Vol. 124, No. 3 Printed in U.S.A.
JouRNAL OF BACTEROLOGY, Dec. 1975, p. 1220-1226 Copyright 0 1975 American Society for Microbiology
On the Question of Integration of Agrobacterium tumefaciens Deoxyribonucleic Acid by Tomato Plants RICHARD S. HANSON AND MARY-DELL CHILTON* Bacteriology Department, University of Wisconsin, Madison, Wisconsin 53706 and Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195*
Received for publication 22 July 1975
Treatment of tomato plants with Agrobacterium tumefaciens causes subsequently administered [3H]thymidine to be preferentially incorporated into a satellite deoxyribonucleic acid (DNA) whose buoyant density is between that of bacterial DNA (p = 1.718 g/cm3) and plant main band DNA (p = 1.692 g/cm3). Satellite DNA upon shearing or sonic treatment releases fragments of higher and lower buoyant density, as reported by earlier investigators. The satellite has no significant base sequence homology with A. tumefaciens DNA, for its rate of reassociation is not accelerated by the addition of high concentrations of the latter. Tomato DNA isolated from shoots or from leaf nuclei accelerates renaturation of labeled satellite DNA. We conclude that the intermediate density labeled DNA is a plant satellite and not the product of covalent joining of bacterial and plant DNA as suggested by earlier investigators.
Exposure of tomato shoots to Agrobacterium tumefaciens deoxyribonuclic acid (DNA) (16) or to living A. tumefaciens administered through the cut stem (15) has been reported to produce dramatic alterations in the labeling pattern of DNA upon subsequent addition of [3H ]thymidine. Labeled precursor was found to be incorporated not only into DNA of plant main band buoyant density (p = 1.692 g/cm3), but also into a new satellite component of variable density intermediate between that of the plant main band DNA and bacterial donor DNA (p = 1.718 g/cm3). In addition, in experiments with living bacteria, a small peak of radioactive DNA is found at donor bacterial DNA density, due presumably to surviving bacteria. Similar findings have been reported using an eggplant system (14). The intermediate density DNA was reported to separate, upon sonic treatment, into components of higher and lower buoyant density (14-16), and this behavior has been interpreted as evidence that the satellite contains donor bacterial DNA covalently associated with plant DNA. The use of buoyant density pattern in CsCl as the sole criterion for characterization of a DNA fraction is subject to criticism (8). Even if the occurrence of false satellites (1) due to contaminating bacteria can be rigorously excluded, it is possible that unknown plant DNA viruses, unknown plant DNA satellites (2, 13), or artifacts in CsCl buoyant density determinations (8, 10) may be responsible for the unusual
buoyant density distribution of DNA labeled after stressing the plant, e.g., by administration of living bacteria. Integration of A. tumefaciens DNA, if it did occur in this experimental system, could have a bearing on the mechanism by which this pathogen transforms plant tissue and produces crown gall tumors. Neither A. tumefaciens DNA (4) nor plasmid DNA from virulent A. tumefaciens (5) has been detected in axenic crown gall tumor tissue, under conditions which would have detected one copy per diploid cell. On the other hand, Yajko and Hegeman (20) have reported indirect evidence for the transfer of A. tumefaciens DNA to carrot tissue shortly after inoculation. Thus, the role of bacterial DNA in the early phases of tumor induction is not clear. The present study was undertaken to determine whether the intermediate density satellite observed by earlier investigators (15) was indeed a product of covalent joining of A. tumefaciens DNA and tomato plant DNA.
MATERIALS AND METHODS Treatment and labeling of tomato plants. Young cherry tomato plants (Lycopersicon esculentum Mill. var. Cerasiforme Hort.) were treated with A. tumefaciens (strain 15955 from the American Type Culture Collection) by the procedure of Stroun et al. (15). Bacteria were cultivated overnight at 30 C with aeration in yeast-extract-peptone broth (4). Bacteria were washed two times in sterile 0.1 x SSC (SSC = 0.15 M NaCl, 0.015 M trisodium citrate) and suspended in sterile Hoagland solution (3). The stem of 1220
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the tomato plant was surface sterilized by scrubbing with 5% sodium hypochlorite, 70% ethanol, and sterile water. After cutting (under sterile water) with a sterile scalpel, the stem of the shoot was immersed in either the bacterial suspension, sterile Hoagland solution, or a solution of A. tumefaciens DNA (200 jig/ml) for 11 to 21 h. The shoot was kept in a sealed chamber to minimize transpiration and to prevent wilting during this treatment. After rinsing with sterile buffer, the terminal portion of the stem (0.5 cm) was removed to eliminate most of the bacteria. In some experiments, plants were next treated with 20 ug of chloramphenicol per ml for 3 h prior to and during labeling. The chloramphenicol treatment was omitted from some of the experiments, as indicated below. The shoot was transferred to sterile Hoagland solution containing 200 to 500 uCi of [3H ]thymidine (New England Nuclear, 40 to 60 Ci/mmol) and 20 jg of chloramphenicol per ml for 7 to 8 h. The plant shoots were removed, and another 0.5-cm portion of the terminal portion stem was discarded. Shoots were placed in a Revco freezer prior to DNA isolation. In some experiments the plants were stored at - 20 C in 95% ethanol, 1% ether for 5 days prior to isolation of the DNA. This procedure prevented the formation of brown pigments during incubation with Pronase. Isolation of DNA. DNA was isolated from labeled tomato plants essentially by the procedure of Stroun et al. (16). Several modifications were tested during the course of this investigation, and in all cases preferential labeling of the intermediate density satellite DNA was observed when shoots were preincubated with a suspension of A. tumefaciens. Isolation of DNA from A. tumefaciens was performed by a modification of Marmur's procedure (12), after lysis of bacteria by digestion with Pronase [500 ug/ml, predigested 90 min at 37 C in 0.05 M tris(hydroxymethyl)aminomethane, 0.02 M ethylenediaminetetraacetate, pH 8] in the presence of 1% sodium dodecyl sulfate at 37 C for 15 to 30 min. Micrococcus lysodeikticus DNA was isolated by Marmur's procedure (12). The isolation of tomato shoot total DNA and nuclear DNA is described elsewhere (M.-D. Chilton, Genetics, in press). Salmon DNA was isolated by the method of Whiteley et al. (18). DNA-filter hybridization. DNA filters were prepared by a modification (6) of the method of McCarthy and McConaughy (11). Labeled DNA in 0.1x SSC was sheared at 12,000 lb/in2 by a French pressure cell (single strand molecular weight about 5 x 10w), adjusted to 2x SSC, heat denatured (100 C, 5 min), and quickly cooled, and 0.2-ml aliquots were placed in reaction tubes (10 by 75 mm). One or two DNA filters containing 15 to 30 Mg of unlabeled DNA was added, samples were overlaid with oil, and tubes were capped with foil and incubated at 67 C overnight (16 to 18 h). Filters were removed from tubes and washed three times in 20 ml of 2x SSC at 67 C, and radioactivity bound was assayed by counting dried filters in toluene-Liquifluor (New England Nuclear) scintillator solution in a Packard Tricarb liquid scintillation spectrometer.
