Assignment<footref rid="foot01">¹</footref> of the poly(ADP-ribose) glycohydrolase gene (PARG) to human chromosome 10q11.23 and mouse chromosome 14B by in situ hybridization

The metabolism of polymers of ADP-ribose (ADP-Rib), a rapid cellular response to DNA damage, involves the concerted action of poly-ADPR polymerase (PARP) and poly-ADPR glycohydrolase (PARG). We have recently described the isolation and characterization of bovine cDNA encoding poly(ADP-ribose) glycohydrolase (PARG) Lin et al. (1997). Surprisingly, this cDNA codes for a protein of 111 kDa, nearly twice the size of the isolated protein from bovine thymus. More recently, we have cloned the human and mouse PARG cDNAs (manuscript in preparation) and analysis of the protein sequences shows that mammalian PARGs are highly conserved proteins constituted by two main domains, a catalytic domain of 65 kDa at the C-terminus and a domain of regulation of 45 kDa located at the N-terminus. Southern hybridization experiments indicated that PARG is encoded by a single copy gene. Thus, mapping of the human and mouse PARG genes may provide important insights to facilitate the study of many, presently unanswered questions pertaining to the biological significance of PARG and the pathway in which it is involved. Chromosomes were prepared from human peripheral blood lymphocyte cultures after BrdU incorporation during the last 7 h before harvesting. Mouse chromosomes were prepared from normal mouse fibroblast cultures. A 2.7-kb fragment of human PARG in vector Lafmid BA (EST H17106) and a 2.8-kb fragment of murine PARG in vector pTZ18R were labeled by nick-translation with biotin-11-dUTP (Sigma, France) and used as probes for FISH. A standard hybridization was performed as described previously (Apiou et al., 1996). The mouse PARG probe was used at a concentration of 15 ng/µl in the presence of 100 fold excess of mouse Cot-1 DNA. The human PARG probe was used at a concentration of 20 ng/µl in 15 µl of hybridization buffer for each slide. For human chromosomes, direct banding of BrdU-substituted chromosomes was obtained by incubation in an alkaline solution of p-phenylenediamine, (PPD11) (Lemieux et al., 1992) and stained with propidium iodide. Mouse chromosomes were stained with DAPI and identified with computer generated reverse DAPI banding. Immunochemical detection of hybridization was performed using goat antibiotin antibodies (Vector laboratories, Burlingame, CA) and rabbit FITC conjugated anti-goat antibodies (Biosys, Compiègne, France). Metaphases were observed under a fluorescent microscope (DMRB, Leica, Germany). Images were captured using a cooled photometrics CCD camera and Quips-smart capture software (Vysis).Probe name: hPARG2.7 human, mPARG2.8 mouseProbe type: cDNAInsert size: 2.7 kb human; 2.8 kb mouseVector: pTZ18R, and Lafmid BAGene reference: GeneBank AF005043 human and AF079557 mouseLocation:10qNumber of cells examined: 25Number of cells with specific signals: 20Most precise assignment:10q11.23Location of background signals (sites with >2 signals): none observedLocation:14Number of cells examined:25Number of cells with specific signals:19Most precise assignment:14BLocation of background signals (sites with >2 signals): none observed (fig. 1)Supported by Fondation pour la Recherche Médicale and by NIH Grant CA43894.