Studies on the two new restriction endonuclease, Cth Ⅰ and Cth Ⅱ, from clostridium thermocellum

Abstract

A set of new restriction endonuclease, Cth I, Cth II, is isolated from Clostridium thermocellum ATCC27405 by the following steps: ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (P-11) column chromatography. Restriction endonuclease, CthI cleaves neither pUC9 nor pBR322 plasmid DNA, while it cleaves SV40 and T7 DNA at one site, Adeno2 DNA at five sites, bacteriophage lamda DNA at eight sites. Restriction endonuclease, CthII, cleaves M13 DNA at two sites, bacteriophage lamda DNA at seventy one sites, Adeno2 DNA at one hundred thirty six sites. The restriction enzyme, R-CthI shows maximum activity at pH 9 to 11, 0 mM Nacl, 2 to 20 mM $Mg^{++}$ and $65\,^\circ\!C$. Restriction enzyme, R-CthII shows maximum activity at pH 8 to 9, 0 mM NaCl, 10 to 20 mM $Mg^{++}$ and $55\,^\circ\!C$. Restriction endonuclease, Cth II recognize the same sequence as Bcl I 5``-TGATCA-3`` and the restriction endonuclease, Cth II recognize the same sequence as BstN I, EcoR II, Zan I 5``-CC$^A_T$GG-3``.