RESUMO
Intact megaplasmids of Rhizobium meliloti 2011 have been isolated and visualized by electron microscopy. The contour lengths of 64 megaplasmid molecules were determined. One definite class of molecules of 400 micron length and a range of larger molecules with lengths of up to 560 micron was observed. The contour lengths of the megaplasmids pRme2011a and pRme2011b were measured after isolation from plasmid-free Agrobacterium strains into which they had been individually transferred. Plasmid pRme2011a corresponds to the 400-micron class of megaplasmids while plasmid pRme2011b belongs to the 560-micron class. Preparatively isolated megaplasmids pRme2011a and b showed completely different restriction patterns. The pattern of total megaplasmid DNA from R. meliloti 2011 is composed of those from pRme2011a and b, suggesting that no more than two different megaplasmids exist. Because the length distributions of measured molecules were broad, R. meliloti 2011 megaplasmids seem to vary in length in vivo. Because only pRme2011a hybridized with a nifHD probe, this is the Sym plasmid. For R. meliloti strain MVII-1, which carries the megaplasmids pRmeMVII-1f and pRmeMVII-1g, pRmeMVII-1f was shown to be the Sym plasmid. Buoyant density determinations of R. meliloti 2011 and MVII-1 megaplasmids gave a value of 1.717 g/cm3 for pSym, which is that of Agrobacterium DNA. The buoyant density of the second megaplasmid was 1.721 g/cm3, corresponding to the density of the R. meliloti chromosome. As determined by reassociation kinetics, pRme2011a and b are unrelated. The degree of relatedness between strains MVII-1 and 2011 was 82%.
Assuntos
Plasmídeos , Rhizobium/genética , Centrifugação Isopícnica , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos , Microscopia Eletrônica , Peso Molecular , Renaturação de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
The entire DNA of Rhizobium meliloti MVII /1 cells was isolated preparatively by gentle lysis and sucrose gradient centrifugation. Fractions of the sucrose gradient were investigated by electron microscopy. We found intact megaplasmids in supercoiled and relaxed form and total chromosomes. The length of the megaplasmid could be measured, which allowed an exact determination of the molecular weight. We found a length of 0.48 +/- 0.019 mm equivalent to a molecular weight of about 1000 X 10(6).
