MOUSE DIHYDROFOLATE REDUCTASE GENE EXPRESSION

这项建议的主要重点是研究对 二氢叶酸还原酶(DHFR)基因表达的变化 细胞的增殖状态。稳态水平和稳定性 将在生长过程中分析不同种类的小鼠dhfr mRNA 放大和非放大状态下的刺激和静止。 使用具有dhfr基因扩增副本的细胞系有几个 优点：i)由于dhfr水平升高，很容易检测到dhfr基因。 在扩增的细胞中的表达和ii)在扩增的 国家扩大了我们对其在肿瘤细胞中的调控的理解 利用基因扩增作为抵抗叶酸的一种方法 甲氨蝶呤等拮抗剂。然而，DHFR基因在一种 未扩增的细胞可能与扩增状态的细胞不同。在那里- 因此，DHFR基因的表达也将在未扩增的状态下进行研究。 以前很难研究dhfr在未扩增的 细胞，但最近发展的定量聚合酶链式反应检测mRNA 使之成为可能。聚合酶链式反应分析的敏感性将使 有机汞分离的硫代尿苷脉冲标记RNA的分析 柱，从而使我们能够研究稳定的和新制造的DHFR 核糖核酸。其次，将在初级结构上做出各种变化 Dhfr基因。酵母遗传学将被用来改变 YAC克隆中包含小鼠dhfr基因。这些YAC构造将是 转移至DHFR缺陷型CHO细胞及其在正常和 致瘤细胞和对dhfr基因作用的进一步认识 在肿瘤细胞耐药发展中的表达。

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.