Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A.
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.
Narva
K E
KE
Department of Plant Pathology, University of Nebraska, Lincoln 68583-0722.
Wendell
D L
DL
Skrdla
M P
MP
Van Etten
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002708:Chlorella / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D007763:Lac Operon; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013604:T-Phages / Q000235:genetics
D011993:Recombinant Fusion Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases