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-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.
Masutani
M
M
Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan.
Nozaki
T
T
Hitomi
Y
Y
Ikejima
M
M
Nagasaki
K
K
de Prati
A C
AC
Kurata
S
S
Natori
S
S
Sugimura
T
T
Esumi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D004330:Drosophila / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases