siml, a new restriction endonuclease that recgnizes non-palindromic sequence 5′-GGGTC-3′(−3/0)

Siml, a type II restriction endonuclease, has been isolated from Staphylococcus iniermedius 6H using heparin and hydrooxyapatite chromatographic steps. The crude extract contained -3000 U Siml per gram of cells. Three cleavage positions of Siml on pUC19 DNA (-999, 1480 and 1768) have been mapped by double digests with enzymes Bgll, /tccl 131 (isoschizomer of Seal), Sail, Pvul, NmGl (isoschizomer of Earn 11051) and Mly 1131 (isoschizomer of Nari). A homology search has revealed that pentanucleotide sequence 5'-GGGTC-3' was located at these positions. The recognition sequence has been confirmed by comparison of cleavage patterns generated with Siml on commonly used DNAs (Fig. 1) with computer predicted ones. The number of Siml sites that occur in lambda, T7, adenovirus-2, pBR322 and pUC19 DNA, are 40, 94, 73, 8 and 3, respectively. The cleavage points of the restriction endonuclease Siml have been determined by sequencing of a-P-labelled Hindlll-Xbal and Xbal-EcoRl fragments of the pMVPRL plasmid (1) by the modified Maxam-Gilbert method (2). The results (Fig. 2) indicate that Siml cleaves the DNA sequence shown below: