Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1).
Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)
Sullivan
M
M
Preclinical Research, Sandoz Pharma Ltd, Basel, Switzerland.
Egerton
M
M
Shakur
Y
Y
Marquardsen
A
A
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000302:isolation & purification; D015870:Gene Expression; D000882:Haplorhini; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009504:Neutrophils / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology
D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D007527:Isoenzymes; D015105:3',5'-Cyclic-AMP Phosphodiesterases