Thermal denaturation profiles of filterbound duplexes were determined as previously described (6). DNA/DNA reassociation measurements. Labeled and unlabeled DNA in 0.1 x SSC were sheared by a French pressure cell (12,000 lb/in2). Unlabeled DNA (400 Mg) was evaporated to dryness and to it was added 1.0 ml of labeled DNA in 0.15 M PB (an equimolar mixture of mono- and dibasic sodium phosphate salts). Redction mixtures were sealed in ten 100-Al capillary pipets, denatured (100 C, 5 min), and incubated at 67 C. Aliquots were removed at various times, and the percentage of double-stranded labeled DNA was assayed by hydroxylapatite fractionation as described previously (4).
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RESULTS
When viable A. tumefaciens is administered to a tomato plant through the cut stem, followed by labeling with [3H]thymidine, the DNA synthesized in the plant exhibits three buoyant density maxima in a CsCl gradient (Fig. 1). Treatment of the plant with chloramphenicol prior to and during labeling eliminates the most dense component (Fig. 2), which presumably is due to surviving A. tumefaciens. The light component coincides in density with tomato main band DNA. The identity of the DNA component of intermediate density is explored in the experiments detailed below. Our first objective was to establish the range of conditions under which intermediate density DNA labeling occurred. In nine experiments in which plants were treated with A. tumefaciens, 50 _
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FIG. 1. CsCI density gradient of labeled DNA isolated from tomato shoots infected with A. tumefaciens. Cut tomato plant shoots were treated with bacteria in Hoagland solution for 21 h, washed, and given 250 MCi of [3H]thymidine for 7 h. DNA from one plant shoot (approximately 25 Mg) was centrifuged in the gradient.
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HANSON AND CHILTON
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FIG. 2. CsCl density gradient of labeled DNA isolated from tomato shoots infected with A. tumefaciens followed by chloramphenicol treatment. The experiment was performed as described for Fig. 1, except that chloramphenicol treatment was administered before and dutring the DNA labeling period, as described in the text. Denatured M. Iysodeikticus DNA (p = 1.745) was added as marker. Symbols: 0, abosrbancy at 260 nm (A2,,); 0, radioactiuity.
the prominent labeling of intermediate density DNA was observed. In several experiments, brown pigments contaminated the DNA preparation so extensively that absorbancy peaks at 260 nm could not be measured in CsCl gradients. DNA preparations isolated from infected shoots by several variations of the procedure described above gave similar radioactivity profiles in CsCl gradients. Contrary to earlier results (8), in the present study we observed prominent labeling of intermediate density DNA when plants were treated with sterile A. tumefaciens DNA for 40 h (Fig. 3). Control plants, treated with sterile Hoagland solution for 40 h and then chloramphenicol, exhibited a shoulder of labeled DNA on the dense side of the main band (Fig. 4). The DNA extracted from six other control plant shoots also shows evidence of satellite components, but to a lesser extent than this control. The DNA isolation procedure used in the present experiments included a Pronase digestion step which was omitted in earlier experiments (8). In addition, different strains of cherry tomato plants were employed. Although the explanation for the discrepancy in results in this laboratory remains obscure, in the present study we have apparently reproduced the phenomenon reported by Stroun and his collaborators (14-16). However, we note that control plants were not entirely free of intermediate density labeled satellite as reported, without presenting data, by earlier investigators (14-16). The intermediate density labeled DNA of Fig. 2 was chosen for further characterization. Earlier investigators have reported (14-16) that upon sonic treatment such DNA separates into
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FIcG. 3. CsCI gradient of labeled DNA isolated from tomato shoots incubated with A. tumefaciens DNA. After incubation with donor DNA (200 Mg/ml) in Hoagland solution for 40 h, chloramphenicol (20,ug) was applied, followed after 3 h by [3H]thymidine labeling for 7 h.
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FIG. 4. CsCI gradient of labeled DNA isolated from tomato shoots incubated with Hoagland solution. This control plant was treated with chloramphenicol and labeled exactly as described for Fig. 3. The incubation with DNA was replaced by an incubation with Hoagland solutuion.
components of higher and lower buoyant density. Figure 5A shows that the intermediate density labeled DNA before sonic treatment rebands unimodally with p = 1.704 g/cm3 and is only slightly contaminated by main band plant DNA. Upon sonic treatment (Fig. 5B), two buoyant density maxima are observed in
VOL. 124, 1975
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A. TUMEFACIENS DNA INTEGRATION
10
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Fraction Number
FIG. 5. Rebanding studies of intermediate density labeled DNA with and without shearing. (A) A portion of fractions 23-26 from Fig. 2 was rebanded with unlabeled marker DNAs: 25 gg of A. tumefaciens DNA (p 1.718) and 25 Mg of Myxobacterium sp. 1.695). (B) A portion of fractions 23-26 DNA (p from Fig. 2 was sheared by a French pressure cell (12,000 Ib/in2) and rebanded with unlabeled marker DNAs as in (A). Symbols: 0, absorbancy at 260 nm (A2,0); 0, radioactivity. =
=
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albumin Keiselguhr column prior to the cesium chloride gradient. The pronounced satellite was present and the pigment was removed from the DNA on the methylated albumin Keiselguhr column. The high density component released from intermediate density satellite DNA upon shearing was rebanded in CsCl gradients, and its homogeneity and buoyant density (1.711 g/cm3) were confirmed in two experiments. We have never observed the release of DNA with a buoyant density as great as that of A. tumefaciens DNA (1.718 g/cm3), except for a portion of a broad band present after very extensive sonic treatment. In other respects, the behavior of the intermediate density labeled DNA in our experiments resembles closely that reported by earlier investigators (14-16). Analysis of intermediate density labeled satellite DNA by DNA filter hybridization. Sheared labeled intermediate density satellite, like that recovered from the gradient illustrated in Fig. 2, was tested for base sequence homology with A. tumefaciens DNA and tomato DNA isolated either from nuclei or from total shoots by DNA filter hybridization as described in Materials and Methods. Data are presented in Table 1. Labeled satellite DNA bound to tomato DNA filters extensively (11 to 14% per 10 yg of DNA on the filter), but the binding to A. tumefaciens DNA filters was low (0.8%). In a separate experiment, DNA from the main peak of the cesium chloride gradient hybridized to tomato DNA filters to the same extent as the satellite DNA. In separate hybridization experiments (data not shown), the satellite was found to have similar binding reaction with control M. lysodeikticus DNA filters (0.8% per
