The commercially available restriction enzymeBspHl is blocked by overlapping methylation

The restriction endonuclease flspHI, commercially available from New England Biolabs (Bishop's Stortford, UK), cleaves the site T/CATGA. This restriction enzyme is of particular use in the construction of gene expression cassettes in the vector pKK233 2 (1) as it is one of the three enzymes along with AflHl and Ncol that has an 'ATG' in its recognition sequence. In this paper we show that BsptQ is Dam methylase (2) sensitive and will not cleave DNA that has the overlapping sequence GATC. The oligonucleotides: OS3142 5'-CTAGATCATGAGCGAACTGATCGTGAACGTG ATC AAAGCTTGGTAC-3' and OS3143 5'-CAAGCTTTGATCACGTTCACGATCAGTTCGCTCATGAT-3' were annealed and ligated into Xbal-Kpnl digested bluescript SKII (Stratagene, Cambridge, UK).Transformation of Escherichia coli TGI with the resulting ligation mix resulted in plasmid pSI3. The presence of an insert was confirmed by DNA sequencing (Fig. 1), and plasmid DNA was prepared by the alkaline lysis method and digested with BspYU. From the published sequence of bluescript SKTJ 3 bands were expected. The resulting agarose gel is shown in Fig. 2; Track (2) shows that only two bands were visible. Examination of the DNA sequence flanking the BspHl site in the insert (Fig. 1) shows that the BsptU. recognition site, TCATGA, overlaps the Dam methylase recognition sequence GATC with the sequence TC ATG ATC. In order to confirm the effect of methylation, plasmid pSI3 DNA was transformed into E. coli BRD511 (dam) (Wellcome Culture Collection). Plasmid DNA was isolated and digested with BsplU; the three expected bands were generated (track 3). This observation supports the idea that AspHI is sensitive to overlapping methylation, and the enzyme should be added to the list (Clal, Hphl, MboU, Nrul, Taql and Xbal) of restriction endonucleases that are inhibited by overlapping Dam methylation.