REGULATION AND CLONING OF D HYDEI TYROSINE AMINOTRANSFER

建议将已在PBR322中生成的克隆 从海岛果蝇多线组织中提取总免疫沉淀TAT RNA 与海德氏和黑腹蛇多线染色体进行原位杂交 以确定这些克隆中是否有包含TAT基因的。纹身 该基因位于海藻唾液多线染色体的第II-48C带。 将Poly A Tat mRNA分离，体外翻译，如果上述 克隆不包含TAT基因，它们将用于cdna合成和 用于克隆的cdna。克隆的TAT基因将作为探针用于 从黑腹盘藻和黑腹盘藻基因组文库中分离出天然基因。 海迪亚。吡哆醇在诱导TAT基因中的作用将是 用克隆的TAT基因和间接检测 用抗体对唾液多线染色体进行免疫荧光 以吡哆醇和脱氧吡哆醇为对照制备。一系列乌木突变体 (位于II-48频段)将检查TAT活动的变化 以及电泳迁移率的变化。终于看到了 海德氏丝虫Tat抗体与大鼠肝匀浆发生交叉反应 将进行一系列初步实验，以确定 海藻Tat克隆可用于从日本血吸虫文库中分离Tat基因 老鼠的DNA。

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.