Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts

Abstract

After a 5 h incubation of protoplasts of N. tabacum L. ''Xanthi'' with 3H-DNA (7.26 .mu.g/ml) from N. tabacum L. ''Xanthi nc'' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE[diethylaminoethanol]-dextran and poly-L-lysine to the incubation medium nearly doubled radioactivity absorption. The absorption was inhibited by 2,4-dinitrophenol, KCN and low temperature (0.degree. C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cv. that was allowed to absorb DNA prepared from a resistant cv. did not show transfer of the virus-resistant gene. A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli 3H-DNA and N. glutinosa protoplasts. In 5-20 h, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60-80% of the absorbed radioactivity resided in the soluble fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA was depolymerized. Of the incoporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 .times. 10-15g donor DNA, or approximately half of the E. coli genome.