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DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Fate of Single-stranded Bacteriophage fd DNA ¹

National Research Council of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan S7N 0W9

Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using ³H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.

Isolated nuclei were incubated with ³H-labeled single-stranded bacteriophage fd DNA. Poly-L-lysine or DEAE-dextran at 0.1 and 1 µg/ml stimulated DNA binding. On the other hand, poly-L-lysine at 1 and 5 µg/ml increased DNA uptake but DEAE-dextran did not. Ten to 20 mM Ca and Mg ions were required for DNA uptake. EDTA (1-20 mM) did not differ from the control or the low levels of Ca or Mg ions. These observations on single-stranded fd DNA differed from those obtained with double-stranded DNA of Salmonella typhimurium. Kinetics of single-stranded DNA binding and uptake also deviated from that of double-stranded DNA.

Analyses of nuclei-bound DNA by sucrose density gradient and CsCI density gradient centrifugation revealed extensive DNA degradation during a 45-minute incubation period. Poly-L-lysine protected against rapid degradation of bound DNA. DEAE-dextran enhanced DNA binding, but bound DNA was cleaved into much smaller polymers than those detected in control experiments or in the presence of poly-L-lysine. Sucrose density gradient and CsCI density gradient centrifugation analyses on DNA taken up by nuclei also showed extensive DNA degradation. Poly-L-lysine slightly inhibited DNA degradation, but DEAE-dextran appeared to enhance degradation of incorporated DNA. Moreover, nonbound DNA in the incubation medium was completely degraded within a 20-minute incubation period.