期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1982
卷号：79
期号：12
页码：3881-3885
DOI：10.1073/pnas.79.12.3881
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Five recombinant DNA plasmids have been constructed that contain structural gene sequences for rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2 ]. Rat pheochromocytoma PC 12 cell line, which contains relatively high levels of catecholamine-synthesizing enzymes, was used to purify RNA. TyrOHase cDNA clones were identified by screening 350 cDNA clones constructed from partially purified TyrOHase mRNA. A rapid and powerful screening of the recombinant clones by differential colony hybridization was possible because TyrOHase is a tissue-specific protein. The final selection relied on the ability of cDNA inserts to hybridize specifically to TyrOHase mRNA as judged by cell-free translation and immunoprecipitation. Blot hybridization analysis of polyadenylylated RNA from PC 12 cells indicated a major mRNA species of 1.9 kilobases. A species of the same size was identified from a human pheochromocytoma tumor, indicating a crossreactivity between rat TyrOHase cDNA and human TyrOHase mRNA.
