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We have isolated Bsp423I, a novel class-II restriction endonuclease from Bacillus species recognizing the palindromic sequence 5'-GCAGC-3' generating 5'-protruding tetranucleotides within the sequence complementary to 5'-GCAGC(N)g-3'. With respect to its isoschizomer Bbvl it can be isolated in higher purity and stability. A comparison of cleavage patterns obtained with Bsp423I using lambda, Ad-2, SV-40, phiX174, M13mpl9, pBR322, pBR328 and pUC18 DNAs of known nucleotide sequence (Figure 1, lanes 3-10) with computer-derived mapping data (1) predicts the sequence 5'-GCAGC-3'. The recognition sequence was confirmed by parallel digestion of lambda DNA with its isoschizomer Bbvl (2) (Figure 1, lane 2) resulting in both cases in fragments of approximately 1750, 1500, 1200, 1000, 970, 900, and numerous smaller bands which correlate with the computerderived length of 1741, 1538, 1523, 1249, 1246, 1242, 1027, 1020, 976, 974, 897, 894. 891 bp for the sequence 5'-GCAGC-3'. The exact positions of the cut 3' of the Bsp423I-recognition site were determined according to the enzymatic sequencing approach described in (3). An pBluescript Sk derivative with an insert containing a Bsp423I cleavage site was used for enzymatic sequencing reactions starting with a 5'-phosphorylated reverse M13 sequencing primer (CAGGAAACAGCTATGACC). In a parallel reaction, the same primer, [P]-endlabelled with T4 PNK and [Y3 P]ATP, was annealed to the template and the labelled primer was extended by treatment with Klenow enzyme and all four dNTPs through the Bsp423I site. The double stranded DNA was used as substrate for Bsp423I to produce an 5'-endlabelled DNA fragment comparable to the sequencing ladder. Samples were analyzed without or with ( / + ) further incubation with T4 DNA polymerase and all four dNTPs by electrophoresis and subsequent autoradiography (Fig. 2). In the Bsp423l reaction the observed single band comigrated with the 12th nucleotide 3' to the recognition sequence; after T4 DNAP treatment the observed band shift refers to the 8th nucleotide 3' to the recognition sequence 5'-GCAGC-3'.

Bsp423I, a novel isoschizomer ofBbvlfromBacillusrecognizing 5′-GCAGC-3′