Stepwise cloning and genetic organization of the seemingly unclonable HgiCII restriction-modification system from Herpetosiphon giganteus strain Hpg9, using PCR technique

Abstract

The genes, hgiCIIR and hgiCIIM, that encode the HgiCII restriction and modification (R-M) system from Herpetosiphon giganteus strain Hpg9, an AvaII isoschizomer recognizing the sequence, GGATCC, were cloned in Escherichia coli. Cloning the respective hgiCIIM gene was achieved via in vitro selection both from a Sau3AI- and an NheI-generated plasmid gene library using AvaII, a commercially available isoschizomer of HgiCII. However, all attempts to clone the closely linked hgiCIIR and M genes in a single step resulted in deletions spanning parts of the coding region of hgiCIIR. Therefore, cloning of the missing 3'-terminal part of this gene was achieved by applying the inverse polymerase-chain-reaction technique. All attempts to construct an enzymatically active R.HgiCII failed; only the inactivated hgiCIIR gene could be cloned. Sequencing of the hgiCIIRM region (carrying predesigned small mutations in the R gene) disclosed three open reading frames (ORFs): one small ORF preceding the methltransferase (MTase)-encoding gene, plus those encoding M.HgiCII (49,620 Da) and R.HgiCII (30,891 Da). M.HgiCII exhibits the common motif of ten conserved amino-acid blocks typically found within the group of m5C-MTases. The R-M system of HgiCII reveals strong homologies to the isoschizomeric R-M system of HgiBI from H. giganteus strain Hpg5, which, in contrast, could be cloned in one step.