FLUOROPYRIMIDINES ON RNA STRUCTURE AND FUNCTION

研究了甲氨蝶呤对5-氟尿嘧啶代谢的影响。 广泛地。大多数暴露于甲氨蝶呤的细胞将积累更多 5-氟尿嘧啶的数量，主要是由于 甲氨蝶呤的抗嘌呤作用及随后的细胞内升高 磷酸核糖焦磷酸的量。这一机制通过它 然而，协同细胞杀伤发生在这个药物序列之后 不知道。可以调查的领域，以寻找原因 这种增加的细胞毒性包括抑制胸苷合成酶， FdUTP掺入DNA、FUDP-糖生产和FUTP掺入 变成了核糖核酸。5-氟尿嘧啶确实干扰RNA加工，然而 5-氟尿嘧啶的RNA效应的确切性质尚不清楚。最近的 基因拷贝数增加的细胞系的可用性 二氢叶酸还原酶，从而合成大量的mRNA 因为这种蛋白质提供了一种途径，通过这种方式5-氟尿嘧啶对 可以对mRNA的结构和功能进行评估。[~3H]的加入 5-氟尿嘧啶转化为RNA类(包括dhfr-mRNAs)及其作用 5-氟尿嘧啶对dhfr-mRNA的作用将通过凝胶电泳法进行检测。 C-DNA探针和S-1作图。可获得性交替的影响 通过抗代谢前处理的嘌呤和嘧啶碱也将 已评估。FUTP被整合到RNA中的速率，而不是总 FUTP公司的数量，我们已经发现与 停止合成RNA。我们计划使用详细的RNA研究作为 概述，希望定义RNA结构似乎对什么敏感 例如，这种快速掺入FUTP的是富含尿嘧啶的小RNA 物种主要受FUTP替代密度的影响，并将 从而导致二级和三级结构的变化。此外，我们还 将评估其他碱基替换对RNA结构和 通过所述的方法发挥作用。具体地说，嘌呤类似物将 被评估。

The effect of methotrexate on 5-fluorouracil metabolism has been studied extensively. Most cells exposed to methotrexate will accumulate greater quantities of 5-fluorouracil which is primarily the consequence of the antipurine effect of methotrexate and subsequent increased intracellular quantities of phosphoribosyl-pyrophosphate. The mechanism by which synergistic cell killing occurs following this drug sequence, however, is not known. The areas that can be investigated to search for the cause of this increased cytotoxicity include inhibition of thymidylate synthetase, FdUTP incorporation into DNA, FUDP-sugar production and FUTP incorporation into RNA. 5-Fluorouracil does interfer with RNA processing, however the precise nature of the RNA effect of 5-fluorouracil is inknown. The recent availability of cell lines which have increased gene copies of dihydrofolate reductase and therefore synthesize enormous amounts of mRNA for this protein provides a means by which the effect of 5-fluorouracil on mRNA structure and function can be evaluated. The incorporation of [3H] 5-fluorouracil into RNA classes (including DHFR-mRNA) and the effect of 5-fluorouracil on DHFR-mRNA will be evaluated by gel electrophetic methods, C-DNA probes and S-1 mapping. The effect of alternating the availability of purine and pyrimidine bases by antimetabolite pretreatment will also be evaluated. The rate at which FUTP is incorporated into RNA, not the total amount of FUTP incorporated, has been found by us to be associated with the cessation of RNA synthesis. We plan to use detailed RNA studies as outlined in hopes of defining what RNA structures appear to be sensitive to this rapid incorporation of FUTP, for example are small uracil rich RNA species primarily affected by the density of FUTP substitution, and will thus result in changes in secondary and tertiary structure. In addition we will evaluate the effects of other base substitution on RNA structure and function by the methods described. Specifically, the purine analogues will be evaluated.