CLONING AND CHARACTERIZATION OF THE UNUSUAL RESTRICTION-MODIFICATION SYSTEM COMPRISING 2 RESTRICTION ENDONUCLEASES AND ONE METHYLTRANSFERASE

Escherichia coli RFL47 DNA fragment containing the Eco47IR and Eco47II restriction-modification (R-M) system has been cloned and sequenced. A clone carrying this system has been selected by its ability to restr ict phage lambda in vivo. The sequence of 5360 bp was determined, and its analysis revealed three major open reading frames (ORF) correspond ing to two restriction endonucleases (ENases) and one DNA methyltransf erase (MTase): R . Eco47II(239 amino acids (aa)), R . Eco47I (230 aa) and M Eco47II (417 aa). The M Eco47II aa sequence possesses all conser ved domains typical for m(5)C MTases and its variable region has a hig h homology with M . Sau96I and M . SinI. The ORF harboring a predicted helix-turn-helix motif upstream from the eco47IR gene has been found. No sequence resembling the eco47IM gene has been detected in the comp lete fragment sequenced, although disrupted ORF, possibly correspondin g to the transposase-encoding gene, has been found in the intergenic a rea between eco47IIM and eco47IR. No homology was found between the EN ases; however, both revealed homology with their isoschizomers, R SinI and R . Sau96I. An Escherichia coli RFL47 DNA fragment containing the Eco47IR and Eco47II restriction-modification (R-M) system has been cl oned and sequenced. A clone carrying this system has been selected by its ability to restrict phage lambda in vivo. The sequence of 5360 bp was determined, and its analysis revealed three major open reading fra mes (ORF) corresponding to two restriction endonucleases (ENases) and one DNA methyltransferase (MTase): R . Eco47II(239 amino acids (aa)), R . Eco47I (230 aa) and M . Eco47II (417 aa). The M . Eco47II aa seque nce possesses all conserved domains typical for m(5)C MTases and its v ariable region has a high homology with M . Sau96I and M . SinI. The O RF harboring a predicted helix-turn-helix motif upstream from the eco4 7IR gene has been found. No sequence resembling the eco47IM gene has b een detected in the complete fragment sequenced, although disrupted OR F, possibly corresponding to the transposase-encoding gene, has been f ound in the intergenic area between eco47IIM and eco47IR. No homology was found between the ENases; however, both revealed homology with the ir isoschizomers, R . SinI and R . Sau96I.