title: Human poly(ADP-ribose) glycohydrolase (PARG) gene and the common promoter sequence it shares with inner mitochondrial membrane translocase 23 (TIM23).
abstract: Poly(ADP-ribosyl)ation is a posttranslational protein modification mediated by members of the poly(ADP-ribose) polymerase (PARP) family. The ADP-ribose polymers, synthesized by the diverse PARP enzymes by cleavage of NAD(+), are involved in the regulation of multiple cellular functions. At present, only a single enzyme, poly (ADP-ribose) glycohydrolase (PARG), has been identified to catalyze ADP-ribose polymer hydrolysis in the cell causing a rapid turnover of the biopolymer which may ultimately result in lethal depletion of cellular NAD(+) pools. In this study, we describe the construction of the first human PARG cDNA clone by reverse transcription of CF3 human fibroblast RNA. Using the NCBI "Genome BLAST" program, the human PARG gene was mapped to chromosome 10 (10q11.23) in agreement to earlier results obtained by in situ hybridization. In vitro coupled transcription and translation of the cDNA yielded several specific bands in the range of 111-85 kDa, indicating possible usage of alternative translation initiation sites. The gene structure was characterized by further detailed computational analyses. The open reading frame consists of 18 exons and 17 introns with exons 9 to 14 forming the catalytic center of the enzyme and exons 1 to 3 encoding the putative regulatory domain. We show that the human PARG gene shares a 470-bp common promoter region with the inner mitochondrial membrane translocase 23 (TIM23). The human bidirectional promoter region was cloned and expression studies in transiently transfected HEK293 cells was performed using an EGFP-luciferase reporter fusion gene (GFL) to quantify transcription activation in both directions. The activity of the promoter was found to be 3.7 fold higher for TIM23 than for PARG, indicating that the two genes are expressed at different levels, although coregulation of the two genes remains an interesting possibility.
authors.affiliation: Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Room 4943 Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724, USA.
authors.forename: Ralph G
authors.initials: RG
authors.lastname: Meyer
authors.forename: Mirella L
authors.initials: ML
authors.lastname: Meyer-Ficca
authors.forename: Elaine L
authors.initials: EL
authors.lastname: Jacobson
authors.forename: Myron K
authors.initials: MK
authors.lastname: Jacobson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005796:Genes / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011993:Recombinant Fusion Proteins; C438387:TIMM23 protein, human; D008156:Luciferases; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase