Studies on Agrobacterium tumefaciens. V. Fate of exogenously added bacterial DNA in Nicotiana tabacum

Physiologizal

Plant Pathology

(1976)

8, 73-82

Studies on Agrobacferium tumefaciens. V. Fate of exogenously added bacterial DNA in Nicofiana tabacum C. I. KADO~

and

P. F. LIJRQUIN

Cell Biochemisty Section, Radiobiology 2400 Mol., Belgium (Acceptedfor

publication

November

Department,

Centre d’Etude

de 1’Energie

NuclLaire,

C.E.3V.IS.C.K.

1975)

DNA.DNA filter hybridization, DNA solution reassociation enrichment and DNA buoyant density analyses in cesium chloride were used to detect Agrobacterium tumxfaciens DNA sequences in exponentially growing tobacco cells that were exposed to the bacterial DNA in axenic culture. The bacterial DNA sequences were not detected in DNA obtained from tobacco callus cells treated with radioactive A. tumeftins DNA after 4 days growth at 27 “C. Furthermore, no radioactive DNA with a buoyant density intermediate between tobacco DNA and bacterial donor DNA was observed. Ultrasonication of DNA from DNA-treated callus cells did not release any presumptive A. tumefaciens DNA. Moreover, A. tumefaciens DNA was not replicated but degraded and re-utilized by the callus cells within the 4 days of incubation. Any traces of presumptive replicated bacterial DNA (
INTRODUCTION The crown gall disease of higher plants results from infection by Agrobacterium tumefacienr (Smith and Town.) Conn and is characterized by the formation of tumors at the site of infection. Because these tumors perpetuate in a non-self-limiting mode of growth eventually in the complete absence of bacteria, it has been long accepted that a “tumor-inducing principle” is elaborated by this pathogenic bacterium [4]. The nature of this tumorigenic substance has not been identified, but recent studies have proposed that a plasmid DNA may play a role in tumorigenesis [25, ,271 and, alternatively, a claim has been made that a DNA associated small RNA is the tumorigenic substance [I]. Such reports seemed, at first hand, to be supported by the apparent presence of bacterial DNA sequences equivalent to one or more genomes of A. tumefaciens in crown gall tumor cells [9, 20, 22,231. However, rigorous analyses of such crown gall tumor cells in axenic culture for these A. tumefaciens DNA sequences showed that little, if any, foreign DNA could be detected by DNA t Visiting NATO Senior Fellow Davis, California 95616, U.S.A.

from

the Department

of Plant

Pathology,

University

of California,

74

C. I. Kado

and

P. F. Lurquin

solution reassociation kinetic, reassociation enrichment and DNA.DNA filter hybridization techniques employing highly purified DNA preparations [S, 71. Moreover, no A. tumefaciensmessenger RNA sequences could be detected in these tumor cells [9], contrary to a preliminary report indicating bacterial-like RNA sequences of a 16 to 18 S RNA fraction [IS]. Therefore, in long-established crown gall tumor cell lines in axenic culture, very little A. tumefaciens DNA molecules seem to persist based on the relatively sensitive techniques used currently. Nevertheless, it is still uncertaih whether or not A. tumefaciensDNA is temporarily incorporated in the host cell during the initial phases of infection and then progressively eliminated thereafter without amplification. Therefore, during the initial phases of infection, 1eveIs of A. tumefaiens DNA may be high enough to permit easy detection. It has been shown that host cells are fully transformed within 2 to 4 days after infection [3, 4, 141 and it may be that within this period the host cell becomes modified by bacterial DNA (or plasmid). Kovoor [12] reported converting host cells into hormone prototrophic cells (a characteristic of crown gall tissues) by the application of purified A. tumefaciensDNA to callus tissue in axenic culture. We have therefore examined the fate of A. tumeftiens DNA applied to tobacco callus tissues in axenic culture within the time period for transformation to take place.

