MECHANISMS REGULATING EXPRESSION OF TGF- MRNA

转化生长因子α是50个氨基酸的促有丝分裂肽 与EGF在结构和功能上具有同源性。 它产生 许多转化细胞，以及发育和正常哺乳动物 组织，表明在自然界中的广泛作用。 然而，我们所知甚少， 关于TGF-α mRNA表达的调节机制。 的 存在由化学衍生的克隆肿瘤细胞系， 大鼠肝上皮细胞的转化提供了一个模型， 探索这些机制。 使用某些细胞系， 血统，这是本提案的长期目标，以阐明 调节TGF-α基因的机制，并确定TGF-α mRNA 表达在不同的语境中会发生变化。 因此，我们将：（1） 进行核连续试验，甲基化和DNA酶I敏感性 测定以确定是否在以下水平进行监管控制： 转录并受基因结构变化的影响;（2） 进一步定义那些代表阳性和阴性的序列 通过CAT测定使用野生型TGF-α启动子的调节元件 和含有推定调控序列的诱变构建体; (3)识别来自细胞提取物的活性， 通过进行凝胶迁移和DNA足迹分析来测定调节序列; (4)检查3'非翻译序列在TGF-α mRNA中的作用 稳定性，因此TGF-α mRNA积累。 这将是 通过制备嵌合构建体来完成，所述嵌合构建体含有不同量的 TGF-α基因的3'非翻译序列， 报告基因。 这些将用于转染实验 并通过S1核酸酶评价所得转录物的稳定性 用以下物质处理转染子后的消化分析 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.