CLONING AND FUNCTIONAL EXPRESSION OF POLY(ADP-RIBOSE) POLYMERASE CDNA FROM SARCOPHAGA-PEREGRINA

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113 033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X(2)-C-X(28)-H-X(2)-C and C-X(2)-C-X(31)-H-X(2)-C)(2) and a basic region in the N-terminal DNA-binding domain recognized in other PARR are conserved. Downstream of the basic region, another cysteine-rich motif (C-X(2)-C-X(13)-C-X(9)-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X(6)L-X(6)-L-X(6)-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.