Cloning and functional expression of poly(ADP‐ribose) polymerase cDNA from Sarcophaga peregrina

Abstract

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 113033 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‐X2‐C‐X28‐H‐X2‐C and C‐X2‐C‐X31‐H‐X2‐C)2 and a basic region in the N‐terminal DNA‐binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine‐rich motif (C‐X2‐C‐X13‐C‐X9‐C), a putative zinc‐finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine‐zipper motif (l‐X6‐l‐X6‐l‐X6‐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. Copyright © 1994, Wiley Blackwell. All rights reserved