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The origin of wound-induced satellite DNA in pea seedlings
Cell Differentiation, 4 (1975) 139--145 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE ORIGIN OF W O U N D - I N D U C E D S A T E L L I T E D N A I N P E A SEEDLINGS
D. BROEKAERT and R. VAN PARIJS
Laboratory of Physiological Chemistry, Faculty of Medicine, and Laboratory of Biochemistry, Faculty of Agricultural Sciences State University of Ghent, Belgium Accepted 6 March 1975 DNA synthesis in wounded pea seedlings was studied by incorporation of [6-3H] thymidine. It was shown that the satellite DNA, synthesized within the first two days after wounding, is not of plant origin, but produced by contaminating bacteria, although a strong sterilizing procedure was used. Main-band DNA synthesized after wounding seems to be the only plant DNA synthesized during the stress-period. T h e p r o b l e m o f D N A s y n t h e s i s in w o u n d e d plants is very actual, especially since i n f e c t i o n o f the w o u n d e d p l a n t s with Agrobacterium tumefaciens results in t h e i n d u c t i o n o f a C r o w n gall t u m o u r . Kupila et al. ( 1 9 6 1 , 1 9 7 1 ) a n d Rasch ( 1 9 6 4 ) o b s e r v e d D N A s y n t h e s i s a f t e r sterile w o u n d i n g o f Vicia faba s t e m s e g m e n t s . L i p e t z ( 1 9 6 7 ) f o u n d t w o waves o f D N A s y n t h e s i s a f t e r w o u n d i n g s t e m tissues o f Kalanchof. W a t a n a b e et al. ( 1 9 7 3 ) r e p o r t e d the n e t synthesis o f D N A a f t e r c u t t i n g p o t a t o t u b e r tissues. 48 hr a f t e r w o u n d i n g a n u c l e a r GC-rich satellite D N A is s y n t h e s i z e d in Solanum lycopersicum, Scorsonera hispanica and Glycine max (Guill~ et at., 1 9 6 8 ) ; m o r e o v e r , s o m e h o m o l o g y was f o u n d b e t w e e n the n u c l e a r h e a v y satellite D N A o f stressed cells a n d D N A o f Agrobacterium tumefaciens (Qu~tier et al., 1969). F r o m these results, a m o d e l f o r the i n t e g r a t i o n o f bacterial D N A i n t o the h o s t p l a n t g e n o m e was d e d u c e d (Guill~ et al., 1970a, b). T h e p u r p o s e o f o u r w o r k was to d e m o n s t r a t e the i n d u c t i o n o f D N A s y n t h e s i s in w o u n d e d p e a seedlings b y using a r a d i o a c t i v e p r e c u r s o r . T h e labelled D N A was c h a r a c t e r i z e d b y d i f f e r e n t m e t h o d s . MATERIALS AND METHODS Dry seeds o f Pisum sativum L. were t h o r o u g h l y sterilized b y s h a k i n g in c h l o r a m i n e T (1%) and e x t e n s i v e washing with sterile water, b e f o r e and a f t e r i m b i b i t i o n (15 hr at 20°C). T h e seeds g e r m i n a t e d d u r i n g 5 or 6 d a y s in sterile Petri dishes, at 25 ° C, in t h e darkness. T h e seedlings were w o u n d e d b y r e m o v i n g t h e c o t y l e d o n s a n d the u p p e r p a r t o f the e p i c o t y l a n d p l a c e d with t h e i r r o o t s in an a q u e o u s s o l u t i o n o f [ 6 - 3 H ] t h y m i d i n e at 25°C. T h e w o u n d z o n e s were excised a f t e r one, t w o or t h r e e days.
