplastic cell lines derived by chemical transformation of a rat liver epithelial cell have provided a model in which to explore these mechanisms. Using certain cell lines from this lineage, it is the long range goal of this proposal to elucidate the mechanisms regulating the TGF-alpha gene and to identify how TGF-alpha mRNA expression is altered in its various contexts. Accordingly, we will: (1) perform nuclear run-on assays, and methylation and DNase I sensitivity assays to ascertain if regulatory control occurs at the level of transcription and is influenced by structural changes in the gene; (2) further define those sequences representing positive and negative regulatory elements of the TGF-alpha promoter by CAT assay using wild-type and mutagenized constructs containing the putative regulatory sequences; (3) recognize activities from cell extracts which bind to identified regulatory sequences by performing gel shift and DNA footprinting assays; (4) examine the role of 3' untranslated sequences in TGF-alpha mRNA stability, and hence TGF-alpha mRNA accumulation. This will be accomplished by preparing chimeric constructs which contain varying amounts of 3' untranslated sequences from the TGF-alpha gene and an easily identified reporter gene. These will be used in a transfection experiments and the stability of the resultant transcripts evaluated by S1 nuclease digestion analysis subsequent to treatment of the transfectants with actinomycin D.