MOUSE DIHYDROFOLATE REDUCTASE GENE EXPRESSION

这项建议的主要重点是研究规管 二氢叶酸还原酶（Dhfr）基因表达的变化过程中， 细胞的增殖状态。 稳态水平和稳定性 将在生长过程中分析各种小鼠Dhfr mRNA种类 在放大和非放大状态下的刺激和静止。 使用具有扩增的Dhfr基因拷贝的细胞系， 优点：i）由于Dhfr mRNA水平升高， 和ii）扩增的细胞中的Dhfr表达 状态扩展了我们对其在肿瘤细胞中的调节的理解， 已经使用基因扩增作为抗叶酸的方法 拮抗剂如甲氨蝶呤。 然而，Dhfr基因表达在一个 未扩增的细胞可以不同于扩增状态。 在那里- 因此，Dhfr基因表达也将在未扩增状态下进行研究。 以前很难在未扩增的细胞中研究Dhfr表达， 细胞，但最近发展的定量PCR检测mRNA， 使这成为可能。 PCR检测的灵敏度将允许 硫尿苷脉冲标记的RNA在有机汞上的分离分析 柱，从而使我们能够研究稳定的和新制作的Dhfr 核糖核酸 第二，初级结构将发生各种变化， Dhfr基因。 酵母遗传学将被用来改变 YAC克隆中含有的小鼠Dhfr基因。 这些YAC结构将是 转移到Dhfr缺陷型CHO细胞中，并在正常和 致瘤细胞，并进一步了解Dhfr基因的作用 在肿瘤细胞耐药性的发展中的表达。

The primary emphasis of this proposal is to study the regulation of dihydrofolate reductase (Dhfr) gene expression during changes in the proliferative state of the cell. The steady state levels and stability of various mouse Dhfr mRNA species will be analyzed during growth stimulation and quiescence in both the amplified and un-amplified states. Using cell lines with amplified copies of the Dhfr gene has several advantages: i)Dhfr MRNA can easily be detected due to the elevated level of expression in amplified cells and ii)Dhfr expression in the amplified state extends our understanding of its regulation in tumor cells which have used gene amplification as a method of resistance to folate antagonists such as methotrexate. However, Dhfr gene expression in an unamplified cell may be different than in the amplified state. There- fore, Dhfr gene expression will also be studied in the unamplified state. It has previously been difficult to study Dhfr expression in unamplified cells, but the recent development of quantitative PCR to detect mRNA has made this feasible. The sensitivity of the PCR assay will allow the analysis of thiouridine pulsed labeled RNA separated on organomercurial columns, thereby enabling us to study both stable and newly made Dhfr RNA. Secondly, various changes will be made in the primary structure of the Dhfr gene. Yeast genetics will be employed to make changes in the mouse Dhfr gene contained in a YAC clone. These YAC constructs will be transferred to Dhfr-deficient CHO cells and Dhfr expression in normal and tumorigenic cells, and additional insight into the role of Dhfr gene expression in the development of drug resistance in neoplastic cell.