MATERIALS AND METHODS Preparation of DNA DNA of A. tumeftiens strain ID135 (ATCC 27912) was isolated and purified as previously described [II]. Radioactive A. tumefaciensDNA was prepared by the same procedure from cells grown in medium E [26] containing rH]-6-thymidine (O-2 @i/ml, 15 Ci/mM, C.E.N./S.C.K., Mol, Belgium). These purified DNA preparations were shaken vigorously at room temperature in the presence of chloroform and stored at 4 “C. Their sterility was checked by plating samples on agar medium 523 [IO]. The molecular weight of A. tumefuciensDNA used was 24 x 10” daltons as determined by viscometry [28]. DNA of Bacillus subtilis strain 168 was purified by the method of Saito & Muira [21]. Tobacco callus cellular DNA was purified by the procedure of Lurquin et al. [15] modified by Kado & Lurquin [ll] involving grinding the callus cells in liquid N,, digestion of the crude DNA with Proteinase K (Merck; 100 pg/ml) for 2 h, phenol extraction at pH 9.0, molecular sieving on Agarose (Sepharose 4B, Pharmacia), chromatography on hydroxylapatite (BioRad) and alkali treatment at 37 “C for 4 h. The yield of the method was 70 to 75% of the DNA originally present in the tissue. D.NA treatment of callus cells Tobacco calluses (Mcotiana tabacum cv. Havana) were grown on agar medium [18] in the dark at 27 “C. Pieces of callus tissue in logarithmic phase of growth (Lin & Kado, in preparation), weighing approximately 3 g were transferred to fresh medium, allowed to grow for 24 h and each piece was then treated with radioactive DNA

Fate

of

A. tumefaciens

DNA

in tobacco

75

ceils

from A. tumefciens (98 650 ct/min/pg, 215 pg/ml) by carefully applying O-2 ml of the DNA solution on top of each tissue sample. After 24 h, the DNA solution was no longer visible on the tissues. The tissues were allowed to grow for 96 more h before they were collected and treated with pancreatic deoxyribonuclease (Worthington) (100 pg/ml) for 20 min at 37 “C in O-14 M-Nacl to 0.01 M-MgCl,. The tissue samples were washed extensively with BE buffer (0.05 M-Na borate to 0.01 M-Na, EDTA), pH 9.0. Control samples consisted of callus tissue treated with 0.2 ml of [sH]-6-thymidine (15 Ci/pM) instead of radioactive DNA, but otherwise they were treated identically to that of the DNA-treated samples. In experiments designed to check for possible synthesis of A. tumefaciens DNA in the plant cell, similar callus cells were treated with 0.3 to 0.5 ml of solution of unlabeled A. tumefaciens DNA (355 pg/ml). Th ese cells were incubated in the dark for 96 h at 27 “C and subsequently labeled with O-2 ml of [sH]-6-thymidine. Control samples, which were not treated with A. tumefaziens DNA, were simply labeled with 0.5 ml of C3H]-6-thymidine. All samples were harvested after 6 days of incubation following the application of the radioactive thymidine. No deoxyribonuclease treatment was used in this experiment. All tissue samples were frozen immediately after harvesting and lyophilized before DNA extraction. DNA. DNA filter hybridization

The methods of hybridization followed those described previously [7, II]. Hybridization reactions were conducted at 46.4 “C with A. tumefaciens DNA immobilized on the filters and at 36-O “C with tobacco DNA filters. B. subtilis DNA filters and blank filters were also included in these reaction mixtures to monitor and estimate non-specific binding of the radioactive DNAs. The reactions were stopped after 24 h incubation and the filters were washed seven times with 5 x SSC in 50% formamide. Radioactivity was measured as before [II]. Thermal

