ENZYME SYNTHESIS AND DEGRADATION IN METABOLIC CONTROL

我们将研究培养的动物细胞成为 DNA选择性扩增导致对甲氨蝶呤的耐药性 二氢叶酸还原酶的编码序列。这项研究针对的是 发生这种情况并导致放大的基本机制(S) 存在于染色体上的DNA序列或存在于 体外分子称为“双小染色体”。基因 放大发生在进化的时间尺度上，以及一种机制 用于蛋白质合成的发育调节。我们希望使用 培养哺乳动物细胞的实验系统研究其作用机制 放大。这项研究将利用培养的Varoius细胞系 汉密尔顿和小鼠细胞。有待研究的机制包括 细胞DNA的摄取-复制-整合，以及跳跃 复制模型。将采用的技术包括使用 用于研究DNA合成的荧光激活细胞分类器，以及 分离二氢叶酸还原酶水平升高的细胞。二氢叶酸 将使用这两种方法检测和分离还原酶DNA序列 目前信使衍生的以及基因组重组DNA克隆 在实验室中可用。最终，我们感兴趣的是研究如何 基因被放大，以及什么内在和外在因素可能会影响 放大过程。

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.