EFFECTS OF ANTIMETABOLITES ON RNA METABOLISM

本申请的目的是扩展以前的研究， 5-氟尿嘧啶（FUra）对RNA代谢的影响， 了解RNA介导的FURA细胞毒性效应。 本研究将 重点研究FURA诱导的两种mRNA代谢异常、前体mRNA剪接 mRNA半衰期 具体建议如下： 完成： 1. 应用聚合酶链反应定量稳态水平 二氢叶酸还原酶（DHFR）RNA剪接底物以及 前体mRNA加工途径中的剪接中间体，在FURA处理的 和对照细胞。 将该试验扩展至非基因扩增细胞， 它最终可以应用于体内。 2. 测量3.5kb DHFR mRNA在生长于培养皿中的细胞中的半衰期。 是否存在FURA。 评估是否增加半衰期后， 在含有FUra的培养基中的细胞生长是对先前的 观察到这种特异性DHFR mRNA的增加。 识别3'非编码 存在于3.5 kb DHFR mRNA中的元件，其似乎介导该效应 的半衰期缺失作图和DNA转染。 3. 氟尿嘧啶预处理对甲氨蝶呤细胞毒性的影响 表达3'非编码区改变的mRNA的细胞， mRNA半衰期与DHFR药物反应性的改变。

The purpose of this application is to extend previous studies of the effects of 5-fluorouracil (FUra) on RNA metabolism in an effort to better understand the RNA mediated effects of FUra cytotoxicity. This study will focus on two FUra induced aberrations of mRNA metabolism, pre-mRNA splicing and mRNA half life. It is specifically proposed that the following be accomplished: 1. Apply the polymerase chain reaction to quantitate steady state levels of dihydrofolate reductase (DHFR) RNA splicing substrates as well as splicing intermediates in the pre-mRNA processing pathway, in FUra treated and control cells. Extend this assay to non-gene amplified cells so that it may eventually be applied in vivo. 2. Measure the half life of 3.5 kb DHFR mRNA in cells grown in the presence or absence of FUra. Assess whether an increased half-life after cell growth in medium containing FUra is an explanation for the previously observed increase in this specific DHFR mRNA. Identify the 3' noncoding element(s) present in 3.5 kb DHFR mRNA which appear to mediate this effect of FUra on half-life deletion mapping and DNA transfection. 3. Study the effect of FUra pretreatment on methotrexate cytotoxicity in cells expressing mRNAs with altered 3' noncoding regions to correlate alterations in mRNA half life with DHFR drug responsiveness.