1. Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A.
- Author
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Narva KE, Wendell DL, Skrdla MP, and Van Etten JL
- Subjects
- Amino Acid Sequence, Base Sequence, Chlorella enzymology, DNA (Cytosine-5-)-Methyltransferases isolation & purification, DNA Mutational Analysis, Escherichia coli genetics, Lac Operon, Molecular Sequence Data, Plant Viruses genetics, Recombinant Fusion Proteins isolation & purification, T-Phages genetics, Chlorella genetics, Cloning, Molecular methods, DNA (Cytosine-5-)-Methyltransferases genetics, Genes
- Abstract
The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E. coli plasmid pUC8. Plasmid (pNC-1A.14.8) encoded M.CviBIII methylates adenine in TCGA sequences both in vivo in E. coli and in vitro. Transposon Tn5 mutagenesis localized the M.CviBIII functional domain to a 1.5 kbp region of pNC-1A.14.8 and also indicated that a virus promoter directs transcription of the gene in E. coli. The 2.1 kbp insert containing the M.CviBIII gene was sequenced and a single open reading frame of 1131 bp was identified within the domain determined by Tn5 mutagenesis. When the M.CviBIII gene was fused in-frame with the 19 amino-terminal codons of lacZ a 45 kD polypeptide was identified in maxicells as predicted by the DNA sequence. The M.CviBIII gene was not essential for virus replication since a virus M.CviBIII deletion mutant also replicated in Chlorella.
- Published
- 1987
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