PMID:1512207

Citation	Tseng, MJ, Hilfinger, JM, He, P and Greenberg, GR (1992) 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)). J. Bacteriol. 174:5740-4
Abstract	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.
Links	PubMed PMC206523
Keywords	Base Sequence; Cloning, Molecular; Genes, Viral; Genetic Vectors; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides/chemistry; Recombinant Proteins/isolation & purification; Ribonucleoside Diphosphate Reductase/genetics; Ribonucleoside Diphosphate Reductase/isolation & purification; T-Phages/enzymology; T-Phages/genetics; Viral Structural Proteins/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:RIR1		GO:0004748: ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor	ECO:0000314:		F		Both nrdA and nrdB were cleaved out of T4 phage genome. They were then put into a plasmid vector (pnrdAB)and implanted into E. coli N4830. Expression was induced by increasing temperature. Ribonucleoside diphosphate reductase was observed to increase in the E. coli strains containing the pnrdAB vector.	complete CACAO 10953
BPT4:RIR1	enables	GO:0004748: ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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