Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93)).
To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products. Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector. The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase. Phage T4 nrdB93, described by Wirak et al. (D. O. Wirak, K. S. Cook, and G. R. Greenberg, J. Biol. Chem. 263:6193-6201, 1988) contains a lesion in exon II of the gene. The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein. An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene.
Tseng
M J
MJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Hilfinger
J M
JM
He
P
P
Greenberg
G R
GR
D001483:Base Sequence; D003001:Cloning, Molecular; D005814:Genes, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000302:isolation & purification; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D012262:Ribonucleoside Diphosphate Reductase