ENZYME SYNTHESIS AND DEGRADATION IN METABOLIC CONTROL

代谢控制中的酶合成和降解

• 批准号: 3268701
• 负责人: ROBERT T SCHIMKE
• 金额: $ 19.43万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268701
• 关键词: DNA; cell sorting; clone cells; dihydrofolate reductase; drug resistance; enzyme induction /repression; enzyme structure; extrachromosomal DNA; genetic crossing over; genetic manipulation; genetic transcription; immunochemistry; messenger RNA; methotrexate; nucleic acid metabolism; radiotracer

We shall be studying the mechanism whereby cultured animal cells become resistant to methotrexate as a result of selective amplification of DNA sequences coding for dihydrofolate reductase. The research is directed at the fundamental mechanism(s) whereby this occurs and results in amplified DNA sequences that are either present on chromosomes, or are present at extrachomosomal elements called "double minute chromosomes." Gene amplification occurs on evolutionary time scale, as well as one mechanism for developmental regulatino of protein synthesis. We wish to use the experimental system of cultured mammalian cells to study the mechanism of amplification. The studies will employ varoius cells lines of cultured hamseter and mouse cells. Mechanisms to be studied include uptake-replicaton-integration of DNA from cells, as well as a saltatory replication model. Techniques to be employed include use of fluorescence-activated cell sorter to study DNA synthesis, as well as isolate cells with elevated dihydrofolate reductase levels. Dihydrofolate reductase DNA sequences will be detected and isolate employing both messenger-derived, as well as genomic recombinant DNA clones presently available in the laboratory. Ultimately, we are interested in studing how genes are amplified, and what intrinsic and extrinsic factors may affect the amplification process.

我们将研究培养的动物细胞变成的机制 DNA 选择性扩增导致对甲氨蝶呤产生耐药性 编码二氢叶酸还原酶的序列。 该研究针对 这种情况发生并导致放大的基本机制 DNA 序列要么存在于染色体上，要么存在于 染色体外元件称为“双微小染色体”。 基因 放大发生在进化时间尺度上，以及一种机制 用于蛋白质合成的发育调节。 我们希望使用 培养哺乳动物细胞实验系统研究机制 放大。 这些研究将使用各种培养的细胞系 仓鼠和小鼠细胞。 要研究的机制包括 细胞 DNA 的摄取-复制-整合，以及突变 复制模型。 所采用的技术包括使用 荧光激活细胞分选仪用于研究 DNA 合成，以及 分离二氢叶酸还原酶水平升高的细胞。 二氢叶酸 还原酶 DNA 序列将使用两者进行检测和分离 目前，信使衍生的 DNA 克隆以及基因组重组 DNA 克隆 可在实验室获得。 最终，我们有兴趣研究如何 基因被放大，哪些内在和外在因素可能会影响 放大过程。