Bentley J K, Kadlecek A, Sherbert C H, Seger D, Sonnenburg W K, Charbonneau H, Novack J P, Beavo J A
Department of Pharmacology, University of Washington, Seattle 98195.
J Biol Chem. 1992 Sep 15;267(26):18676-82.
Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.
基于肽序列的部分简并寡核苷酸被用于分离编码63 kDa牛脑钙调蛋白刺激的磷酸二酯酶（CaM-PDE）同工酶的cDNA。获得了一个412碱基对的聚合酶链反应片段，并将其与寡核苷酸一起用于分离几个cDNA，每个cDNA编码的序列与63 kDa CaM-PDE的已知肽序列相同。最大的cDNA包含一个全长开放阅读框（ORF），编码一个534个氨基酸、61,005道尔顿的蛋白质。它与61 kDa牛脑CaM-PDE有59%的氨基酸同一性，并且包括一个含有PDE催化结构域共有序列的羧基末端保守结构域。氨基末端区域符合钙调蛋白结合结构域的标准。当它在pCDM8载体上由巨细胞病毒启动子驱动在COS-7细胞中表达时，该cDNA编码一种具有催化活性、受钙调蛋白刺激的PDE。用一个包含大部分保守PDE催化结构域的探针，对来自几个组织的RNA进行Northern分析，结果显示只有一条4.0千碱基的条带。在中枢神经系统几个区域的mRNA中可见杂交信号，在基底神经节中信号最强。在包括肾乳头和肾上腺髓质在内的其他组织中也可见强信号。反义RNA探针用于核糖核酸酶保护试验，以寻找多种63 kDa CaM-PDE转录本的证据。来自大脑皮层、基底神经节、小脑、海马体、肾上腺髓质和肾乳头的RNA完全保护了一个催化结构域探针。然而，一个针对氨基末端区域的探针仅被脑和肾上腺髓质RNA完全保护，这表明肾乳头中该区域可能存在一种或多种不同的同工酶。
