Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain.
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.
Bentley
J K
JK
Department of Pharmacology, University of Washington, Seattle 98195.
Kadlecek
A
A
Sherbert
C H
CH
Seger
D
D
Sonnenburg
W K
WK
Charbonneau
H
H
Novack
J P
JP
Beavo
J A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004247:DNA; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D015105:3',5'-Cyclic-AMP Phosphodiesterases