REGULATION AND CLONING OF D HYDEI TYROSINE AMINOTRANSFER

D 型酪氨酸氨基转移的调控和克隆

• 批准号: 3286901
• 负责人: THOMAS E BRADY
• 金额: $ 16.4万
• 依托单位: HAMILTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1987-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286901
• 关键词: RNA; chromosome aberrations; electron microscopy; endonuclease; enzyme induction /repression; gel electrophoresis; genetic manipulation; genetic mapping; genetic regulation; genetic translation; immunofluorescence technique; immunoprecipitation; messenger RNA; molecular cloning; nucleic acid structure; pyridoxine; tissue /cell culture; tyrosine transaminase

It is proposed that the clones which have already been generated in PBR322 to total immunoprecipitated TAT RNA from Drosophila hydei polytene tissue be in situ hybridized to D. hydei and D. melanogaster polytene chromosomes to determine whether any of these clones contain the TAT gene. The TAT gene is located at band II-48C in D. hydei salivary polytene chromosomes. Poly A TAT mRNA will be isolated, in vitro translated and if the above clones do not contain the TAT gene they will be used for cDNA synthesis and the cDNA used for cloning. The cloned TAT cDNA will be used as probes to isolate the native gene from genomic libraries of D. melanogaster and D. hydei. The role of pyridoxine in the induction of the TAT gene will be examined using the cloned TAT genes as well as with indirect immunofluorescence to the salivary polytene chromosomes using antibodies prepared against pyridoxine and deoxpyridoxine. A series of ebony mutants (located at the II-48 band) will be examined for alteration in TAT activity as well as for alterations in electrophoretic mobility. Finally seeing that the D. hydei TAT antibody cross reacts with rat liver homogenates we will perform a series of preliminary experiments to determine whether the D. hydei TAT clones may be used to isolate the TAT gene from libraries of rat DNA.

建议将已经在PBR 322中产生的克隆 与果蝇多线期组织中总免疫沉淀的达特RNA 与D. hydei和D.黑腹多线染色体 以确定这些克隆中是否含有达特基因。 的达特 基因定位于D.多线染色体 将分离Poly A达特mRNA，体外翻译，如果上述结果不正确， 克隆不含达特基因，它们将用于cDNA合成， 用于克隆的cDNA。 克隆的达特cDNA将用作探针， 从D.黑腹果蝇D. 海迪。 吡哆醇在诱导达特基因中的作用将被 使用克隆的达特基因以及间接的 使用抗体对唾液多线染色体的免疫荧光 针对吡哆醇和脱氧吡哆醇制备。 一系列乌木变种人 将检查（位于II-48带）是否存在达特活性的变化 以及电泳迁移率的改变。 终于看到 该D. hydei达特抗体与大鼠肝匀浆交叉反应， 将进行一系列初步实验，以确定 D. hydei达特克隆可用于从以下文库中分离达特基因： 老鼠的DNA