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Nucleic Acids Research, 1989, Vol. 17, No. 23 9783-9796
© 1989

MOLECULAR BIOLOGY

Cloning, characterization and heterologous expression of the SmaI restriction-modification system

Stefan Heidmann, Wolfgang Seifert, Christoph Kessler1 and Horst Domdey

Laboratorium für Molekulare Biologie, Genzentrum, der Ludwig-Maximilians-Universität München Am Klopferspitz 18a, D-8033 Martinsried 1Boehringer Mannheim GmbH, Biochemical Research Center, Department of Genetics and Molecular Biology Nonnenwald 2, D-8122 Penzberg, FRG

Received August 8, 1989. Revised October 27, 1989. Accepted October 27, 1989.

The genes coding for the class-II Serratia marcescens restnction-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 insertionmutation experiment the 876 bp open reading flame 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 a 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 N6 A-methyltransferases M HinfI and the N4C-methyltransferase M PvuII.


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