ANALYSIS OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION

二氢叶酸还原酶基因表达分析

• 批准号: 3271244
• 负责人: Lawrence Allen Chasin
• 金额: $ 29.8万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271244
• 关键词: RNA splicing; X ray; aminoacid; dihydrofolate reductase; drug resistance; enzyme mechanism; gel electrophoresis; gene expression; gene mutation; genetic mapping; genetic markers; genetic transcription; genetic translation; hamsters; linkage mapping; messenger RNA; molecular cloning; mutagens; nucleic acid sequence; radiotracer; site directed mutagenesis; temperature sensitive mutant; tissue /cell culture; transcription factor; transfection; transposon /insertion element; tritium; ultraviolet radiation

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.

这个建议围绕着二氢叶酸还原酶的使用