Repaske D R, Swinnen J V, Jin S L, Van Wyk J J, Conti M
Division of Pediatric Endocrinology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1992 Sep 15;267(26):18683-8.
Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.
环核苷酸磷酸二酯酶（PDEs）的多种同工酶在哺乳动物组织中同时表达。为了鉴定和克隆这些PDEs，开发了一种聚合酶链反应（PCR）策略，使用设计用于与高度保守的PDE DNA结构域杂交的简并寡核苷酸引物。从大鼠肝脏、小鼠K30a - 3.3淋巴瘤细胞系和人下丘脑cDNA文库中克隆出了已知和新型的PDEs，表明这些PCR引物可用于扩增多种PDE同工酶的cDNA。发现一个独特的小鼠PDE克隆编码的多肽与配套文章中报道的牛脑63 kDa钙调蛋白依赖性PDE的相应部分相同（Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676 - 18682）。这个小鼠克隆用作探针，从大鼠脑cDNA文库中筛选全长克隆。所得大鼠克隆的核苷酸序列的概念性翻译有一个535个氨基酸的开放阅读框，与牛63 kDa钙调蛋白依赖性PDE保持高度同源性，表明该蛋白可能是63 kDa钙调蛋白依赖性PDE的大鼠同源物。全长克隆在大肠杆菌中的表达产生了一种cGMP水解活性，该活性受到钙调蛋白的数倍刺激。Northern印迹分析表明，编码这种PDE的mRNA在大鼠脑中以及S49.1 T淋巴细胞系中高度表达。这些数据表明，所述的PCR方法是分离不同家族PDE同工酶已知和新成员cDNA克隆的可行策略。这些PDEs的分子克隆将为研究这些同工酶在调节细胞内环核苷酸浓度中的作用提供有价值的工具。