chromatograbhic

analyses of rajidly

renatured DNA

Radioactive tobacco DNA from A. turn&&ens DNA-treated callus cells (4.39 pg/ml) or from untreated control cells (6.46 kg/ml) was mixed with an excess of unlabeled A. tumefaciens DNA (225 pg/ml) in 0.12 M-Na phosphate buffer, pH 6.8 (equimolar). The mixture was sonicated for 10 min with a MSE Mullard Ultrasonic Power Unit to a molecular weight of 300 000 daltons as determined by sedimentation velocity (M. Janowski, personal communication). The DNA solutions were then denatured for 15 min in boiling water and immediately transferred to a water bath at 69 “C. Kenaturation was allowed to proceed to C,t of 200 [5] for A. tumefciens DNA. Under these conditions, Cot values of 3.9 and 5.7 were obtained for the DNA of DNA-treated and untreated tobacco callus cells. A control sample consisted of a mixture of sonicated and denatured A. tumefaciens radioactive DNA (1.9 pg/ml; 98 650 ct/min/pg) and excess unlabeled A. tumefaciens DNA (225 pg/ml) that was renatured along with the above test samples under identical conditions. After renaturation, the double-stranded DNA was obtained by hydroxylapatite chromatography at 69 “C I.51 and then characterized by thermal chromatography c171.

76

C. 1. Kado

and

P. F. Lurquin

Density analysis of DNA

The DNA samples were centrifuged to equilibrium in 3 ml CsCl density gradients at 25 “C for 63 h at 33 000 rev/min in a Spinco SW 50.1 rotor in the presence of 5 pg each of two unlabeled reference DNAs of known density: StreptomycescoelicolorDNA (p = 1.730 g cm-3) and E. coli DNA (p = l-710 g cm-3). The gradients were fractionated from the top and optical density at 254 nm was automatically recorded with an ISCO density gradient fractionator connected to a BD8 Kipp and Zonen recorder. Radioactivity was determined with 1 ml of distilled water, and countings were made in a Packard liquid scintillation spectrometer, model 2425. RESULTS Uptake of A. tumefaciens DNA

To determine the initial fate of A. tumefaciens DNA in tobacco cells growing in axenic culture, sterile radioactive DNA (43 pg DNA/sample; total input = 4.2 x lo6 ct/min sample) or [3H]-thymidine was dispensed onto callus tissue samples. The samples were allowed to continue growth for 4 days and were then harvested, treated with deoxyribonuclease, washed and processed for DNA extraction and purification as described in Materials and Methods. Equivalent callus tissue samples were also treated with A. tumefaciens tritiated DNA and at daily intervals, processed for total tissue DNA as above. The total radioactivity recovered by precipitation of the DNA with 5% trichloroacetic acid increased to 1*S% of the total ct/min added after the first day of incubation and then to 3.2% after the second day of incubation. The amount of precipitable DNA remained relatively constant at this level thereafter. The labeled DNA from callus tissue samples, that were either treated with A. tumefaciens rH]-DNA or rH]-thymidine and incubated for 4 days (a period which acid-precipitable radioactivity stabilized), was hybridized at 20 “C below ‘T, in 5 x SSC and 50% formamide to A. tumefaciens DNA and tobacco DNA immobilized on nitrocellulose filters. The results of this assay are summarized in Table 1. It can be seen that no significant amount of DNA from the samples treated withA. tumefaciens radioactive DNA bound to A, tumefaciens DNA filters. On the other hand, these DNA preparations readily hybridized with tobacco DNA filters (Table 1) indicating that most, if not all, of the labeled DNA sequences were tobacco DNA. The buoyant density of the radioactive DNA molecules was that of tobacco DNA as judged from density analysis in CsCl density gradients [Fig. 1 (a) and (b)]. All of the radioactivity banded at the position of tobacco reference DNA (p = 1.696 g cmd3) regardless of the treatment, i.e. whether the callus cells were treated with rH]-thymidine [Fig. o radioactive DNA peak was observed 1 (a) ] or A. tumefaciens rH]-DNA [Fig. 1 (b)]. N possessing a density intermediate between tobacco DNA and bacterial donor DNA, and no free A. tumefaciens DNA (p = 1.718 g cm-3) was detected. It has been claimed that ultrasonication of high molecular weight DNA can release presumably integrated bacterial DNA sequences after incubating plants with radioactive bacterial DNA [I.!?, 241. However, ultrasonication of our DNA preparations showed no such release of bacterial DNA when examined by density analysis in CsCl [Fig. 1(c) and (d)]. These results, together with the above hybridization data, suggest that no recognizable sequences of A. tumefaciens [3H]-DNA remained in the tobacco tissue after 4 days of incubation.

