ABSTRACT
A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.
Subject(s)
Phosphoric Diester Hydrolases/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 6 , Cloning, Molecular , DNA, Complementary , Humans , Molecular Sequence Data , Phosphoric Diester Hydrolases/chemistry , RNA Splicing , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino AcidABSTRACT
Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.
Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Aorta/chemistry , Aorta/cytology , Aorta/metabolism , Base Sequence , Blotting, Northern , Cattle , Chromosome Mapping , Chromosomes, Human, Pair 4/genetics , Cyclic Nucleotide Phosphodiesterases, Type 5 , DNA, Complementary/chemistry , Gene Expression/genetics , Genetic Variation/genetics , Humans , Molecular Sequence Data , Muscle, Smooth/chemistry , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
A type II restriction endonuclease, named CviJI, was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA containing virus IL-3A. CviJI is the first restriction endonuclease to recognize the sequence PuGCPy; CviJI cleaves DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. CviJI cleaved DNA into smaller but defined fragments in the presence of ATP. This "star" activity was stimulated by dithiothreitol and/or S-adenosylmethionine but did not occur under conditions which favor "star" activity of other restriction endonucleases.
Subject(s)
Chlorella/enzymology , DNA Restriction Enzymes/analysis , Genes, Viral , Adenosine Triphosphate/pharmacology , Base Sequence , DNA/analysis , DNA Restriction Enzymes/isolation & purification , Magnesium/pharmacology , Methylation , Nucleotide MappingABSTRACT
An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.
