DIRECT SELECTION OF BINDING PROFICIENT CATALYTIC DEFICIENT VARIANTS OF BAMHI ENDONUCLEASE

Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placi ng a BamHI recognition sequence, GGATCC, positioned as an operator seq uence in an antisense promoter far the aadA gene (spectinomycin resist ance). Repression of this promoter relieved the inhibition of expressi on of spectinomycin resistance. This system was then used to select ne w binding proficient/cleavage deficient BamHI variants. The BamHI endo nuclease gene was mutagenized either by exposure to hydroxylamine or b y PCR. The mutagenized DNA was reintroduced into E.coli carrying the a adA gene construct, and transformants that conferred spectinomycin res istance were selected. Twenty Sp(r) transformants were sequenced. Thir teen of these were newly isolated variants of the previously identifie d D94 and E113 residues which are known to be involved in catalysis. T he remaining seven variants were all located at residue 111 and the gl utamate 111 residue was shown to be involved with catalysis.