1.711 g/cm3 and p = 1.699 this DNA, at p g/cm 3. In each of six experiments, the component of highest density in sheared intermediate density DNA exhibited p = 1.710 to 1.712 g/cm3, whether sheared by a French pressure cell or by sonic treatment. The component with the lowest buoyant density was variable TABLE 1. Reaction of intermediate density labeled in its behavior in CsCl gradients. When reDNA with filter-bound DNAsa leased by shearing intermediate density DNA in the French pressure cell (four experiments), Input 'H 3Hel-e the light component resembled that of Fig. 5B. labeled bound Input per DNA When released by sonic treatment (two experi10 jsg of 3H bound Filter-bound DNA ments), the low density component formed DNA on (% a hypersharp peak with a buoyant density of filter (%) min) 1.695 g/cm3. In contrast, labeled DNA from the main band of CsCl gradients such as that of A.tumefaciens 2.8, 2.4 0.88, 0.75 31,24 15955b Fig. 2, upon shearing, was observed to produce 14.1 19.5 215 Tomato shootc a single broad peak in CsCl gradients. Thus, 11.3 17.3 190 nuclearc Tomato intermediate density the separation of the labeled DNA into two components upon sheara Input labeled intermediate density DNA (1,100 ing does not appear to be an artifact due to counts/min per 0.2 ml) was incubated with DNA impurities in the DNA preparation (8). To avoid filters at 67 C in 2x SSC as described in the text. problems associated with contamination of the b Two filters were used per vial, and duplicate vials DNA by various compounds, DNA from an were prepared. c One filter was used per vial. infected plant was purified on a methylated =
(bounds
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HANSON AND CHILTON
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mediate density satellite DNA and A. tumefaciens DNA, a trace of labeled satellite DNA was allowed to reassociate in the presence of a high concentration of unlabeled salmon DNA (control), A. tumefaciens DNA, or tomato DNA (either nuclear or total). The addition of bacterial DNA did not accelerate the reassociation of the labeled satellite, whereas addition of either preparation of tomato DNA caused a dramatic increase in rate (Fig. 7). From the rate of reassociation of labeled satellite in the presence of driving concentrations of tomato DNA, some inference can be drawn concerning the concentration and complexity of these DNA sequences in the plant. Such a calculation is not possible from the rate of reassociation of labeled satellite alone, because its concentration was too low to be measured meaningfully. The observed C.t, value of labeled satellite (with respect to conK00 centration of the tomato driver DNA) is 101 to
FIG. 6. Melting profiles of filter-bound duplexes formed between labeled intermediate density DNA and DNA filters containing tomato DNA or A. tumefaciens DNA. Duplexes described in Table 1 were subjected to thermal dissociation in 1 x SSC as described. Symbols: 0, labeled intermediate density DNA x A. tumefaciens DNA filter; A, labeled intermediate density DNA x tomato DNA filter; 0, authentic 3H-labeled A. tumefaciens DNA x A. tumefaciens DNA filter.
2 x 101.
DISCUSSION The experiments presented above strongly suggest that we have isolated an intermediate density satellite similar to that reported by Stroun and his collaborators (14, 15). The unusual behavior of this satellite DNA upon sonic treatment or shearing, i.e., the release of components of high and low buoyant density, characterizes this DNA fraction uniquely. We offer no explanation of this unexpected property, but presume that it is due to unusual base pair distribution in the satellite.
10 Ag of filterbound DNA). Such direct binding values are difficult to evaluate without further controls, because of the occurrence of variable extents of nonspecific background binding in DNA-filter reactions (6). Thermal dissociation profiles of duplexes 10-'~~~~~~0 (Fig. 6) show that the duplexes formed between 10' 100 ,,X,,, ,,,, ,,,, labeled satellite and A. tumefaciens DNA do not Lu exhibit the thermal stability of true bacterial Z, 80 i ° DNA duplexes (shown on the same graph for crcomparison). Satellite duplexes exhibit a Tm 0 (midpoint of thermal dissociation) of 85 C, U60 z whereas authentic A. tumefaciens duplexes 40 have Tm of 92 C. The duplexes formed be- z tween labeled satellite and tomato DNA exhibit 20 a Tm of 86 C. This is an unusually high value ai for plant duplexes: labeled main band tomato 100 10l 1o2 103 DNA under these conditions forms duplexes TIME (MINUTES) with a Tm of 76.5 C (data not shown), as exFIG. 7. Reassociation kinetics of labeled intermepected for mismatched duplexes formed by diate density DNA with unlabeled driver DNAs. redundant DNA under filter hybridization conReassociation measurements were performed as deditions. scribed. The Cjt scale refers to C. (moles of nucleotide Analysis of intermediate density labeled per liter) of the driver DNA multiplied by time satellite DNA by reaction with unlabeled (seconds). Unlabeled driver DNAs used were: 0, total driver DNAs in solution. In another attempt DNA from tomato; 0, nuclear DNA from tomato; A, to discern homology between labeled inter- salmon DNA; 0, A. tumefaciens 15955 DNA.
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A. TUMEFACIENS DNA INTEGRATION
Our two hybridization studies have failed to provide any evidence for a bacterial DNA component in this satellite. In DNA filter reactions using authentic labeled A. tumefaciens 15955 DNA and homologous DNA filters, we observe about 10% of the input radioactivity bound per 10 ug of filter-bound tomato DNA in different experiments under the reaction conditions used here (6). If this intermediate density satellite were composed of approximately equal parts of bacterial and plant DNA, we would expect approximately 5% of input satellite radioactivity to bind to bacterial DNA filters, and we observe background levels of less than 1% binding in three experiments. Moreover, the Tm of the duplexes formed is 70 lower than that observed for authentic bacterial DNA duplexes. We conclude that this low level of binding is nonspecific background binding (6), and that the satellite DNA has failed to exhibit homology with bacterial DNA in filter reactions. Still more convincing is the evidence afforded by DNA-driven reactions of labeled satellite DNA. If half of the radioactivity of the satellite were in bacterial DNA, half of the labeled satellite 'should reassociate rapidly in the presence of 400 Mg of bacterial DNA per ml, which is 80% reassociated by 100 min under these conditions. The failure of any labeled satellite to exhibit homology with bacterial DNA under these circumstances conclusively rules out the model proposed by Stroun and his collaborators (14, 15). It does not rule out the possibility of any bacterial DNA in the satellite; the presence of up to 3% cannot be ruled out by this experiment. However, our findings invalidate the only evidence presented by earlier investigators supporting the model of DNA integration under these conditions. If the intermediate buoyant density of the labeled DNA, as well as its density segregation upon sonic treatment, were due to bacterial DNA integration, about 50% of it would have to be bacterial DNA, a possibility which our hybridization data rule out definitively. We conclude that there is no valid evidence for A. tu,,&efaciens DNA integration into tomato DNA upon treatment of cut stems with bacterial suspensions. We further add the example of this unusual labeled plant DNA satellite to the list of pitfalls in the use of CsCl buoyant density to characterize labeled plant DNA components (8). Separation of a labeled DNA into components of higher and lower density upon shearing occurs in a satellite which we have now characterized as a plant DNA component. This type of evidence for