140 The DNA was extracted following the m e t h o d of Stern ( 1 9 6 8 ) u s i n g deproteinization in 3 to 4 M NaC104 and furt her purified by preparative CsC1 gradient centrifugation (Flamm et al., 1966). Pea seedling DNA, when used in excess, was isolated from chromatin (Coucke et al., 1972). B u o y a n t densities were determined in CsC1 gradients in a Spinco Model E analytical centrifuge, using Micrococcus lysodeikticus DNA as a reference (Schildkraut et al., 1962). Renaturation of sonicated DNA was done in 0.12 M phosphate buffer (pH 6.8), 0.3 M NaC1 and 0.1% sodium lauryl sulphate, at 62 and 72°C, respectively 20 to 25°C under the Tm of pea and bacterial DNA in 1.0 SSC (0.15 M NaC1 -- 0.015 M Na citrate). The reaction mixture was analysed on a h y d r o x y l a p a t i t e (HAP)-column (McCallum et al., 1 9 6 7 ) a t different Cotvalues (Britten et al., 1966). The renaturated DNA was tested for thermal stability by using a HAP-column at 55 ° C, which was subsequently brought at higher temperatures. At each interval of 2.5°C the denatured DNA was eluted. All samples tested for radioactivity were diluted and c o u n t e d in Insta-gel scintillator (Packard), using the Packard Tri-Carb 3380 scintillation counter. The sterility of the medium and the wounded plants were tested by incubation o f diluted aliquots on agar plates enriched with general bacterial and yeast media. Bacterial DNA was isolated following the m e t h o d of Marmur
(1961). RESULTS The preparative CsC1 gradient profiles from several DNA preparations of l-day-old wound tissues were not exactly reproducible. In most cases a labelled satellite DNA peak (5 = 1.712--1.716 g/cm 3) was found at the heavy side o f the weakly labelled main-band DNA (Fig. la). The characteristics of r en atu r atio n at 62 ° (Table Ia) and 72°C (Table Ib) of the total DNA, isolated f r o m w o u n d e d tissues (OD260 measurement) and of the labelled satellite DNA (radioactivity counting) were different. When renaturation of labelled w o u n d tissue satellite DNA was studied by renaturation--hybridization at 62°C in the presence of a 100- to 1000-fold excess of pea seedling DNA (Table Ic) we could n o t d e t e c t any significant hybridization between w o un d tissue satellite DNA and the excess pea seedling DNA. These negative results, together with some observations by other authors, led to a more critical study. Obviously, a small a m o u n t of c o n t a m i n a t e d seedlings at the beginning of the incubation period were responsible for the clear c o n t a m i n a t i o n at the end of the incubation period. Mostly, 2 or 3 contaminating bacteria were isolated, sometimes only one contaminating bacterium was found. This organism most closely resembled an Erwinia, as established in the L a b o r a t o r y for Microbiology, Faculty of Sciences, State University of Ghent. From these contaminating bacteria, mixed with carrier pea seedlings, the total DNA was isolated. The CsC1 gradient profile and
141
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Fig. 1. P r e p a r a t i v e CsCl g r a d i e n t c e n t r i f u g a t i o n of a) o n e - d a y - o l d w o u n d tissue D N A isolated f r o m c o n t a m i n a t e d seedlings; b) r a d i o a c t i v e bacterial D N A isolated in the prese n c e of carrier p l a n t tissues; c) o n e - d a y - o l d w o u n d tissue D N A isolated f r o m asceptic seedlings. ~3 --~J, U V - m e a s u r e m e n t at 260 nm. v. e, r a d i o a c t i v i t y m e a s u r e m e n t .
density of this labelled bacterial DNA was identical to the profile and density of w o u n d tissue satellite DNA. The isolated bacterial species was cultured on large scale. Fr om these cultures a sample was transferred to a liquid medium, enriched with [6-3 H] thymidine. Labelled bacterial DNA was again isolated in the presence of carrier plant tissues; the density of this 'satellite' DNA (Fig. l b ) was identical to the density of w o u n d tissue satellite DNA. Thermally denatured, labelled satellite DNA from w ound tissues and a 100- to 1000-fold excess of single-stranded bacterial DNA wery incubated together: the excess bacterial DNA favoured the renat urat i on at 72°C of the w o u n d tissue satellite DNA (Table Id). In the control experiment, labelled bacterial DNA incubated in the presence of excess unlabelled bacterial DNA r e n atu r ed roughly to the same e x t e n t as the unlabelled DNA (Table Ie). The thermal stability and the melting t e m p e r a t u r e (Tm) of the di fferent DNAs
142 TABLE I Renaturation of total DNA isolated from wounded tissues, and of labelled wound tissue satellite DNA and bacterial DNA in the presence of excess unlabelled DNA of different origin (HAP-chromatography).
