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, GG T ACC, 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 (49620 Da) and R· HgiCII (30891 Da). M· HgiCII exhibits the common motif of ten conserved amino-acid blocks typically found within the group of m 5C-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.