foreign DNA integration is therefore subject to doubt. From the rate of reassociation of labeled satellite in the presence of driving concentrations of unlabeled tomato DNA, some inferences can be drawn about its complexity and concentration in tomato DNA. The occurrence of a visible satellite in tomato DNA at a buoyant density of 1.704 g/cm3 (7; Chilton, in press) suggests a likely candidate for the labeled satellite. However, the concentration and reassociation rates of the isolated satellite (Chilton, in press) are so high that in unfractionated tomato DNA under the renaturation conditions used here (0.15 M PB, 67 C), the visible satellite component would be over 75% reassociated by Cot = 0.2 (i.e., earlier than the first kinetic point in Fig. 5). Thus, although up to 20% of the radioactivity in labeled satellite DNA could be in the visible satellite, the bulk of the radioactivity is in some other component. Because DNA coding for ribosomal ribonucleic acid forms a part of the visible tomato satellite (Chilton, in press), such DNA is ruled out as the source of the intermediate density labeled satellite. Another reasonable candidate for labeled satellite would appear to be mitochondrial DNA, whose buoyant density in the case of several dicotyledonous plants appears to be 1.705 to 1.707 g/cm 3 (9, 17, 19), similar to that of the labeled satellite. If mitochondrial DNA has a genome size of about 70 x 106 (9), its CO;t if isolated, under our reaction conditions, would be approximately 10- l. However, if it represents approximately 1% of the total plant DNA, it would exhibit a Cot% of 10 with respect to concentration of total DNA. This is similar to the value observed for the DNA which is homologous to the bulk of labeled satellite DNA. Although nuclear DNA appears to contain about 2.5-fold lower concentration of the homologous DNA sequences, it is not free from them. If the labeled satellite DNA is in fact mitochondrial in origin, it appears that nuclear DNA either is heavily contaminated with or contains copies of mitochrondrial DNA in this plant. Repeated attempts to purify DNA from tomato mitochondria by several published procedures were unsuccessful. Thus, we were unable to test this hypothesis definitively. ACKNOWLEDGMENTS This research was supported by Public Health Service research grant number CA13015 from the National Cancer Institute. One of us (R.S.H.) was the recipient of Public
Health Service Senior Postdoctoral Fellowship number 1 F03
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HANSON AND CHILTONJJ. BACTERIOL.
CA53022-01 from the National Cancer Institute and was partially supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison. We thank Eugene W. Nester for making his laboratory facilities available for this investigation. LITERATURE CITED 1. Bendich, A. J. 1972. Effect of contaminating bacteria on the radiolabeling of nucleic acids from seedlings: false DNA "satellites." Biochim. Biophys. Acta 272: 494-503. 2. Bendich, A. J., and P. Filner. 1971. Uptake of exogenous DNA by pea seedlings and tobacco cells. Mutat. Res. 13:199-214. 3. Bonner, J., and A. W. Galston. 1959. Principles of plant physiology, p. 55. W. H. Freeman, San Francisco. 4. Chilton, M.-D., T. C. Currier, S. K. Farrand, A. J. Bendich, M. P. Gordon, and E. W. Nester. 1974. Agrobacterium tumefaciens DNA and PS8 DNA not detected in crown gall tumors. Proc. Natl. Acad. Sci. U.S.A. 71:3672-3676. 5. Chilton, M.-D., S. K. Farrand, F. Eden, T. Currier, A. J. Bendich, M. P. Gordon, and E. W. Nester. 1975. Is there foreign DNA in crown gall tumor DNA?, p. 297-311. In R. Markham, D. R. Davies, D. A. Hopwood, and R. W. Horne (ed.), Second Annual John Innes Symposium. North Holland/Elsevier, New York. 6. Farrand, S. K., F. C. Eden, and M.-D. Chilton. 1975. Attempts to detect Agrobacterium tumefaciens and bacteriophage PS8 DNA in crown gall tumors by DNA- DNA-filter hybridization. Biochim. Biophys. Acta 390:264-275. 7. Ingle, J., G. G. Pearson, and J. Sinclair. 1973. Species distribution and properties of nuclear satellite DNA in higher plants. Nature (London) New Biol. 242: 193-197. 8. Kleinhofs, A., F. C. Eden, M.-D. Chilton, and A. J. Bendich. 1975. Exogenous bacterial DNA not integrated or replicated in plants. Proc. Natl. Acad. Sci. U.S.A. 72:2748-2752.
9. Kolodner, 10. 11.
12. 13.
14.
R., and K. K. Tewari. 1972. Physicochemical characterization of mitochondrial DNA from pea leaves. Proc. Natl. Acad. Sci. U.S.A. 69:1830-1834. Ledoux, L., and P. Charles. 1972. In L. Ledoux (ed.), Uptake of information molecules by living cells, p. 29-46. North Holland, New York. McCarthy, B. J., and B. L. McConaughy. 1968. Related base sequences in the DNA of simple and complex organisms. I. DNA/DNA duplex formation and the incidence of partially related base sequences in DNA. Biochem. Genet. 2:37-53. Marmur, J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol. 3:208-218. Parenti, R., E. Guill6, J. Grisvard, M. Durante, L. Giorgi, and M. Buiatti. 1973. Transcient DNA satellite in dedifferentiating pith tissue. Nature (London) New Biol. 246:237-239. Stroun, M., and P. Anker. 1971. Bacterial nucleic acid synthesis in plants following bacterial contact. Mol. Gen. Genet. 113:92-98.
Stroun, M., P. Anker, P. Gahan, A. Rossier, and H. Greppin. 1971. Agrobacterium tumefaciens ribonucleic acid svnthesis in tomato cells and crown gall induction. J. Bacteriol. 106:634-639. 16. Stroun, M., P. Anker, and L. Ledoux. 1967. DNA replication in Solanum lycopersicum esc. after ab15.
17.
sorption of bacterial DNA. Curr. Mod. Biol. 1:231-234. Vedel, F., and F. Quetier. 1974. Physico-chemical
characterization of mitochondrial DNA from potato tubers. Biochim. Biophys. Acta 340:374-387. 18. Whiteley, A. H., B. J. McCarthy, and H. R. Whiteley. 1966. Changing populations of messenger RNA during sea urchin development. Proc. Natl. Acad. Sci. U.S.A. 55:519-525. 19. Wong, F. Y.. and S. G. Wildman. 1972. Simple procedure for isolation of satellite DNAs from tobacco leaves in high yield and demonstration of minicircles. Biochim. Biophys. Acta 259:5-12. 20. Yajko, D. M., and G. D. Hegeman. 1971. Tumor induction by Agrobacterium tumefaciens: specific transfer of bacterial deoxyribonucleic acid to plant tissue. J. Bacteriol. 108:973-979.