Cot
0.12 M fraction
0.16 M fraction
0.30 M fraction
OD26o (%)
cpm (%)
OD260 (%)
cpm (%)
OD26o (%)
cpm (%)
a) 1.10 -I 1.10 ° 1.10 J 7.7.101
63.3 33.0 26.8 21.1
90.1 75.9 59.7 52.7
12.5 19.9 15.7 14.9
5.9 8.5 13.4 14.5
24.2 47.1 57.5 64.0
4.0 i5.6 26.9 32.8
b) 1.10 -1 1.10 o 1.101 7.7.101
71.6 41.8 32.8 28.9
90.7 77.5 60.2 54.2
11.5 14.5 12.3 17.4
5.6 7.9 14.2 9.9
16.9 43.7 54.9 61.7
3.7 14.6 25.6 35.9
c) 1.101 1.102 1.103 *
36.6 28.5 21.7
88.6 79.8 65.7 72.5
11.9 I 1.0 7.1 --
4.3 8.5 10.1 6.3
51.5 60.5 71.2 --
7.1 11.7 24.2 21.1
d) 5.102 *
1.5 --
3.4 77.5
0.5 --
0.2 10.2
98.0 --
96.4 12.3
e) 4.5.102 *
4.6 --
8.9 96.0
0.8 --
0.6 1.0
94.6 --
90.5 3.0
Fractionation of the reaction mixture was done following the method of McCallum et al. (1967); the OD260 values obtained for the single-stranded DNA (0.12 M fraction) were corrected for hyperchromicity (--30%); partially renatured DNA was eluted with 0.16 M phosphate buffer; the 0.30 M fraction contains the renatured DNA. * Control reaction in which single-stranded labelled DNA was incubated in the same concentration and for the same time, as for the incubation in the presence of excess DNA. were c o m p a r e d and f o u n d to be closely related: 9 3 . 0 0 ° C for bacterial D N A and 92.50°C for wound tissue satellite DNA; 88.75°C for the renatured d u p l e x s t r u c t u r e s at Cot 500 w h e n w o u n d tissue satellite D N A or labelled b a c t e r i a l D N A r e n a t u r e d in t h e p r e s e n c e o f e x c e s s b a c t e r i a l D N A . By a d d i n g the f o l l o w i n g a n t i b i o t i c s to the r a d i o a c t i v e i n c u b a t i o n m e d i u m : penicillin (100 U/ml), streptomycin (100 pg/ml) and chloramphenicol (50 p g / m l ) ( S k o o d e t al., 1 9 7 2 ) , o n l y a c c i d e n t a l b a c t e r i a l c o n t a m i n a t i o n t o a s m a l l e x t e n t c o u l d be d e m o n s t r a t e d . N o s a t e l l i t e D N A was p r e s e n t in lab e l l e d D N A p r e p a r a t i o n s o f 1, 2 o r 3 - d a y - o l d w o u n d t i s s u e s ( F i g . l c ) . T h e labelling pattern coincided with the UV-absorbing fractions of the main-band D N A in p r e p a r a t i v e CsC1 g r a d i e n t c e n t r i f u g a t i o n . M i c r o d e n s i t o m e t e r t r a c i n g s of U V - a b s o r p t i o n p h o t o g r a p h s o f D N A isolated f r o m w o u n d tissues, after a n a l y t i c a l CsC1 g r a d i e n t a n a l y s i s r e v e a l s o n e s y m m e t r i c a l D N A b a n d
143
Q.
Ref.
\A
Fig. 2. M i e r o d e n s i t o m e t e r tracings of U V - a b s o r p t i o n p h o t o g r a p h s of 3 t o 4 /.tg pea D N A a n d i pg r e f e r e n c e D N A ( M i c r o c o c c u s l y s o d e i k t i c u s , 5 = 1.731 g / c m 3 ) in CsCI ( S u p r a p u r - - Merck). a) D N A isolated f r o m c o n t r o l 12-day-old u n w o u n d e d e p i c o t y l tissues; b) D N A i s o l a t e d f r o m 1-day-old w o u n d tissues; c) D N A i s o l a t e d f r o m 2-day-old w o u n d tissues; d) D N A isolated f r o m 3-day-old w o u n d tissue; M = meniscus.
