GENETIC REGULATION OF PYRIDINE DEHYDROGENASES

吡啶脱氢酶的基因调控

• 批准号: 3438552
• 负责人: JOHN H. WILLIAMSON
• 金额: $ 5万
• 依托单位: DAVIDSON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438552
• 关键词: affinity chromatography; complementary DNA; cytogenetics; endonuclease; gel electrophoresis; gene expression; genetic regulation; genetic transcription; immunoprecipitation; messenger RNA; molecular cloning; molecular genetics; nucleic acid sequence; spectrometry

Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme share a common pyridine nucleotide (NADP) as coenzyme. Activities of these enzymes are coordinated to generate adequate pools of NADPH for lipid biosyntheses. Biochemical comparisons of the parameters of pyridine-linked dehydrogenases suggest a common evolutionary origin of these enzymes. In Drosophila melangaster there appears to be two forms of NADP-isocitrate dehyrogenase and there may be two genes controlling the activity of this enzyme. A molecular characterization of this gene(s) and the transcript(s) made from it is proposed as the initial step in understanding regulation of this gene-enzyme system. Expression libraries of Drosophila cDNA will be screened for sequences specific for IDH. The identity of these cloned sequences will be verified by in situ hybridization to salivary gland polythene chromosomes and immunoprecipitation of in vitro translated hybrid selected mRNA. Clones which are identified by these techniques will be used to isolate corresponding sequences of genomic DNA. Restriction analyses will compare cDNA and genomic sequences. The developmental expression of these sequences will be determined by Southern analysis. Hybridization of strand specific transcripts of cDNA to "Northern" blots of appropriately staged pupal mRNA will allow determiniation of the orientation of transcription. Future experiments would include S1 nuclease mapping and primer extension analysis.

葡萄糖6-磷酸脱氢酶 脱氢酶、异柠檬酸脱氢酶和苹果酸酶份额 一种常见的吡啶核苷酸（NADP）作为辅酶。 活动 这些酶被协调以产生足够的 NADPH用于脂质生物合成。 生化比较 吡啶连接的酶的参数表明一个共同的 这些酶的进化起源。 在黑腹果蝇中， NADP-异柠檬酸脱氢酶可能有两个基因 控制这种酶的活性。 分子 该基因和由其制备的转录物的表征 建议作为理解这一规则的第一步， 基因-酶系统 果蝇cDNA表达文库 将筛选IDH特异性序列。 的身份 这些克隆的序列将通过原位杂交进行验证， 唾液腺聚乙烯染色体和免疫沉淀 体外翻译的杂交选择的mRNA。 克隆体， 这些技术将用于分离 相应的基因组DNA序列。 限制分析 将比较cDNA和基因组序列。 发育 这些序列的表达将通过Southern杂交确定。 分析. 将cDNA的链特异性转录物杂交至 适当分期的蛹mRNA的"北方"印迹将允许 确定转录方向。 未来 实验将包括S1核酸酶图谱和引物 可拓分析