Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene.
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.
Lamm
N
N
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität Bochum, Federal Republic of Germany.
Wang
Y
Y
Mathews
C K
CK
Rüger
W
W
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D005786:Gene Expression Regulation; D005796:Genes; D005815:Genetic Code; D019877:Hydroxymethyl and Formyl Transferases; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000032:analysis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D014166:Transferases / Q000032:analysis / Q000235:genetics*
D010454:Peptide Termination Factors; D004247:DNA; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C100226:deoxycytidylate hydroxymethyltransferase