144 (5 = 1.695 g/cm 3) in all preparations (Fig. 2). The main-band DNA, synthesized after wounding is actually under investigation. DISCUSSION Our data concerning DNA synthesis in wounded peas clearly show the bacterial origin of the satellite DNA and emphasize the essentiality of efficient precautions in labelling experiments. No satellite DNA could be demonstrated using asceptic conditions. Contaminating bacteria which poorly contribute to UV-absorption of nucleic acids are of great importance in studies with radioactive precursors and could lead to extremely different interpretations (e.g. Lonberg-Holm, 1967; Bendich, 1972). Several authors {e.g. Green et al., 1966; Meyer et al., 1967; Sobota et al., 1968; Pearson et al., 1972; Sarrouy-Balat et al., 1973; Delsemy, 1974) recall different results in the field of satellite DNA and the so-called metabolic DNA, because of the objection cited above. The DNA synthesized in wounded pea seedlings is undoubtedly characterized as main-band DNA. Preparative CsC1 gradient centrifugation of aseptically cultured wounded pea seedlings demonstrates only main-band DNA synthesis; analytical CsC1 centrifugation reveals a symmetrical DNA band with a density of 1.695 g/cm 3 as observed by Beridze et al. (1967) and Ingle et al. (1973). The satellite DNA (6 = 1.707 g/cm 3) observed by Beridze et al. {1967) is probably of mitochondrial origin (Kolodner et al., 1972a). Also pea chloroplast DNA bands as a satellite peak (5 = 1.698 g/cm 3) (Kolodner et al., 1972b). Both organelle DNAs are not present in our preparations, or at least masked in the main-band DNA. Up till now, pea nuclear satellite DNA is u n k n o w n . These results differ from the results of Guill6 et al. (1968) who developed a model for the integration of bacterial DNA into the host plant genome (Guill6 et al., 1970a, b), based on the synthesis of a nuclear GC-rich DNA, partly homologous to Agrobacterium tumefaciens DNA (Qu6tier et al., 1969). This model implies the presence of A. tumefaciens DNA in the gehome of transformed cells, an assumption lacking convincing evidences since recent attempts to demonstrate this were unsuccessful (Chilton et al., 1974; Drlica et al., 1974; Eden et al., 1974). The proposed model is not only premature but certainly not a general one. The synthesis of a nuclear GC-rich DNA is probably not an obligatory step in Crown gall induction. We suppose that wounding plants rather induces DNA synthesis of the same type as during the onset of the germination, instead of a specific type of DNA synthesis. ACKNOWLEDGEMENTS We wish to thank Prof. Dr. J. De Ley and Mr. R. Tytgat (Laboratory of Microbiology, Faculty of Sciences, State University of Ghent, Belgium) for the analytical CsCl gradient
145 experiments and for fruitful discussions; Dr. P. Coucke for aid and advice in chromatin isolation.
REFERENCES
Bendich, A.J.: Biochim. Biophys. Acta 272, 494- 503 (1972). Beridze, T.G. and M.S. Odintsova: Dokl. Akad. Nauk SSSR 173, 1209--1211 (1967). Britten, R.J. and D.E. Kohne: Carnegie Inst. Wash. Yearbook 65, 78- 106 (1966). Chilton, M-D., T.C. Currier, S.K. Farrand, A.J. Bendich, M.P. Gordon and E.W. Nester: Proc. Natl. Acad. Sci. U.S. 71, 3672--3676 (1974). Coucke, P. and R. van Parijs: Arch. Int. Physiol. Biochim. 80, 956--957 (1972). Delsemy, M.: Abstract Book of the International Summer School on Genetic Manipulations with Plant Materials, Li6ge, August 4--17, in press (1974). Drlica, K.A. and C.I. Kado: Proc. Natl. Acad. Sci. U.S. 71, 3 6 7 7 - 3 6 8 1 (1974). Eden, F.C., S.K. Farrand, J.S. Powell, A.J. Bendich, M.D. Chilton, E.W. Nester and M.P. Gordon: J. Bact. 119, 547---553 (1974). Flamm, W.G., H.E. Bond and H.E. Burr: Biochim. Biophys. Acta 129, 310--319 (1966). Green, B.R. and M.P. Gordon: Science 152, 1071--1074 (1966). Guill~, E., F. Qu6tier and T. Huguet: C. R. Acad. Sci. Paris 266, 836 838 (1968). Guill6, E. and F. Qu6tier: Bull. Cancer 57, 217--238 (1970a). Guill~, E. and F. Qu6tier: C. R. Acad. Sci. Paris 270, 3307--3310 (1970b). Ingle, J., G.G. Pearson and J. Sinclair: Nature New Biol. 2 4 2 ,1 9 3 - - 197 (1973). Julien, R. and M. Delsemy: Biochem. Biophys. Res. Commun. 