77

Fate of A. fumefaciens DNA in tobacco cells

F 20 Qx .; IO 1 u =. 0 .E .$ x 2g 6

0.4 -2 0.2 f z O .t; B 5.;” a 0.4 O

14 r, 2

0.2

,O 0

IO

20

30

40

0

Fraction

IO

20

30

40

Number

FIG. 1. Density analyses in cesium chloride gradients of DNA purified from tobacco callus cells treated as follows: (a) callus labeled with [sH]-thymidine; (b) callus labeled with A. tumeficienc rH’j-DNA; (c) callus labeled with PHI-thymidine and its DNA sonicated; (d) callus labeled with A. tumefuciens rH]-DNA and the callus DNA sonicated. E. coli. DNA and S. co&color DNA were used as density markers (see text). (O-O) fH]-Radioactivity (ct/min x 10”).

TABLE

DNA.DNA Treatment callus

of

[sH]-thymidine

A. tumefaciens

1

jilter hybridization analysis of tobaccocallus cell D.NA after exposure to A. tumefaciens [aHI-DNA

Specific radioactivity (ctjmin pg) of callus 116 863 11 558

DNA

Input (ct/min) 183 525 25 716

pg DNA

hybridized/

pg A. tumefuciensDNA

,ug DNA@ hybridized/ ng tobacco DNA

0 0

0.12 0.23

0.0005b

0.24

[sH]-DNA

A. tumefacieenr

4499

9245

[sH]-DNA 0 At least 12% of the input ct/min hybridized. Figures based on the ct/min after non-specific and non-homologous background ct/min were subtracted. b This value may be insignificant since a balance of only 2.1 ct/min remained cellulose filter after non-specific and non-homologous background ct/min were

remaining per nitrosubtracted.

Analysis of A. tumefaciens D3vA synthesis in tobacco callus cells

The specific radioactivity of DNA obtained from the bacterial [3H]-DNA-treated callus cells was about 14-fold lower than that of DNA obtained from cells treated with C3H]-thymidine and therefore may be insufficient for detecting very small amounts (less than 0.5% of the total radioactivity in DNA) of A. tumefuciens DNA in these callus cells. To determine if any trace amounts of A. tumefaciens DNA were present and replicated in the DNA-treated callus cells, experiments were performed

78

C. 1. Kado

and

P. F. Lurquin

by incubating tobacco callus tissue samples with unlabeled A. tumefaciens DNA and with [sH]-thymidine, and analyzing the tissue DNA by filter hybridization after allowing sufficient time for any possible uptake and integration (see Materials and Methods). In this experiment, the specific radioactivity was sufficiently high to detect extremely small quantities of A. tumefaciens DNA in the treated tobacco callus DNA preparations. The lower limit of detecting A. tumefaciens DNA was estimated from calibration data using known amounts of A. tumejbciens DNA (Table 2). Taking into account the input ct/min and specific radioactivities, the limit of detection was at least 0.01 o/o apparent homology in total tobacco callus cell DNA. Hybridization analyses of the DNA from five samples of tobacco calluses treated with A. tumefaciens DNA and [sH]-thymidine showed the complete absence of A. tumefaciens DNA sequences (Table 2).

D.NA.DNA

Jilter

Specific radioactivity Wmin fe)

Treatment Calibration input (vg A. tumefaciens DNA) o-192 1.925 4.810 9.625 Callus

treated None None

A. tumftiens

TABLE 2 hybridization ana&is of tobacco callus cell DNA labeled with after exposure to unlabeled A. tumefaciens DNA

98 98 98 98

650 650 650 650

Input (ct/min)

18 189 474 949

743 408 506 013

pg hybridized/Kg A. tumefackns DNA

0.002 0.028 0.072 0.105

[3H]-thymidine

ct/min bound pg A. tumfaciens DNA”

