A novel gene-fusing vector: construction of a 5'-GGmCC-specific chimeric methyltransferase, M.BspRI/M.BsuRI

Abstract

A vector was designed to allow predetermined and precise fusion between two cloned genes by constructing a cassette with two unique class-IIS restriction sites, 5'-ACCTGC-3' (BspMI) and 5'-CCGGATG-3' (FokI overlapping with MspI), arranged back-to-back in a divergent manner and inserted at the HincII site of a multiple cloning site (MCS) in plasmid pUC18 or analogous vehicle. Two DNA fragments or genes to be precisely fused are cloned into the MCS parts located on each side of the cassette containing the two unique class-IIS restriction sites. The BspMI and MspI/FokI sites are used to generate unidirectional deletions of the genes as previously described [Hasan et al., Gene 50 (1986) 55-62; Pósfai and Szybalski, Nucleic Acids Res. 16 (1988) 6245]. The precisely trimmed genes are ligated after the cassette containing the unique class-IIS restriction sites are excised with BspMI + FokI and the termini were blunted with mung-bean nuclease. This method was used to construct a hybrid methyltransferase (MTase) from the M.BspRI and M.BsuRI MTases, which share a high degree of overall homology (about 65%) and have the identical sequence specificity (5'-GGmCC-3'). A hybrid MTase composed of the N-terminal part of M.BspRI and the C-terminal part of M.BsuRI was constructed and found to be fully functional.