Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.
By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.
Yoshihara
K
K
Department of Biochemistry, Nara Medical University, Japan.
Itaya
A
A
Hironaka
T
T
Sakuramoto
S
S
Tanaka
Y
Y
Tsuyuki
M
M
Inada
Y
Y
Kamiya
T
T
Ohnishi
K
K
Honma
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016172:DNA Fingerprinting; D015342:DNA Probes; D005822:Genetic Vectors; D006801:Humans; D007939:Leukemia L1210 / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D015342:DNA Probes; D011065:Poly(ADP-ribose) Polymerases