FgoI, a type II restriction endonuclease from the thermoanaerobe Fervidobacterium gondwanense AB39(T)

Restriction endonuclease activity was detected in 11 out of 13 Fervidobacte rium isolates, including F. islandicum H21(T), F. gondwanense AB39(T), and nine other Fervidobacterium-like strains isolated from the Great Artesian B asin of Australia. The restriction endonuclease from F. gondwanense AB39(T) was partially purified and designated FgoI. FgoI recognized a 4 nucleotide sequence 5'-CTAG-3' and cleaved between nucelotides C and T to produce a 2 base 5' overhang (5'-C/TAG-3'). As predicted from the enzyme recognition a nd cleavage specificity, FgoI was found to cleave lambda DNA 13 times, phiX 174 three times, pBR322 five times, pUC18 four times, and PSK Six times. Fg oI exhibited a broad temperature optimum range (between 60 to 70 degrees C) and was active at pH6.5 to 8.5, but not at pH9.0. Manganese could replace magnesium as a cofactor for activity, but not cobalt chloride, calcium chlo ride, cupric chloride, or zinc chloride. The restriction endonuclease was c ompletely inactivated by phenol/chloroform extraction and was heat inactiva ted at 80 degrees C for 60 min or at 100 degrees C for 15 min. FgoI has bee n identified as a heat stable isoschizomer of the Type II restriction endon ucleases, MaeI and BfaI. (C) 1998 Academic Press.