RESUMEN
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.
Asunto(s)
Hidrolasas Diéster Fosfóricas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 6 , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/química , Empalme del ARN , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Aorta/química , Aorta/citología , Aorta/metabolismo , Secuencia de Bases , Northern Blotting , Bovinos , Mapeo Cromosómico , Cromosomas Humanos Par 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , ADN Complementario/química , Expresión Génica/genética , Variación Genética/genética , Humanos , Datos de Secuencia Molecular , Músculo Liso/química , Músculo Liso/citología , Músculo Liso/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , GMP Cíclico/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario , Activación Enzimática , Humanos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Genetically engineered fibroblasts have been successfully used to produce therapeutic proteins in animals, but sustained production of the proteins has not been achieved. This limits the potential of fibroblast-mediated gene therapy in humans. We have studied the phenomenon of decreased production in rats by using retroviral vectors carrying genes encoding human adenosine deaminase and neomycin phosphotransferase. While transplanted skin fibroblasts containing vector sequences persisted at constant levels for at least 8.5 mo, vector expression decreased by greater than 1500-fold after 1 mo. Cellular or antibody-mediated immune responses were not detected in transplanted animals, and expression could not be restored in fibroblasts recultivated from the grafts. This phenomenon is reminiscent of sequence-specific gene inactivation observed in other cell types. Because genetic manipulation and expression of foreign proteins did not affect survival of the transplanted cells, effective long-term therapy may be possible with the use of alternative gene regulatory elements.
Asunto(s)
Adenosina Desaminasa/genética , Fibroblastos/trasplante , Ingeniería Genética , Trasplante de Piel/fisiología , Piel/citología , Adenosina Desaminasa/metabolismo , Animales , Secuencia de Bases , Supervivencia Celular , Células Cultivadas , ADN/genética , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/fisiología , Terapia Genética , Vectores Genéticos , Supervivencia de Injerto , Humanos , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas F344 , Secuencias Repetitivas de Ácidos Nucleicos , Virus 40 de los Simios/genética , Trasplante de Piel/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.
