Isolation and characterization of the rat tryptophan oxygenase gene.
Isolation and characterization of the rat tryptophan oxygenase gene.
复制标题
大鼠色氨酸加氧酶基因的分离和表征。
作者：
W. Schmid;G. Scherer;Ulrich Danesch;H. Zentgraf;Patrick Matthias;Carolyn M. Strange;Walter Rowekamp;Gunther Schuitz
来自大鼠肝脏的色氨酸加氧酶（TO，EC 1.13.1.12）受到糖皮质激素和发育控制。为了研究调控机制，克隆了TO mRNA序列和染色体TO基因。从从富含TO mRNA的大鼠肝脏poly（A）+ RNA制备的cDNA文库中，通过杂交选择的RNA的翻译和用针对TO的抗体的免疫沉淀来鉴定含有TO cDNA序列的重组质粒。该cDNA克隆与地塞米松可诱导的2000个碱基长的mRNA杂交。用该克隆作为探针，我们从噬菌体λ大鼠DNA文库中分离出基因组克隆，它们一起跨越32个内切酶对（kb）的区域。异源双链分析表明，该基因延伸超过19 kb，并被至少11个内含子中断。为了表征推定的控制区，测定了TO基因5′端周围的DNA序列。S1核酸酶保护实验揭示了该区域内TO mRNA转录的两个独立的起始位点。
Tryptophan oxygenase (TO, EC 1.13.1.12) from rat liver is subject to glucocorticoid and developmental control. To study the mechanism of regulation, TO mRNA sequences and the chromosomal TO gene were cloned. From a cDNA library prepared from rat liver poly(A)+ RNA enriched for TO mRNA, a recombinant plasmid containing TO cDNA sequences was identified by translation of hybrid‐selected RNA and immunoprecipitation with antibodies directed against TO. This cDNA clone hybridizes to a mRNA 2000 bases long that is inducible by dexamethasone. With this clone as probe we isolated from a bacteriophage lambda rat DNA library genomic clones which together span a region of 32 kilobase pairs (kb). Heteroduplex analysis revealed that the gene extends over 19 kb and is interrupted by at least 11 introns. To characterize the presumptive control region the DNA sequence around the 5′ end of the TO gene was determined. S1 nuclease protection experiments revealed two separate start sites for TO mRNA transcription within this region.