262 2841 7113 10 415

Apparent homology (%lb

-

with 81 500 48 900

287 800 316 100

0 0

Od 0

0 0

0 0 0 0 8.4

0 0 0 0 0901

DNA 10 2 3 4 5

69 94 137 72 93

900 500 600 300 000

306 229 163 269 68

700 800 200 900 000

0 0 0 0 0~00009

@ 10 vg of A. tumefacienr DNA immobilized on 5 mm nitrocelloluse filters (Sartorius, O-2 pg pore size). b Figures based on the specific radioactivities, total input ct/min, calibration input ct/min and the amount of DNA on each filter after measuring the ct/min bound. An equivalent synthesis of bacterial putative DNA with that of the tobacco callus DNA was assumed. 0 Numbers represent different tobacco callus samples treated with non-radioactive A. tumefacienr DNA and subsequently labeled with [3H]-6-thymiclme as described in Materials and Methods. d Ct/min remaining after non-specific and non-homologous ct/min were subtracted. The 8.4 ct/min bound in sample 5 may be considered insignificant and within the error of the experiment.

The results of these analyses were further supported by DNA sequence enrichment and characterization of the enriched DNA. Radioactive callus DNA from DNA-treated and [3H]-thymidine-treated tobacco callus was allowed to reassociate in the presence of excess unlabeled A. tumefaciens DNA to Cot = 200 for the bacterial

Fate

of

A. fumefaciens DNA

in tobacco

79

cells

DNA. Under these conditions 75% of the bacterial DNA renatured as determined by fractionation of single-stranded and double-stranded DNA on hydroxylapatite at 69 “C. At the same time, only 3.3 and 4.6% of the total tobacco DNA from DNA-treated and [3H]-thymidine-treated tissue respectively were found in the double-stranded DNA fraction. Thermal stabilities and melting characteristics of these double-stranded DNAs were examined by thermochromatography on hydroxylapatite. Figure 2(a) is an example of the melting characteristics of these renatured DNA samples. Note that the derivative melting curves of these enriched DNAs from DNA-treated and [3H]-thymidine-treated tissues are superimposed and clearly

b)

I

80 Temperature

90

I

10(

PC)

FIG. 2. Melting profiles of rapidly renatured tobacco DNA and renatured A. tumefciemDNA. (a) Derivative melting of rH]-DNA from A. tumfaciem (O-O) ; [sH]-DNA from callus tissue treated with A. tumefacienr DNA (O----O) ; and [3H]-DNA from control callus tissue (A-A). (b) Cumulative melting curves (normalized to %) based on the radioactivity values in (a). Symbols identical to those in (a).

80

C. I. Kado

and

P. F. Lurquin

distinct from that of the reassociated A. tumefaciens DNA. Likewise, there were no detectable DNA sequences displaying a T* higher than that of tobacco DNA [Fig. 2(b)] after renaturation of the latter DNA in the presence of excess A. tumefaciens DNA. DISCUSSION

This study was undertaken to determine the fate of A. tumefaciens DNA in tobacco callus cells since transformation by adding purified A. tumefaciens DNA to callus tissue has been claimed [ 121. Contrary to this report, our results clearly showed that purified A. tumefaciens DNA was extensively degraded when added to tobacco callus cells growing in axenic culture. The DNA degradation products were re-utilized by these cells for their own DNA synthesis within 4 days of growth (a time period sufficient for transformation to take place [3, 4, 141). Although we did not measure deoxyribonuclease activity of the tobacco callus cells, similar experiments performed with Arubidopsis thaliana callus cells in axenic culture showed that bacterial DNA is extensively degraded and reutilized for endogenous recipient cellular DNA synthesis as a result of nuclease-mediated production of very high amounts of acid-soluble nucleotides (Lurquin, unpublished data). Also, it has been reported that deoxyribonuclease is apparently excreted by tobacco cells in suspension culture [2]. Bendich & Filner [Z] reported that tobacco cells in suspension culture were able to take up Pseudomonas aeruginosa DNA and homologous tobacco DNA, both of which peristed for about 12 h. However, they observed that if the DNA-treated cells were transferred to fresh medium and incubated for an additional 35 to 72 h, no donor DNA remained as judged from density analysis in CsCl. All of the radioactivity banded at the position of the recipient tobacco cell DNA. This recipient cellular DNA was not analyzed by hybridization for the possible presence of small amounts of donor bacterial DNA. Based on density analyses in C&l, Heyn & Schilperoort [8] showed that 0.025% of the input A. tumefacienr DNA was associated with tobacco protoplasts even after 24 h incubation. However, it was uncertain whether there was actual uptake, penetration or simply adsorption of the bacterial DNA. We have employed sensitive assay procedures to demonstrate that no more than 0.01 y0 of the total tobacco callus cell DNA could contain bacterial DNA sequences and that almost all of the added DNA was digested and re-utilized. Thus, the possibility seems remote to genetically modify or transform plant cells in axenic culture, such as tobacco callus cells by direct incubation with purified foreign DNA unless very minute amounts of DNA not detectable by present methods survived the attack by plant nucleases and make its way to target sites culminating in genetic modification. The use of foreign DNA protected by natural nuclease inhibitors may warrant further study, but it appears that plant cells in axenic culture are highly refractory to the direct uptake, integration and replication of purified bacterial DNA and that the efficiency of transformation, if any, would be extremely low. The authors would like to express their thanks to Lucien Ledoux for providing generous laboratory facilities and for his interest and discussions in the preparation

