DNA analysis of the thermophilic Campylobacter by pulsed-field gel electrophoresis.
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概要
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In order to determine the suitable conditions for gaining distribution of DNA fragments useful for the chromosomal DNA analysis of the thermophilic Campylobacter, C. coli, C. jejuni and C. laridis, we were interested in the poor G+C content of the chromosomal DNAs and the intact DNAs embedded in agarose blocks were digested with 15 restriction enzymes which can recognize sequences containing only G and C nucleotides (NotI, SfiI, ApaI, BglI, SacII, SmaI and HpaII), sequences rich with G and C (BamHI, KpnI, PstI, SalI and XhoI) and some other sequences (DraI, EcoRI and HindIII) respectively. The resulted restriction fragments were fractionated by pulsed-field gel electrophoresis.When the chromosomal DNAs prepared from the 3 Campylobacter strains were digested with two restriction enzymes (NotI and SfiI) available with 8-base recognition, electrophoretic profiles of the digested chromosomal DNAs were shown to be very similar to those of each undigested chromosomal DNAs, respectively.The DNAs digested with EcoRI migrated at the same level as each undigested chromosomal DNAs by unknown reason(s).Twelve other restriction enzymes were able to cut the DNAs from the 3 Campylobacter strains with the exception that KpnI did not cut the DNA from the C. jejuni strain. Among the enzymes, ApaI, SalI and SmaI cut the DNAs from the 3 Campylobacter strains into a relatively limited number of restriction fragments in the range of approximately 50-1, 500kb in length.Thus, three restriction enzymes, ApaI, SalI and SmaI, were found to produce distributions of DNA fragments useful for chromosomal DNA analysis of the 3 Campylobacter strains by pulsed-field gel electrophoresis.Moreover, the genome size of the 3 bacterial strains were preliminarily estimated to be approximately 2, 000kb for C. coli JCM 2529T, 1, 900kb for C. jejuni JCM 2013 and 1, 700kb for C. laridis JCM 2530T in length, respectively.
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