Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleoti de sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/ T)GG sequences. These modifications which introduce 6-methyladenine and 5-m ethylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of B-methylcytosine at dinucleotides -CG- is involved in silencing gene expres sion, bud the possible consequences of the presence of the bacterial G(m)AT C and (CC)-C-m(A/T)GG sequences in the plasmids used in gene transfer exper iments are presently unknown. Since the possibility exists to obtain plasmi d DNA lacking this specific bacterial pattern of methylation by using (dam( -), dcm(-)) bacteria we performed experiments to compare in vitro and in vi vo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obt ained demonstrated that the presence of 6-methyladenine in GATC sequences a nd 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the sam e transfection performed with a plasmid amplified in the mutated JM110 (dam (-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene tran sfer efficiency. However, the persistence of the use of (dam(+) dcm(+)) bac teria in order to amplify plasmid DNA raises the question of the possible b iological consequences of the introduction of the bacterial G(m)ATC and (CC )-C-m(A/T)GG sequences in eukaryotic cells or organisms. (C) 2000 Academic Press.