RIBOFLAVIN AND FLAVOPROTEIN GENE EXPRESSION

核黄素和黄素蛋白基因表达

• 批准号: 3470063
• 负责人: NATHAN S ROSS
• 金额: $ 9.22万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3470063
• 关键词: acyl coA dehydrogenases; cofactor; complementary DNA; enzyme induction /repression; flavoproteins; gene expression; genetic regulatory element; genetic transcription; human age group; laboratory rabbit; laboratory rat; messenger RNA; molecular cloning; neoplastic cell culture for noncancer research; nutrition related tag; riboflavin; riboflavin deficiency; transfection; translation factor; transposon /insertion element

The long term objective of the proposed research is to determine the role of cofactor availability in the regulation of gene expression. The specific aims are to use in vivo and in vitro models of riboflavin deficiency to study the role of that vitamin in regulating the expression of the short-chain acyl-CoA dehydrogenase (SCAD) gene. SCAD is an FAD dependent dehydrogenase of the mitochondrial beta-oxidation pathway. In young rats, this enzyme undergoes a characteristic post weaning maturation, during which its activity increases by approximately 4-fold over a 3-4 day period. Riboflavin deficiency blocks this development. To examine further the mechanisms by which riboflavin availability regulates the activity of the enzyme, a hepatoma cell culture system of riboflavin deficiency was developed in which treatment with riboflavin causes a 50-100 fold increase in SCAD activity. Preliminary data suggests that the increased SCAD activity reflects increased enzyme mass. The specific aims of the proposed project are to 1) determine the level at which SCAD gene expression is influenced by riboflavin availability and 2) characterize the SCAD gene sequences that mediate the response to either riboflavin or a riboflavin-derived factor. These aims will be approached by isolating a cDNA clone encoding SCAD and using it as a probe to measure SCAD mRNA levels during the developmental period in weanling rats, to compare the levels in riboflavin-deficient and normally fed rats and to assess changes in mRNA level in cell culture when riboflavin-deficient cells are treated with the vitamin. The cDNA clone will then be used to estimate SCAD genomic clones. The long range goal of the research is to identify gene sequences that may comprise a riboflavin-response element. This will be approached by subcloning putative regulatory sequences into a vector containing the chloramphenicol acetyl transferase structural gene, transfecting the vector into the cultured cells and examining the expression of CAT in response to riboflavin treatment.

拟议研究的长期目标是确定作用 基因表达调控中辅助因子的可用性。 这 具体目标是使用核黄素的体内和体外模型 缺乏研究维生素在调节表达中的作用 短链酰基辅酶A脱氢酶（SCAD）基因。 SCAD 是一种时尚 线粒体β-氧化途径的依赖脱氢酶。 在 年轻的老鼠，这种酶经历了断奶后的特征性成熟， 在此期间，其活性在 3-4 天内增加约 4 倍 时期。 核黄素缺乏阻碍了这一发展。 进一步检查 核黄素可用性调节活性的机制 酶，核黄素缺乏的肝癌细胞培养系统 开发出核黄素治疗可导致 50-100 倍增加 在 SCAD 活动中。 初步数据表明，SCAD 的增加 活性反映了酶质量的增加。 拟议项目的具体目标是 1) 确定水平 其中 SCAD 基因表达受核黄素可用性的影响，2) 表征介导对任一反应的 SCAD 基因序列 核黄素或核黄素衍生因子。 这些目标将被实现 通过分离编码 SCAD 的 cDNA 克隆并将其用作探针来测量 断奶大鼠发育期间的 SCAD mRNA 水平 比较核黄素缺乏和正常喂养的大鼠的水平，并 评估核黄素缺乏时细胞培养物中 mRNA 水平的变化 用维生素处理细胞。 然后 cDNA 克隆将用于 估计 SCAD 基因组克隆。 该研究的长期目标是 鉴定可能包含核黄素反应元件的基因序列。 这将通过将假定的调控序列亚克隆到 含有氯霉素乙酰转移酶结构基因的载体， 将载体转染至培养细胞并检查 核黄素处理后 CAT 的表达。