ANALYSIS OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION

该提案围绕二氢叶酸还原酶的使用展开。 模式哺乳动物中国仓鼠卵巢细胞(CHO)的(DHFR)基因座 基因对于体内基因表达的突变扰动。我们的 最近的研究表明，无义突变之间存在着一种奇怪的关系 和RNA处理。我们计划测试一个将翻译与 利用外显子特异性从细胞核剪接和输出信使核糖核酸 蛋白质合成抑制剂(氨基醇)及RNA分析 离体核中的新陈代谢。有几个项目的重点是镇压，因为 一种揭示迄今未知功能的基因的方法 转录和拼接。干扰转录的突变或 剪接将在体外被引入到DHFR微型基因中。之后 将微型基因转移到DHFR缺乏的宿主中，即转染体 克隆将被诱变并选择用于dhfr基因的返回 被压制的活跃度。第二个位点突变的抑制应该是 揭示有助于理解DNA和/或RNA的顺式相互作用 细胞内的构象。外部抑制因子将定义基因 反式作用转录和/或剪接因子。这些基因将 然后根据它们抑制dhfr的能力进行克隆- 缺乏表型。另一个项目针对的是诱变 供体/受体剪接位点对。整合的dhfr基因包含一种 插入内含子的可选标记将用于分离所有 可能导致剪接中断的单碱基变化。这种饱和度 突变应该提供碱基是什么的详细图像 在基因组中原位产生的转录本的剪接所必需的。 这样的信息可能有助于形成RNA结构的模型，该模型 在拼接中发挥作用。在其他项目中，由框架诱导的突变 将从低水平的DHFR酶活性中筛选出转移诱变剂； 基因蛋白质编码区的突变。放射性氨基 酸将被用作针对dhfr基因区域的诱变剂 结合特定的蛋白质。最后，一系列错义突变体和 将收集返回体以探讨DHFR酶的作用机制 活动和结构。

This proposal revolves around the use of the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells as a model mammalian gene for the in vivo mutational perturbation of gene expression. Our recent work has shown a curious relationship between nonsense mutations and RNA processing. We plan to test a model linking translation to the splicing and export of mRNA from the nucleus, using exon-specific protein synthesis inhibitors (amino alcohols), and analysis of RNA metabolism in isolated nuclei. Several projects focus on suppression as a means to reveal genes for hitherto unknown functions related to transcription and splicing. Mutations that disrupt transcription or splicing will be introduced into a dhfr minigene in vitro. After transfer of the minigene into a DHFR-deficient host, the transfectant clones will be mutagenized and selected for the return of dhfr gene activity by suppression. Suppression by second site mutations should reveal cis interactions that will aid in understanding DNA and/or RNA conformations in the cell. External suppressors will define genes for trans-acting transcriptional and/or splicing factors. These genes will then be cloned on the basis of their ability to suppress the DHFR- deficient phenotype. Another project is directed at the mutagenesis of a donor/acceptor splice site pair. An integrated dhfr gene containing a selectable marker inserted into an intron will be used to isolate all possible single base changes that disrupt splicing. This saturation mutagenesis should provide a detailed picture of what bases are necessary for splicing of transcripts generated in situ in the genome. Such information may aid in formulating models of RNA structures that play a role in splicing. In other projects, mutants induced by a frame shift mutagen will be screened fro low level DHFR enzyme activity; such mutants in the protein coding region of the gene. Radioactive amino acids will be used as mutagens targeted to regions of the dhfr gene that bind specific proteins. Finally, a series of missense mutants and revertants will be collected to probe the mechanism of DHFR enzyme activity and structure.