When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65^80³C, which is far above the lethal temperature for the host microorganism (40³C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely : PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsaI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60³C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon. ß : S 0 3 7 8 -1 0 9 7 ( 0 1 ) 0 0 1 2 9 -X
When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.
PamI and PamlI are a type II restriction endonucleases from the Cyanobacterial strain Phormidium ambiguum GOM (CCALA Hindak 1965/117). PamI and PamlI are isoschizomers of MstI and Acyl respectively (1). The enzymes were purified using two chromatographic steps: 1) phosphocellulose, 2) DEAE-sephadex G-25. The enzymes were free of contaminating nucleases activity. All digestions were performed at 37°C in a buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl. Parallel digestion of a standard substrate (Figure 1) and sequencing (Figures 3 and 4) confirmed that PamI is an isoschizomer of MstI (5'-TGC/GCA-3') and PamlI is an isoschizomer of Acyl (5'-GR/CGYC-3'). The cleavage sites of PamI and PamIl were determined as follows: PamI: Two fragments (of about 1000 and 1663 bp) were isolated from pUC19 digested with PamI enzyme (Figure 2), and both fragments were treated with PolIK and dNTP. Both untreated-and filled-in DNA fragments were used to clone into SmaI in M13mpl8 and both DNA fragments were clonable. The enzyme must therefore generate blunt ends. The clones containing the inserts were sequenced by the Sanger et al. (1977) method. The sequence immediately after 5'-CCC of the SmaI cut site was always 5'-GCA and that before 5'-GGG of the SmaI cut was 5'-TGC. The recognition site must therefore be 5'-TGCGCA-3' and the cut site is in the middle of the sequence, resulting in blunt ends as the sequences of both ends of the cut are symmetric (Figure 3).
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