Fate

of A. tumefaciens

DNA

in tobacco

cells

81

of this manuscript; to Karl A. Drlica and S. Deeb for critical reading of the manuscript; and special thanks to Joseph Gerits for preparing radioactive A. tumefaciens cells. Portions of the experiments were carried out in the laboratory of Noboru Sueoka, University of Colorado, Boulder, for which we gratefully acknowledge. This research was supported by NIH grant CA-I 1526 from the National Cancer Institute. REFERENCES 1. BELJANSKI, M., AARON-DA CUNHA, M. I., BELJANSKI, P., MANIGAULT, P. & BOIJRGAREL, P. (1974). Isolation of the tumor-inducing RNA from oncogenic and nononcogenic Agrobacterium tumefaciens. Proceedings of the JVational Academy of Sciences U.S.A. 71, 1585-1589. 2. BENDICH, A. J. & FILNER, P. (1971). Uptake of exogenous DNA by pea seedlings and tobacco cells. Mutation Research 13, 199-214. 3. BRAUN, A. C. (1943). Studies on tumour inception in the crown-gall disease. American Journal of Botany 30, 674677. 4. BRAUN, A. C. (1947). Thermal studies on the factors responsible for tumor initiation in crown gall. AmeriGan 3ournal of Botany 34, 234240. 5. BRIITEN, R. J. & KOHNE, D. E. (1965). Nucleotide sequence repetition in DNA. Carnegie Institution Year Book 65, 78-106. 6. CHILTON, M. D., CURRIER, T. C., FARRAND, S. K., BENDICH, A. J., GORDON, M. P. & NESTER, E. W. (1974). Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proceedin,os of the National Academy of Sciences U.S.A. 71, 3672-3676. 7. DRLICA, K. A. & KAIJO, C. I. (1974). Quantitative estimation of Agrobacterium tumefaciens DNA in crown sall tumor cells. Proceedines of the National Academv of Sciences U.S.A. 71, 3677-3681. 8. HEYN, R. “F. & SCHILPEROORT, R. A. “(1973). The use of*ppotoplasts to follow-the fate of Agrobacterium tumeftiens DNA on incubation with tobacco cells. Colloques Internationaux C.N.R.S. No. 212,385-395. 9. Ado C. I. (1974). Studies on Agobacterium tumefackns. III. A concept on the role of Agrobacterium tumefaiens DNA in plant tumorigenesis. In Proceedings of the First Intersectional Congress of the International Association of Mirrobiological Societies, Tokyo, September 1974, Vol. 1, pp. 100-130. 10. KADO, C. I., HESKETT, M. G. & LANGLEY, R. A. (1972). Studies on Agrobacterium tumefaciens. I. Characterization of strains lD135 and B6, and analysis of the bacterial chromosome, transfer RNA and ribosome for tumor-inducing ability. Physiological Plant Pathology 2,47-57. 11. KADO, C. I. & LURQIJIN, P. F. (1975). Studies on Agrobacterium tumefaciens. IV. Nonreplication of the bacterial DNA in mung bean (Phaseolus uureus). &chemical and Biophysical Research Communicatiotls 64, 175-183. 12. KOVOOR, A. (1967). Sur la transformation de tissus normaux de Scorsonhe provoquee in vitro par l’acide dboxyribonucltique d’Agroba&rium tumefaciens. Comptes rendus hebdomadaire des seances de I’Academie 265, 1623-1629. 13. LEDOUX, L. & HUART, R. (1969). Fate of exogenous bacterial deoxyribonucleic acids in barley seedlings. Journal of Molecular Biology 43, 243-262. 14. LIPPINCOTT, J. A. & LIPPINCOTT, B. B. (1967). Time required for tumour initiation by Agrobacterium tumefaciens on pinto bean leaves. Nature 213, 596-598. 15. LURQUIN, P. F., TSHITENGE, G., DELAUNOIT, G. & LEDOIJX, L. (1975). Isolation of DNA from plant cells by gel filtration on agarose. Analytical Biochemistry 65, l-10. 16. MILO, G. & SRIVASTAVA, B. I. S. (1969). RNA:DNA hybridization studies with crown gall bacteria and the tobacco tumor tissue. Biochemical and Biophysical Research Communications 34, 196-199. 17. MIYAZAWA, Y. & THOMAS, C. A., JR (1965). Nucleotide composition of short segments of DNA molecules. 3ournal of Molecular Biology 11, 223-237. 18. MUFCASHIGE, T. & SKOOG, F. ( 1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia plantarum 15, 473-497. 19. PATILLON, M. (1974). Quantitative evaluation by reassociation kinetics of Agrobacterium DNA sequences residing in the genome of a crown gall tissue culture. Journal of Experimental Botany 25,860-870. 20. QU&ER, F., HUGEUT, T. & GUILLO, E. (1969). Induction of crown gall: partial homology between tumor cell DNA bacterial DNA and the GC-rich DNA of stressed normal cells. Biochemical and Biophysical Research Communications 34, 128-133. 6

