ENZYME SYNTHESIS AND DEGRADATION IN METABOLIC CONTROL

我们将研究培养的动物细胞 由于DNA的选择性扩增而对甲氨蝶呤耐药 编码二氢叶酸还原酶的序列。 该研究旨在 发生这种情况并导致放大的基本机制 存在于染色体上或存在于 被称为“双微染色体”的染色体外成分。“基因 放大发生在进化的时间尺度上，以及一种机制， 蛋白质合成的发育调节。 我们希望使用 培养的哺乳动物细胞的实验系统，以研究 放大 这些研究将使用培养的varoius细胞系， 仓鼠和小鼠细胞。 有待研究的机制包括 从细胞中摄取-复制-整合DNA，以及突变 复制模型 将采用的技术包括使用 荧光激活细胞分选仪研究DNA合成，以及 分离具有升高的二氢叶酸还原酶水平的细胞。 氢叶酸 还原酶DNA序列将被检测和分离， 信使衍生的以及基因组重组DNA克隆目前 在实验室中可用。 最终，我们有兴趣研究 基因被放大，哪些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.