Rmal, a type II restriction endonuclease from Rhodothermus marinus which recognizes 5' CTAG 3'

Abstract

RmaI, an isoschizomer of MaeI (1) has been isolated from Rhodothermus marinus (2). RmaI recognizes the palindromic sequence 5'-CTAG-3'. Like its isoschizomer RmaI cleaves the sequence, C/TAG, generating 5'-protruding TA-dinucleotides.The recognition sequence of RmaI was determined using double digestions on pBR322and phiXI74 DNAs (figure 1, lanes 2-6 and 8-12). The cleavage patterns obtained were compared with computer-derived mapping data (3). The data predicts the sequence 5'-CTAG-3'. The sequence was also tested by digesting Lambda-and Ml3mpl8 DNAs with RmaI (figure 1, lanes 13 and 14). The fragments obtained matched the computer predicted fragments that would be produced when cleaving at 5'-CTAG-3'. RmaI has the following number of recognition sites on these commonly used DNAs: pUC19 (4), pBR322 (5), phiX174 (3), SV40 (12), Ml3mpl8 (5), T7 (60), Lambda (13) and Adeno2 (54).The cleavage site of RmaI was determined by cleavage of primed synthesis reaction (4). M13mpl9 DNA containing the recognition site of RmaI was used as a template. Using a M13 sequencing primer the DNA was sequenced according to Sanger et al. ( 5). In addition to the four standard reactions a fifth reaction was performed which was extended through the RmaI recognition site. The reaction was terminated by heat treatment and the product was cleaved with RmaI. The cleaved product was divided in two. The addition of Klenow to one part resulted in a band migrating with the A-band (figure 2, lane +). The cleaved product (figure 2, lane -) migrated with the C-band. Thus, the cleavage site for RmaI is: 5'-C/TA-G-3' 3'-G-AT/C-5'