JouRNAL OF BACTEROLOGY, Dec. 1975, p. 1220-1226 Copyright 0 1975 American Society for Microbiology
On the Question of Integration of Agrobacterium tumefaciens Deoxyribonucleic Acid by Tomato Plants RICHARD S. HANSON AND MARY-DELL CHILTON* Bacteriology Department, University of Wisconsin, Madison, Wisconsin 53706 and Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195*
Received for publication 22 July 1975
Treatment of tomato plants with Agrobacterium tumefaciens causes subsequently administered [3H]thymidine to be preferentially incorporated into a satellite deoxyribonucleic acid (DNA) whose buoyant density is between that of bacterial DNA (p = 1.718 g/cm3) and plant main band DNA (p = 1.692 g/cm3). Satellite DNA upon shearing or sonic treatment releases fragments of higher and lower buoyant density, as reported by earlier investigators. The satellite has no significant base sequence homology with A. tumefaciens DNA, for its rate of reassociation is not accelerated by the addition of high concentrations of the latter. Tomato DNA isolated from shoots or from leaf nuclei accelerates renaturation of labeled satellite DNA. We conclude that the intermediate density labeled DNA is a plant satellite and not the product of covalent joining of bacterial and plant DNA as suggested by earlier investigators.
Exposure of tomato shoots to Agrobacterium tumefaciens deoxyribonuclic acid (DNA) (16) or to living A. tumefaciens administered through the cut stem (15) has been reported to produce dramatic alterations in the labeling pattern of DNA upon subsequent addition of [3H ]thymidine. Labeled precursor was found to be incorporated not only into DNA of plant main band buoyant density (p = 1.692 g/cm3), but also into a new satellite component of variable density intermediate between that of the plant main band DNA and bacterial donor DNA (p = 1.718 g/cm3). In addition, in experiments with living bacteria, a small peak of radioactive DNA is found at donor bacterial DNA density, due presumably to surviving bacteria. Similar findings have been reported using an eggplant system (14). The intermediate density DNA was reported to separate, upon sonic treatment, into components of higher and lower buoyant density (14-16), and this behavior has been interpreted as evidence that the satellite contains donor bacterial DNA covalently associated with plant DNA. The use of buoyant density pattern in CsCl as the sole criterion for characterization of a DNA fraction is subject to criticism (8). Even if the occurrence of false satellites (1) due to contaminating bacteria can be rigorously excluded, it is possible that unknown plant DNA viruses, unknown plant DNA satellites (2, 13), or artifacts in CsCl buoyant density determinations (8, 10) may be responsible for the unusual
buoyant density distribution of DNA labeled after stressing the plant, e.g., by administration of living bacteria. Integration of A. tumefaciens DNA, if it did occur in this experimental system, could have a bearing on the mechanism by which this pathogen transforms plant tissue and produces crown gall tumors. Neither A. tumefaciens DNA (4) nor plasmid DNA from virulent A. tumefaciens (5) has been detected in axenic crown gall tumor tissue, under conditions which would have detected one copy per diploid cell. On the other hand, Yajko and Hegeman (20) have reported indirect evidence for the transfer of A. tumefaciens DNA to carrot tissue shortly after inoculation. Thus, the role of bacterial DNA in the early phases of tumor induction is not clear. The present study was undertaken to determine whether the intermediate density satellite observed by earlier investigators (15) was indeed a product of covalent joining of A. tumefaciens DNA and tomato plant DNA.
MATERIALS AND METHODS Treatment and labeling of tomato plants. Young cherry tomato plants (Lycopersicon esculentum Mill. var. Cerasiforme Hort.) were treated with A. tumefaciens (strain 15955 from the American Type Culture Collection) by the procedure of Stroun et al. (15). Bacteria were cultivated overnight at 30 C with aeration in yeast-extract-peptone broth (4). Bacteria were washed two times in sterile 0.1 x SSC (SSC = 0.15 M NaCl, 0.015 M trisodium citrate) and suspended in sterile Hoagland solution (3). The stem of 1220
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A. TUMEFACIENS DNA INTEGRATION
the tomato plant was surface sterilized by scrubbing with 5% sodium hypochlorite, 70% ethanol, and sterile water. After cutting (under sterile water) with a sterile scalpel, the stem of the shoot was immersed in either the bacterial suspension, sterile Hoagland solution, or a solution of A. tumefaciens DNA (200 jig/ml) for 11 to 21 h. The shoot was kept in a sealed chamber to minimize transpiration and to prevent wilting during this treatment. After rinsing with sterile buffer, the terminal portion of the stem (0.5 cm) was removed to eliminate most of the bacteria. In some experiments, plants were next treated with 20 ug of chloramphenicol per ml for 3 h prior to and during labeling. The chloramphenicol treatment was omitted from some of the experiments, as indicated below. The shoot was transferred to sterile Hoagland solution containing 200 to 500 uCi of [3H ]thymidine (New England Nuclear, 40 to 60 Ci/mmol) and 20 jg of chloramphenicol per ml for 7 to 8 h. The plant shoots were removed, and another 0.5-cm portion of the terminal portion stem was discarded. Shoots were placed in a Revco freezer prior to DNA isolation. In some experiments the plants were stored at - 20 C in 95% ethanol, 1% ether for 5 days prior to isolation of the DNA. This procedure prevented the formation of brown pigments during incubation with Pronase. Isolation of DNA. DNA was isolated from labeled tomato plants essentially by the procedure of Stroun et al. (16). Several modifications were tested during the course of this investigation, and in all cases preferential labeling of the intermediate density satellite DNA was observed when shoots were preincubated with a suspension of A. tumefaciens. Isolation of DNA from A. tumefaciens was performed by a modification of Marmur's procedure (12), after lysis of bacteria by digestion with Pronase [500 ug/ml, predigested 90 min at 37 C in 0.05 M tris(hydroxymethyl)aminomethane, 0.02 M ethylenediaminetetraacetate, pH 8] in the presence of 1% sodium dodecyl sulfate at 37 C for 15 to 30 min. Micrococcus lysodeikticus DNA was isolated by Marmur's procedure (12). The isolation of tomato shoot total DNA and nuclear DNA is described elsewhere (M.-D. Chilton, Genetics, in press). Salmon DNA was isolated by the method of Whiteley et al. (18). DNA-filter hybridization. DNA filters were prepared by a modification (6) of the method of McCarthy and McConaughy (11). Labeled DNA in 0.1x SSC was sheared at 12,000 lb/in2 by a French pressure cell (single strand molecular weight about 5 x 10w), adjusted to 2x SSC, heat denatured (100 C, 5 min), and quickly cooled, and 0.2-ml aliquots were placed in reaction tubes (10 by 75 mm). One or two DNA filters containing 15 to 30 Mg of unlabeled DNA was added, samples were overlaid with oil, and tubes were capped with foil and incubated at 67 C overnight (16 to 18 h). Filters were removed from tubes and washed three times in 20 ml of 2x SSC at 67 C, and radioactivity bound was assayed by counting dried filters in toluene-Liquifluor (New England Nuclear) scintillator solution in a Packard Tricarb liquid scintillation spectrometer.
