Nucleic Acids Research: VOLUME 40 ISSUE 5 2012

A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized. This plasmid, pLB1, is 6.2 kb long and carries only the Eco RV genes. A subclone of 3 kb has been inserted in pBR322. The relative positions of the endonuclease and the methylase genes were determined by the construction of a 3et of overlapping deletions generated by Bal31 resection. The DNA sequence of a 2.2 kb fragment containing the two genes was determined. The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis. Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed. INTRODUCTION Interest in the type II restriction enzymes apart from their broad use in genetic engineering has increased lately, and there have been several reports of the cloning of such systems in Escherlchia coll (1-6). Apart from the obvious advantage of overproducing these enzymes, their cloning in Escherichia coli provides the means to study more fundamental questions. First the potential lethality of these systems brings up the problem of their establishment and maintenance. It is known that plasmids coding for restriction and modification systems can be transformed in bacterial hosts deprived of any protecting methylase. This would imply at least some control of the expression of the endonuclease. Until now no such control mechanism has been identified. The second most interesting feature of these enzymes is their ability to recognize and interact either by cutting or methylating with specific DNA sequences generally 4 to 6 bp long. They can thu3 © IRL Press Limited, Oxford, England. 3659 Nucleic Acids Research provide an interesting model for studying protein-DNA interactions. Until now only the Eco RI endonuclea3e has been crystallized (7), and little is known about the restriction endonucleases structure. It would then be of great interest to have available different restriction systems to allow their comparison at the structural level and determine whether, as in the case of other DNA binding proteins (8-9), some general features can be found for restriction enzymes. The recent description of the Eco RV restriction and modification system opened a new possible field of investigation. The Eco RV endonuclease, like the Eco RI endonuclease recognizes a 6 bp long target. The sites of action of those two enzymes are similar GATATC and GAATTC respectively, but while Eco RI generates 5 overhanging cohesive ends, Eco RV generates blunt ends (R. Brown, L. Bougueleret; manuscript in preparation). It was therefore interesting to investigate the relationship between these two systems. We report here the cloning, the characterization and the DNA sequence of the genes encoding the Eco RV restriction and modification system. MATERIAL AND METHODS Strains: Strains J62(pGL7U) was a gift from Noreen Murray. Strain K514 (C600, r~ m£) was used as an indicator strain for X tests and as a recipient strain for transformations. The cells were grown in L. Broth or on L. Agar. When necessary ampicillin was added to the medium at a final concentration of 100 yug/ml. Phage tests were done on BBL Agar plates supplemented with MgS04 0.1 M final concentration. Restriction and Modification Te3ts: Phage X vir was used. Restriction spot test: dilutions of a non methylated X vir.O stock (10 pfu/ml) were spotted on a lawn of the cells to be tested, as well as on an indicator non restricting strain (K514). The efficiencies of plating were compared and the ratio eop(K5TO/eop(cell) gives the efficiency of restriction. Hethylation test: 0.1 ml of an appropriate dilution of X vir (= 10^ to 10* pfu/ml for non restricting and