Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue

Abstract

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, cv. ''Mammoth Russian'') wound-inoculated with A. tumefaciens (Smith and Townsend) Conn strain B6. Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8.degree. C) and melting profiles in 25 mM sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm-3), between DNA isolated from crude nuclear preparations of the 4 tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs. Heterologous DNA renaturation kinetic analyses were performed in 0.14 M sodium phosphate (pH 6.8) at 70.degree. C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B6 DNA (MW, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed 2nd order kinetics, with a Cot1/2 [nucleotide concentration .times. incubation time] of 2.8, identical to that observed when B6 DNA was reannealed in the absence of foreign DNA. Reannealing rates of B6 DNA in the presence of 5400-fold excesses of DNA from 2 lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic DNase I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with RNase A or DNase I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids. Tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random 1/5 of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per 5 diploid plant cell DNA equivalents. The possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.