MECHANISMS REGULATING EXPRESSION OF TGF- MRNA

TGF-mRNA 表达调节机制

• 批准号: 3086200
• 负责人: LINDA G LEVIN
• 金额: $ 8.77万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3086200
• 关键词: DNA footprinting; DNA methylation; chemical carcinogenesis; chloramphenicol acetyltransferase; deoxyribonuclease I; exonuclease; gel mobility shift assay; gene expression; gene induction /repression; genetic regulation; genetic regulatory element; genetic transcription; messenger RNA; nuclear runoff assay; nucleic acid sequence; phorbols; reporter genes; restriction mapping; southern blotting; tissue /cell culture; transcription factor; transfection; transforming growth factors

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.

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)检测TGF-α mRNA中3'非翻译序列的作用 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