A Type II restriction endonuciease, Mmel, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-pallndromic recognition sequence 5' T C C Pu A C (N)2o " 3' 3' A G G Py T G (N) 1 8 " 5' and to cleave (as Indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved U3lng two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules. INTRODUCTION Type II restriction endonucleases that cut DNA molecules at, or near, defined nucleotide sequences, have now been obtained from a wide variety of bacterial species (1) and have become essential tools in molecular biology. The sequence-speclflcity of an endonuciease can often be determined by analysis of the ends generated by the action of the enzyme. It is also possible to define a target sequence by analysis of the pattern of cleavage of DNA molecules of known nucleotide sequence (2,3,1),5). Here we describe the isolation of a new restriction enzyme, Mmel, from the Gram-negative bacterium Methylophilus methylotrophus, an obligate methylotroph and source of commercial single cell protein (SCP). A computer program, RECOG, which includes an algorithm not used in similar programs (2, 3,4,5), was developed to determine the recognition sequence of the enzyme using accurate mapping data on a 3equenced DNA, pBR322 (6). (The pBR322 sequence revision, adding one base pair to the TcR gene (7), has a negligible effect on the analyses described below, so the original coordinates are used throughout.) Mmel restriction generates a complex © IRL Press Limited, Oxford, England. 5255 at Y le U niersity on Jne 6, 2015 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research mixture of partial and complete digest products; therefore, another computer procedure, GELSIM, was written to simulate graphically the migration of DNA fragments during agarose gel electrophoresis, simplifying analysis of Mnel gel patterns. Computer methods described below are generally applicable to the problem of characterizing newly isolated restriction enzymes, especially preparations too crude to be analysed biochemically. MATERIALS AND METHODS Methylotroph culture conditions M.methylotrophus is an obligate methylotroph used commercially as a 3ource of single cell protein (8). Cells were grown aeroblcally at 37°C in medium containing 0.9J (v/v) methanol, and (per litre), NaH2P0i| d.'tg), K2HPOi4 (1.9g), (NHi))2SOi) (1.8g), MgSOi, (0.2g) with frace elements CuSOn-SHjO (0.02mg), MnSOi4.4H2O (0.1mg), FeCl3 (0.98mg), ZnSOn-TH^ (0.1mg) and CaCO3 (1.8mg). Substrate DNAs and enzymes Plasmid pBR322 and M13mp7 RF DNAa were prepared by a scaled-up alkallne/SDS Iysl3 method followed by purification on a CsCl/ethidlum bromide gradient (9, 10). The E.coll strain GMH8 (11) (dam-3 dcm-6 gal ara lac thr leu thl tonA tax) was transformed with pBR322 and was used as a source of "dam*"" plasmid DNA. Phage Lambda (cj_857, Sam7) DNA was prepared from a suitable lysogen by a standard technique (12). SV10, PhiX17H RF and H13 RF DNAs were gifts from (respectively) B.K. Ely, G.E. Blair and J.G.G. Schoenmakers. Restriction enzymes P3_tI, Sau3A, TaqI, EcoRI and BamHI were from BRL Inc. and used as recommended. Symbols for degenerate nucleotldes The programs described here were developed before any standard symbols for degenerate nucleotlde positions were accepted. Therefore, as well as the commonly used symbols X purlne, Y pyrimidine, N A, C, G or T, new ones were devised mnemonically to describe degeneracies found in many recognition sequences. These are: 1 A or C ("ACe"); 8 A or T ("ATe") and their "arithmetic complements" 9 G o r T ; 2 G o r C ( 1 + 9 2 + 8 10). Under this system, for example, AccI recognizes GT19AC and Hael recognizes