Cloning, characterization and heterologous expression of the SmaI restriction-modification system.
The genes coding for the class-II Serratia marcescens restriction-modification system have been cloned and expressed in E. coli. Recombinant clones, restricted incoming phage only poorly; the recombinant plasmids, however, became fully modified in vivo, i.e. completely resistant against digestion with R.SmaI. The determined nucleotide sequence of the cloned system revealed three open reading frames with lengths of 252 bp, 741 bp, and 876 bp. Through various deletion experiments and an insertion-mutation experiment the 876 bp open reading frame could be assigned to the SmaI DNA modification enzyme and the 741 bp open reading frame to the SmaI restriction endonuclease. Mapping of the transcription start sites of the genes revealed that the SmaI endonuclease is transcribed as polycistronic mRNA together with a 252 bp long preceding open reading frame of unknown function. No homology was found when comparing the amino acid sequence of M.SmaI with the published sequences of m5C-specific DNA modification methyltransferases. On the other hand, a stretch of 14 amino acids in the C-proximal region of M.SmaI shows a significant homology to the C-proximal amino acid sequences of the N6A-methyltransferases M.HinfI and M.DpnIIA and the N4C-methyltransferase M.PvuII.
Heidmann
S
S
Laboratorium für Molekulare Biologie, Genzentrum, der Ludwig-Maximilians-Universität München, FRG.
Seifert
W
W
Kessler
C
C
Domdey
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010957:Plasmids; D015183:Restriction Mapping; D012706:Serratia marcescens / Q000201:enzymology / Q000235:genetics*; D012984:Software
D015345:Oligonucleotide Probes; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific