MOLECULAR CHARACTERIZATION OF THE RESTRICTION-ENDONUCLEASE GENE (SCRFIR) ASSOCIATED WITH THE SCRFI RESTRICTION MODIFICATION SYSTEM FROM LACTOCOCCUS-LACTIS SUBSP CREMORIS UC503/

The nucleotide sequence of the chromosomally encoded type II ScrFI res triction/modification system from Lactococcus lactis subsp, cremoris U C503 was completed. The ScrFI restriction endonuclease (ENase) has pre viously been University College, Cork, shown to specifically recognize 5' CCNGG 3' sites, cleaving after the second Ireland cytosine and the degenerate central base. The ENase gene (scrFIR; 862 bp) was located between, and co-directionally transcribed with, two formerly character ized 5-methylcytosine methyltransferase genes, which encode proteins t hat independently confer protection against ScrFI digestion, scrFIR co des for a protein of 272 amino acids with a predicted molecular mass o f 31470 Da, which agrees favourably with a previously estimated molecu lar mass of 34 kDa for this enzyme. The deduced sequence of this prote in did not show any significant homology with known protein sequences, including the isoschizomeric Ssoll ENase from Shigella sonnei, The EN ase gene was cloned and expressed in Escherichia coil and Lactococcus; however, no in vivo restriction of phage was observed, suggesting tha t expression of the ENase gene may be repressed, or that the appropria te expression signals may be absent in the cloned constructs, The abil ity of ScrFI to cleave non-canonically modified 5' CCNGG 3' sequences suggested that some ScrFI sites may require complex modifications to f ully impair digestion by this enzyme.