ANALYSIS OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION

这项建议围绕着使用二氢叶酸还原酶 （dhfr）基因座在中国仓鼠卵巢（CHO）细胞中作为模型哺乳动物 基因表达的体内突变扰动。 我们 最近的研究表明，无义突变之间存在着一种奇怪的关系， RNA加工 我们计划测试一个将翻译与 剪接和出口的mRNA从细胞核，使用外显子特异性 蛋白质合成抑制剂（氨基醇）和RNA分析 在孤立的细胞核中进行代谢。 几个项目的重点是抑制， 一种揭示基因未知功能的方法， 转录和剪接。 破坏转录的突变或 剪接将在体外引入DHFR小基因。 后 将小基因转移到DHFR缺陷型宿主中， 克隆将被诱变并选择用于dhfr基因的返回。 通过镇压活动。 第二个位点突变的抑制应该 揭示顺式相互作用，有助于理解DNA和/或RNA 细胞中的构象。 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， 缺陷型 另一个项目是针对 供体/受体剪接位点对。 一个整合的dhfr基因， 插入内含子的选择标记将用于分离所有 可能的单碱基改变破坏剪接。 这种饱和 诱变应该提供一个详细的图片，什么碱基是 基因组中原位产生的转录物剪接所必需的。 这些信息可以帮助制定RNA结构的模型， 起到拼接的作用。 在其他项目中，由框架诱导的突变体 将筛选低水平DHFR酶活性转换诱变剂; 基因的蛋白质编码区中的突变体。 放射性氨基 酸将用作靶向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 将收集回复突变体以探索DHFR酶的机制 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.