Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.
Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.
复制标题
儿茶酚胺生物合成基因：通过表达和鉴定大鼠多巴胺β-羟化酶的cDNA进行克隆。
DOI：
10.1073/pnas.80.8.2161
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发表时间：
1983
影响因子：
11.1
通讯作者：
Kedes,L
中科院分区：
文献类型：
--
作者：
O'Malley,KL;Mauron,A;Raese,J;Barchas,JD;Kedes,L
多巴胺β-羟化酶[3，4-二羟基苯乙胺，抗坏血酸盐：氧氧化还原酶（β-羟化），EC 1.14.17.1]的mRNA已从大鼠嗜铬细胞瘤细胞系分离的poly（A）+ mRNA中部分纯化。酪氨酸羟化酶和多巴胺β-羟化酶之间共享的抗原决定簇使我们能够通过用酪氨酸羟化酶抗血清免疫沉淀翻译的mRNA产物来获得多巴胺β-羟化酶mRNA的富集部分。富集的多巴胺β-羟化酶mRNA用于合成相应的cDNA，然后将其克隆到pBR 322的Pst I位点。通过原位菌落免疫测定和杂交选择翻译来表征重组菌落。从一个重组克隆中选择的mRNA的体外翻译产生了与真正的多巴胺β-羟化酶共迁移的75，000道尔顿的蛋白质。真实的多巴胺β-羟化酶和重组克隆编码的蛋白质的部分蛋白水解产生相同的肽模式。
mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.