CLONING AND CHARACTERIZATION OF THE GENES OF THE CEQI RESTRICTION-MODIFICATION SYSTEM

Two gents from Corynebacterium equii, a Gram-positive bacterium produc ing the CegI restriction-modification enzymes were cloned and sequence d. III vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltra nsferase enzymes. However, when the two genes are expressed in E. coli , practically no enzyme activity can be detected in the supernatants o f sonicated cells, Based on the DNA sequence data CeqI restriction end onuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with th e previously measured 32 +/- 2 kDa, The methyltransferase is 517 resid ues long (approx. 60 kDa). The two genes are in opposite orientation a nd overlap by 37 base pairs on the chromosome, The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unu sual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II me thyltransferases. (C) 1997 Elsevier Science Ltd.