Thermal denaturation profiles of filterbound duplexes were determined as previously described (6). DNA/DNA reassociation measurements. Labeled and unlabeled DNA in 0.1 x SSC were sheared by a French pressure cell (12,000 lb/in2). Unlabeled DNA (400 Mg) was evaporated to dryness and to it was added 1.0 ml of labeled DNA in 0.15 M PB (an equimolar mixture of mono- and dibasic sodium phosphate salts). Redction mixtures were sealed in ten 100-Al capillary pipets, denatured (100 C, 5 min), and incubated at 67 C. Aliquots were removed at various times, and the percentage of double-stranded labeled DNA was assayed by hydroxylapatite fractionation as described previously (4).
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RESULTS
When viable A. tumefaciens is administered to a tomato plant through the cut stem, followed by labeling with [3H]thymidine, the DNA synthesized in the plant exhibits three buoyant density maxima in a CsCl gradient (Fig. 1). Treatment of the plant with chloramphenicol prior to and during labeling eliminates the most dense component (Fig. 2), which presumably is due to surviving A. tumefaciens. The light component coincides in density with tomato main band DNA. The identity of the DNA component of intermediate density is explored in the experiments detailed below. Our first objective was to establish the range of conditions under which intermediate density DNA labeling occurred. In nine experiments in which plants were treated with A. tumefaciens, 50 _
cP
0~~~~~~~~~~~~
30
I
20
0
10
30 20 Fraction Number
40
FIG. 1. CsCI density gradient of labeled DNA isolated from tomato shoots infected with A. tumefaciens. Cut tomato plant shoots were treated with bacteria in Hoagland solution for 21 h, washed, and given 250 MCi of [3H]thymidine for 7 h. DNA from one plant shoot (approximately 25 Mg) was centrifuged in the gradient.
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TJ. BACTERIOL.
HANSON AND CHILTON
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Fraction Number
FIG. 2. CsCl density gradient of labeled DNA isolated from tomato shoots infected with A. tumefaciens followed by chloramphenicol treatment. The experiment was performed as described for Fig. 1, except that chloramphenicol treatment was administered before and dutring the DNA labeling period, as described in the text. Denatured M. Iysodeikticus DNA (p = 1.745) was added as marker. Symbols: 0, abosrbancy at 260 nm (A2,,); 0, radioactiuity.
the prominent labeling of intermediate density DNA was observed. In several experiments, brown pigments contaminated the DNA preparation so extensively that absorbancy peaks at 260 nm could not be measured in CsCl gradients. DNA preparations isolated from infected shoots by several variations of the procedure described above gave similar radioactivity profiles in CsCl gradients. Contrary to earlier results (8), in the present study we observed prominent labeling of intermediate density DNA when plants were treated with sterile A. tumefaciens DNA for 40 h (Fig. 3). Control plants, treated with sterile Hoagland solution for 40 h and then chloramphenicol, exhibited a shoulder of labeled DNA on the dense side of the main band (Fig. 4). The DNA extracted from six other control plant shoots also shows evidence of satellite components, but to a lesser extent than this control. The DNA isolation procedure used in the present experiments included a Pronase digestion step which was omitted in earlier experiments (8). In addition, different strains of cherry tomato plants were employed. Although the explanation for the discrepancy in results in this laboratory remains obscure, in the present study we have apparently reproduced the phenomenon reported by Stroun and his collaborators (14-16). However, we note that control plants were not entirely free of intermediate density labeled satellite as reported, without presenting data, by earlier investigators (14-16). The intermediate density labeled DNA of Fig. 2 was chosen for further characterization. Earlier investigators have reported (14-16) that upon sonic treatment such DNA separates into
n
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-
FIcG. 3. CsCI gradient of labeled DNA isolated from tomato shoots incubated with A. tumefaciens DNA. After incubation with donor DNA (200 Mg/ml) in Hoagland solution for 40 h, chloramphenicol (20,ug) was applied, followed after 3 h by [3H]thymidine labeling for 7 h.
0)
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FIG. 4. CsCI gradient of labeled DNA isolated from tomato shoots incubated with Hoagland solution. This control plant was treated with chloramphenicol and labeled exactly as described for Fig. 3. The incubation with DNA was replaced by an incubation with Hoagland solutuion.
components of higher and lower buoyant density. Figure 5A shows that the intermediate density labeled DNA before sonic treatment rebands unimodally with p = 1.704 g/cm3 and is only slightly contaminated by main band plant DNA. Upon sonic treatment (Fig. 5B), two buoyant density maxima are observed in
VOL. 124, 1975
5
A. TUMEFACIENS DNA INTEGRATION
10
15
20
25
30
Fraction Number
FIG. 5. Rebanding studies of intermediate density labeled DNA with and without shearing. (A) A portion of fractions 23-26 from Fig. 2 was rebanded with unlabeled marker DNAs: 25 gg of A. tumefaciens DNA (p 1.718) and 25 Mg of Myxobacterium sp. 1.695). (B) A portion of fractions 23-26 DNA (p from Fig. 2 was sheared by a French pressure cell (12,000 Ib/in2) and rebanded with unlabeled marker DNAs as in (A). Symbols: 0, absorbancy at 260 nm (A2,0); 0, radioactivity. =
=
1223
albumin Keiselguhr column prior to the cesium chloride gradient. The pronounced satellite was present and the pigment was removed from the DNA on the methylated albumin Keiselguhr column. The high density component released from intermediate density satellite DNA upon shearing was rebanded in CsCl gradients, and its homogeneity and buoyant density (1.711 g/cm3) were confirmed in two experiments. We have never observed the release of DNA with a buoyant density as great as that of A. tumefaciens DNA (1.718 g/cm3), except for a portion of a broad band present after very extensive sonic treatment. In other respects, the behavior of the intermediate density labeled DNA in our experiments resembles closely that reported by earlier investigators (14-16). Analysis of intermediate density labeled satellite DNA by DNA filter hybridization. Sheared labeled intermediate density satellite, like that recovered from the gradient illustrated in Fig. 2, was tested for base sequence homology with A. tumefaciens DNA and tomato DNA isolated either from nuclei or from total shoots by DNA filter hybridization as described in Materials and Methods. Data are presented in Table 1. Labeled satellite DNA bound to tomato DNA filters extensively (11 to 14% per 10 yg of DNA on the filter), but the binding to A. tumefaciens DNA filters was low (0.8%). In a separate experiment, DNA from the main peak of the cesium chloride gradient hybridized to tomato DNA filters to the same extent as the satellite DNA. In separate hybridization experiments (data not shown), the satellite was found to have similar binding reaction with control M. lysodeikticus DNA filters (0.8% per
1.711 g/cm3 and p = 1.699 this DNA, at p g/cm 3. In each of six experiments, the component of highest density in sheared intermediate density DNA exhibited p = 1.710 to 1.712 g/cm3, whether sheared by a French pressure cell or by sonic treatment. The component with the lowest buoyant density was variable TABLE 1. Reaction of intermediate density labeled in its behavior in CsCl gradients. When reDNA with filter-bound DNAsa leased by shearing intermediate density DNA in the French pressure cell (four experiments), Input 'H 3Hel-e the light component resembled that of Fig. 5B. labeled bound Input per DNA When released by sonic treatment (two experi10 jsg of 3H bound Filter-bound DNA ments), the low density component formed DNA on (% a hypersharp peak with a buoyant density of filter (%) min) 1.695 g/cm3. In contrast, labeled DNA from the main band of CsCl gradients such as that of A.tumefaciens 2.8, 2.4 0.88, 0.75 31,24 15955b Fig. 2, upon shearing, was observed to produce 14.1 19.5 215 Tomato shootc a single broad peak in CsCl gradients. Thus, 11.3 17.3 190 nuclearc Tomato intermediate density the separation of the labeled DNA into two components upon sheara Input labeled intermediate density DNA (1,100 ing does not appear to be an artifact due to counts/min per 0.2 ml) was incubated with DNA impurities in the DNA preparation (8). To avoid filters at 67 C in 2x SSC as described in the text. problems associated with contamination of the b Two filters were used per vial, and duplicate vials DNA by various compounds, DNA from an were prepared. c One filter was used per vial. infected plant was purified on a methylated =
