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Isolates of thermophilic bacteria from desert soil in Kuwait, heavily contaminated with crude oil, have been screened for the presence of restriction endonuclease activity. One of the isolates (B7S), identified as Bacillus stearothermophilus, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda bacteriophage DNA at 65 degrees C, A type II restriction endonuclease (BstB7SI) has been partially purified from this isolate. BstB7SI SI recognises the six-base sequence RCCGGY (R = A or G; Y = T or C) and hydrolyses the phosphodiester bond in both strands of the DNA substrate between the first and second bases of the recognition sequence 5’-R down arrow CCGGY-3’ producing four-base 5’ overhangs. BstB7SI is therefore an isoschizomer of the mesophilic prototype restriction endonuclease Cfr10I. BstB7SI has a pH optimum of 9.7, requires 10 mM MgCl2 and 75 mM NaCl for maximum activity, and retains full enzyme activity when incubated for 5 min at temperatures up to 70 degrees C. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.