Deoxycytidylate hydroxymethylase gene of bacteriophage T4
- 3 March 1988
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 172 (3), 553-563
- https://doi.org/10.1111/j.1432-1033.1988.tb13925.x
Abstract
We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.This publication has 53 references indexed in Scilit:
- Studies on transformation of Escherichia coli with plasmidsJournal of Molecular Biology, 1983
- Molecular cloning of the DNA ligase gene from bacteriophage T4Journal of Molecular Biology, 1979
- Molecular cloning of the DNA ligase gene from bacteriophage T4Journal of Molecular Biology, 1979
- T4 phage-coded deoxycytidylate hydroxymethylase: Purification and studies on intermolecular interactionsBiochemical and Biophysical Research Communications, 1977
- Screening λgt Recombinant Clones by Hybridization to Single Plaques in SituScience, 1977
- Simple Isolation Method and Assay for T4 DNA Ligase and Characterization of the Purified EnzymeEuropean Journal of Biochemistry, 1977
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- The N-Terminal Amino-Acid Sequences of DNA Polymerase I from Escherichia coli and of the Large and the Small Fragments Obtained by a Limited ProteolysisEuropean Journal of Biochemistry, 1974
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- The enzymic synthesis of 5-hydroxymethyldeoxycytidylic acidBiochimica et Biophysica Acta, 1957