(bounds
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z U
J. BACTERIOL.
HANSON AND CHILTON
60
-
z
H-40z LiJ
20
60
0
I'll
0
70
80 90 TEMPERATURE (°C)
mediate density satellite DNA and A. tumefaciens DNA, a trace of labeled satellite DNA was allowed to reassociate in the presence of a high concentration of unlabeled salmon DNA (control), A. tumefaciens DNA, or tomato DNA (either nuclear or total). The addition of bacterial DNA did not accelerate the reassociation of the labeled satellite, whereas addition of either preparation of tomato DNA caused a dramatic increase in rate (Fig. 7). From the rate of reassociation of labeled satellite in the presence of driving concentrations of tomato DNA, some inference can be drawn concerning the concentration and complexity of these DNA sequences in the plant. Such a calculation is not possible from the rate of reassociation of labeled satellite alone, because its concentration was too low to be measured meaningfully. The observed C.t, value of labeled satellite (with respect to conK00 centration of the tomato driver DNA) is 101 to
FIG. 6. Melting profiles of filter-bound duplexes formed between labeled intermediate density DNA and DNA filters containing tomato DNA or A. tumefaciens DNA. Duplexes described in Table 1 were subjected to thermal dissociation in 1 x SSC as described. Symbols: 0, labeled intermediate density DNA x A. tumefaciens DNA filter; A, labeled intermediate density DNA x tomato DNA filter; 0, authentic 3H-labeled A. tumefaciens DNA x A. tumefaciens DNA filter.
2 x 101.
DISCUSSION The experiments presented above strongly suggest that we have isolated an intermediate density satellite similar to that reported by Stroun and his collaborators (14, 15). The unusual behavior of this satellite DNA upon sonic treatment or shearing, i.e., the release of components of high and low buoyant density, characterizes this DNA fraction uniquely. We offer no explanation of this unexpected property, but presume that it is due to unusual base pair distribution in the satellite.
10 Ag of filterbound DNA). Such direct binding values are difficult to evaluate without further controls, because of the occurrence of variable extents of nonspecific background binding in DNA-filter reactions (6). Thermal dissociation profiles of duplexes 10-'~~~~~~0 (Fig. 6) show that the duplexes formed between 10' 100 ,,X,,, ,,,, ,,,, labeled satellite and A. tumefaciens DNA do not Lu exhibit the thermal stability of true bacterial Z, 80 i ° DNA duplexes (shown on the same graph for crcomparison). Satellite duplexes exhibit a Tm 0 (midpoint of thermal dissociation) of 85 C, U60 z whereas authentic A. tumefaciens duplexes 40 have Tm of 92 C. The duplexes formed be- z tween labeled satellite and tomato DNA exhibit 20 a Tm of 86 C. This is an unusually high value ai for plant duplexes: labeled main band tomato 100 10l 1o2 103 DNA under these conditions forms duplexes TIME (MINUTES) with a Tm of 76.5 C (data not shown), as exFIG. 7. Reassociation kinetics of labeled intermepected for mismatched duplexes formed by diate density DNA with unlabeled driver DNAs. redundant DNA under filter hybridization conReassociation measurements were performed as deditions. scribed. The Cjt scale refers to C. (moles of nucleotide Analysis of intermediate density labeled per liter) of the driver DNA multiplied by time satellite DNA by reaction with unlabeled (seconds). Unlabeled driver DNAs used were: 0, total driver DNAs in solution. In another attempt DNA from tomato; 0, nuclear DNA from tomato; A, to discern homology between labeled inter- salmon DNA; 0, A. tumefaciens 15955 DNA.
