PRESENCE OF ONE LINEAR AND ONE CIRCULAR CHROMOSOME IN THE AGROBACTERIUM-TUMEFACIENS C58 GENOME

Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed- field gel electrophoresis (PFGE) reveals four replicons: two large mol ecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200- kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybri dize with different restriction fragments, showing that these replicon s are two independent genetic entities. A 16S rRNA probe and genes enc oding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature . In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1 -Mb chromosome always generated an intense band, while the 3-Mb band w as barely visible. After linearization of the DNA by X-irradiation, th e intensity of the 3-Mb band increased while that of the 2.1-Mb remain ed constant. This suggests that the 3-Mb chromosome is circular and th at the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomi c DNA, trapped in an agarose plug, was first submitted to PFGE to remo ve any linear DNA present. The plug was then recovered, and the remain ing DNA was digested with either PacI or SwaI and then separated by PF GE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained , further proof of the linear nature of the 2.1-Mb chromosome.