82

C. I. Kado

and

P. F. Lurquin

2 1. SAITO, H. & M~URA, K.-I. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochimica et biophrsica acta 72, 619-629. 22. SCHILPEROORT,R. A., VAN SITTERT, S. J. & SCHELL, J. (1973). The presence of both phage PS8 and Agrobackrium tumefaciens DNA base sequences in AG-induced sterile crown gall tissue cultured in vitro. EuroPean Journal of Biochemistry 33, l-7. 23. SRIVASTAVA,B. I. S. (1970). DNA:DNA hybridization studies between bacterial DNA, crown gall tumor DNA and normal cell DNA. Life Sciences 9 (II), 889-892. 24. STROUN, M., ANKER, P. & LEDOUX, L. (1967). DNA replication in Solanum lysoperticum esc after absorption of bacterial DNA. Currents in Modern Bioloa 1, 231-234. 25. VANLAREBEKE,N.,ENGLER,G.,HOLSTER, M.,VANDEN ELSAOKER,S.,ZAENEN,I.,SCHILPEROORT, R. A. & SCHELL, J. (1974). Large plasmid in Agrobacterium tumefciens essential for crown gall inducing ability. Nature 252, 169-l 7 1. 26. VOGEL, H. J. & BONNER, D. M. (1956). Acetylornithinase of Escherichia coli partial purification and some properties. Journal of Biological Chemistry 218,97-106. 27. ZAENEN, I., VAN LAREBEKE, N., TEUCHY, H., VAN MONTAGU, M. & SCHELL, J. (1974). Supercoiled circular DNA in crown gall inducing Agrobacterium tumefaciens strains. Journal of Molecular Biology 86, 109-127. 28. ZIMM, B. H. & CROTHERS,D. M. (1962). Simplified rotating cylinder viscometer for DNA. Proceedings of the JVational Academy of Sciences U.S.A. 48, 905-911.