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VOL. 124, 1975
A. TUMEFACIENS DNA INTEGRATION
Our two hybridization studies have failed to provide any evidence for a bacterial DNA component in this satellite. In DNA filter reactions using authentic labeled A. tumefaciens 15955 DNA and homologous DNA filters, we observe about 10% of the input radioactivity bound per 10 ug of filter-bound tomato DNA in different experiments under the reaction conditions used here (6). If this intermediate density satellite were composed of approximately equal parts of bacterial and plant DNA, we would expect approximately 5% of input satellite radioactivity to bind to bacterial DNA filters, and we observe background levels of less than 1% binding in three experiments. Moreover, the Tm of the duplexes formed is 70 lower than that observed for authentic bacterial DNA duplexes. We conclude that this low level of binding is nonspecific background binding (6), and that the satellite DNA has failed to exhibit homology with bacterial DNA in filter reactions. Still more convincing is the evidence afforded by DNA-driven reactions of labeled satellite DNA. If half of the radioactivity of the satellite were in bacterial DNA, half of the labeled satellite 'should reassociate rapidly in the presence of 400 Mg of bacterial DNA per ml, which is 80% reassociated by 100 min under these conditions. The failure of any labeled satellite to exhibit homology with bacterial DNA under these circumstances conclusively rules out the model proposed by Stroun and his collaborators (14, 15). It does not rule out the possibility of any bacterial DNA in the satellite; the presence of up to 3% cannot be ruled out by this experiment. However, our findings invalidate the only evidence presented by earlier investigators supporting the model of DNA integration under these conditions. If the intermediate buoyant density of the labeled DNA, as well as its density segregation upon sonic treatment, were due to bacterial DNA integration, about 50% of it would have to be bacterial DNA, a possibility which our hybridization data rule out definitively. We conclude that there is no valid evidence for A. tu,,&efaciens DNA integration into tomato DNA upon treatment of cut stems with bacterial suspensions. We further add the example of this unusual labeled plant DNA satellite to the list of pitfalls in the use of CsCl buoyant density to characterize labeled plant DNA components (8). Separation of a labeled DNA into components of higher and lower density upon shearing occurs in a satellite which we have now characterized as a plant DNA component. This type of evidence for
foreign DNA integration is therefore subject to doubt. From the rate of reassociation of labeled satellite in the presence of driving concentrations of unlabeled tomato DNA, some inferences can be drawn about its complexity and concentration in tomato DNA. The occurrence of a visible satellite in tomato DNA at a buoyant density of 1.704 g/cm3 (7; Chilton, in press) suggests a likely candidate for the labeled satellite. However, the concentration and reassociation rates of the isolated satellite (Chilton, in press) are so high that in unfractionated tomato DNA under the renaturation conditions used here (0.15 M PB, 67 C), the visible satellite component would be over 75% reassociated by Cot = 0.2 (i.e., earlier than the first kinetic point in Fig. 5). Thus, although up to 20% of the radioactivity in labeled satellite DNA could be in the visible satellite, the bulk of the radioactivity is in some other component. Because DNA coding for ribosomal ribonucleic acid forms a part of the visible tomato satellite (Chilton, in press), such DNA is ruled out as the source of the intermediate density labeled satellite. Another reasonable candidate for labeled satellite would appear to be mitochondrial DNA, whose buoyant density in the case of several dicotyledonous plants appears to be 1.705 to 1.707 g/cm 3 (9, 17, 19), similar to that of the labeled satellite. If mitochondrial DNA has a genome size of about 70 x 106 (9), its CO;t if isolated, under our reaction conditions, would be approximately 10- l. However, if it represents approximately 1% of the total plant DNA, it would exhibit a Cot% of 10 with respect to concentration of total DNA. This is similar to the value observed for the DNA which is homologous to the bulk of labeled satellite DNA. Although nuclear DNA appears to contain about 2.5-fold lower concentration of the homologous DNA sequences, it is not free from them. If the labeled satellite DNA is in fact mitochondrial in origin, it appears that nuclear DNA either is heavily contaminated with or contains copies of mitochrondrial DNA in this plant. Repeated attempts to purify DNA from tomato mitochondria by several published procedures were unsuccessful. Thus, we were unable to test this hypothesis definitively. ACKNOWLEDGMENTS This research was supported by Public Health Service research grant number CA13015 from the National Cancer Institute. One of us (R.S.H.) was the recipient of Public
Health Service Senior Postdoctoral Fellowship number 1 F03
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HANSON AND CHILTONJJ. BACTERIOL.
CA53022-01 from the National Cancer Institute and was partially supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison. We thank Eugene W. Nester for making his laboratory facilities available for this investigation. LITERATURE CITED 1. Bendich, A. J. 1972. Effect of contaminating bacteria on the radiolabeling of nucleic acids from seedlings: false DNA "satellites." Biochim. Biophys. Acta 272: 494-503. 2. Bendich, A. J., and P. Filner. 1971. Uptake of exogenous DNA by pea seedlings and tobacco cells. Mutat. Res. 13:199-214. 3. Bonner, J., and A. W. Galston. 1959. Principles of plant physiology, p. 55. W. H. Freeman, San Francisco. 4. Chilton, M.-D., T. C. Currier, S. K. Farrand, A. J. Bendich, M. P. Gordon, and E. W. Nester. 1974. Agrobacterium tumefaciens DNA and PS8 DNA not detected in crown gall tumors. Proc. Natl. Acad. Sci. U.S.A. 71:3672-3676. 5. Chilton, M.-D., S. K. Farrand, F. Eden, T. Currier, A. J. Bendich, M. P. Gordon, and E. W. Nester. 1975. Is there foreign DNA in crown gall tumor DNA?, p. 297-311. In R. Markham, D. R. Davies, D. A. Hopwood, and R. W. Horne (ed.), Second Annual John Innes Symposium. North Holland/Elsevier, New York. 6. Farrand, S. K., F. C. Eden, and M.-D. Chilton. 1975. Attempts to detect Agrobacterium tumefaciens and bacteriophage PS8 DNA in crown gall tumors by DNA- DNA-filter hybridization. Biochim. Biophys. Acta 390:264-275. 7. Ingle, J., G. G. Pearson, and J. Sinclair. 1973. Species distribution and properties of nuclear satellite DNA in higher plants. Nature (London) New Biol. 242: 193-197. 8. Kleinhofs, A., F. C. Eden, M.-D. Chilton, and A. J. Bendich. 1975. Exogenous bacterial DNA not integrated or replicated in plants. Proc. Natl. Acad. Sci. U.S.A. 72:2748-2752.
9. Kolodner, 10. 11.
12. 13.
14.
R., and K. K. Tewari. 1972. Physicochemical characterization of mitochondrial DNA from pea leaves. Proc. Natl. Acad. Sci. U.S.A. 69:1830-1834. Ledoux, L., and P. Charles. 1972. In L. Ledoux (ed.), Uptake of information molecules by living cells, p. 29-46. North Holland, New York. McCarthy, B. J., and B. L. McConaughy. 1968. Related base sequences in the DNA of simple and complex organisms. I. DNA/DNA duplex formation and the incidence of partially related base sequences in DNA. Biochem. Genet. 2:37-53. Marmur, J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol. 3:208-218. Parenti, R., E. Guill6, J. Grisvard, M. Durante, L. Giorgi, and M. Buiatti. 1973. Transcient DNA satellite in dedifferentiating pith tissue. Nature (London) New Biol. 246:237-239. Stroun, M., and P. Anker. 1971. Bacterial nucleic acid synthesis in plants following bacterial contact. Mol. Gen. Genet. 113:92-98.
Stroun, M., P. Anker, P. Gahan, A. Rossier, and H. Greppin. 1971. Agrobacterium tumefaciens ribonucleic acid svnthesis in tomato cells and crown gall induction. J. Bacteriol. 106:634-639. 16. Stroun, M., P. Anker, and L. Ledoux. 1967. DNA replication in Solanum lycopersicum esc. after ab15.
17.
sorption of bacterial DNA. Curr. Mod. Biol. 1:231-234. Vedel, F., and F. Quetier. 1974. Physico-chemical
characterization of mitochondrial DNA from potato tubers. Biochim. Biophys. Acta 340:374-387. 18. Whiteley, A. H., B. J. McCarthy, and H. R. Whiteley. 1966. Changing populations of messenger RNA during sea urchin development. Proc. Natl. Acad. Sci. U.S.A. 55:519-525. 19. Wong, F. Y.. and S. G. Wildman. 1972. Simple procedure for isolation of satellite DNAs from tobacco leaves in high yield and demonstration of minicircles. Biochim. Biophys. Acta 259:5-12. 20. Yajko, D. M., and G. D. Hegeman. 1971. Tumor induction by Agrobacterium tumefaciens: specific transfer of bacterial deoxyribonucleic acid to plant tissue. J. Bacteriol. 108:973-979.
