Characterization of restriction endonuclease activities in tetracycline producing strains ofStreptomyces aureofaciens

We have tested several strains of Streptomyces aureofaciens from different sources for restriction endonuclease. In all tested tetracycline producing strains (B-96, NMU, 16, R8/26) restriction endonuclease was isolated and characterized (SauLPl, SauNl, SauSl, SauHPl respectively). Previously described restriction endonuclease from strain S. aureofaciens R8/26I (1) was renamed for SauHPl in this report. Endonucleases were purified from cell extracts by phosphocellulose and heparin-sepharose chromatography according to (2) with minor modifications. The restriction endonucleases were free of contaminating nucleases activity. All isolated restriction endonucleases produced the same cleavage patterns on tested DNA substrates. Computer-derived mapping data predict the sequence 5'-GCCGCC-3'. The comparison of cleavage patterns of pBR322 by S.aureofaciens restrictases to Nael confirmed the recognition sequence (Figure 1). M13mpl8 derivative with cloned DNA fragment containing Nael site (unpublished results) was used for determination of cleavage site. Sequencing reactions were performed as described by Sanger et al. (3). Fifth reaction containing no dideoxy termination was extended through the Nael site. The double stranded DNA recovered from the fifth reaction was used as substrate for isolated endonucleases. The cleaved products resulted in single band which comigrates with the second C (underlined) in the sequence 5'-GCCGCC-3'. After the blunting reaction with Klenow fragment, the migration distance of cleaved products was not changed. Results obtained in this way are documented in Figure 2. Thus, SauLPl, SauNl, SauSl and SawHPI recognizes and cleaves 5'-GCCGCC-3' and are true isoschizomers of Nael (4).