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Available from: Didier Wion, Oct 10, 2015
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    ABSTRACT: The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide 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-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial GmATC and CmC(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam-, dcm-) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam+, dcm+) or JM110 (dam-, dcm-) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 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 same 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 transfer efficiency. However, the persistence of the use of (dam+, dcm+) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial GmATC and CmC(A/T)GG sequences in eukaryotic cells or organisms.
    Biochemical and Biophysical Research Communications 11/2000; 276(3):1261-4. DOI:10.1006/bbrc.2000.3603 · 2.30 Impact Factor
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    ABSTRACT: Mutation is a fundamental biological process occurring in each living organism. Plasmid DNA which is used in gene therapy protocols or DNA vaccination passes through two different living cells which are, respectively, the producing cell (bacterial) and the target cell (eukaryotic). Hence, modifications in the nucleotide sequence of plasmids are likely to occur both in bacteria during the amplification step of plasmid DNA and in eukaryotic cells following gene transfer. In addition to these biological modifications resulting from the physical passage of the plasmid into two different living organisms, an additional source of sequence alteration resides in our mode of representation of the nucleotide sequence of plasmid DNA which uses a four letters code, whereas, bacterial DNA is made of six different nucleosides. Indeed, the therapeutic DNA paradigm seems to have neglected the qualitative importance of these DNA sequence alterations. In this review we discuss the importance and the role of these DNA sequence modifications in the context of non-viral gene therapy approaches.
    Medical Hypotheses 06/2003; 60(5):711-5. DOI:10.1016/S0306-9877(03)00043-4 · 1.07 Impact Factor