48, 578--584 (1972). Kolodner, R. and K.K. Tewari: Proc. Natl. Acad. Sci. U.S. 69, 1830--1834 (1972a). Kolodner, R. and K.K. Tewari: J. Biol. Chem. 247, 6355--6363 (1972b). Kupila, S. and H. Stern: Plant Physiol. 36, 216 219 (1961). Kupila, S. and E. Therman: Physiol. Plant. 24, 23--26 (1971). Lipetz, J.: Ann. N.Y. Acad. Sci. 144, 320--325 (1967). Lonberg-Holm, K.K.: Nature 213, 454--457 (1967). Marmur, J.: J. Mol. Biol. 3, 208--218 (1961). McCallum, M. and P.M.B. Walker: Biochem. J. 105, 163- 169 (1967). Meyer, W.W. and J.A. Lippincott: Plant Physiol. 42, 5 3 - 5 4 (1967). Pearson, G.G. aod J. Ingle: Cell Differentiation 1, 43--51 (1972). Qu6tier, F., T. Huguet and E. Guill6: Biochem. Biophys. Res. Commun. 34, 128--133 (1969). Rasch, E.M.: Exptl. Cell Res. 3 6 , 4 7 5 - - 4 8 6 (1964). Sarrouy-Balat, H., M. Delsemy and R. Julien: Plant Science Lett. 1, 287--292 (1973). Schildkraut, C.L., J. Marmur and P. Dory: J. Mol. Biol. 4, 430--443 (1962). Skood, C.K. and D.T.N. Pillay: Z. Pflanzenphysiol. 67, 10--18 (1972). Sobota, A.E., C.J. Leaver and J.L. Key: Plant Physiol. 43, 907--913 (1968). Stern, H.: Methods Enzymol. 12B, 100--112 (1968). Watanabe, A. and H. Imaseki: Palnt Physiol. 51, 772--776 (1973).
THE ORIGIN OF W O U N D - I N D U C E D S A T E L L I T E D N A I N P E A SEEDLINGS
D. BROEKAERT and R. VAN PARIJS
Laboratory of Physiological Chemistry, Faculty of Medicine, and Laboratory of Biochemistry, Faculty of Agricultural Sciences State University of Ghent, Belgium Accepted 6 March 1975 DNA synthesis in wounded pea seedlings was studied by incorporation of [6-3H] thymidine. It was shown that the satellite DNA, synthesized within the first two days after wounding, is not of plant origin, but produced by contaminating bacteria, although a strong sterilizing procedure was used. Main-band DNA synthesized after wounding seems to be the only plant DNA synthesized during the stress-period. T h e p r o b l e m o f D N A s y n t h e s i s in w o u n d e d plants is very actual, especially since i n f e c t i o n o f the w o u n d e d p l a n t s with Agrobacterium tumefaciens results in t h e i n d u c t i o n o f a C r o w n gall t u m o u r . Kupila et al. ( 1 9 6 1 , 1 9 7 1 ) a n d Rasch ( 1 9 6 4 ) o b s e r v e d D N A s y n t h e s i s a f t e r sterile w o u n d i n g o f Vicia faba s t e m s e g m e n t s . L i p e t z ( 1 9 6 7 ) f o u n d t w o waves o f D N A s y n t h e s i s a f t e r w o u n d i n g s t e m tissues o f Kalanchof. W a t a n a b e et al. ( 1 9 7 3 ) r e p o r t e d the n e t synthesis o f D N A a f t e r c u t t i n g p o t a t o t u b e r tissues. 48 hr a f t e r w o u n d i n g a n u c l e a r GC-rich satellite D N A is s y n t h e s i z e d in Solanum lycopersicum, Scorsonera hispanica and Glycine max (Guill~ et at., 1 9 6 8 ) ; m o r e o v e r , s o m e h o m o l o g y was f o u n d b e t w e e n the n u c l e a r h e a v y satellite D N A o f stressed cells a n d D N A o f Agrobacterium tumefaciens (Qu~tier et al., 1969). F r o m these results, a m o d e l f o r the i n t e g r a t i o n o f bacterial D N A i n t o the h o s t p l a n t g e n o m e was d e d u c e d (Guill~ et al., 1970a, b). T h e p u r p o s e o f o u r w o r k was to d e m o n s t r a t e the i n d u c t i o n o f D N A s y n t h e s i s in w o u n d e d p e a seedlings b y using a r a d i o a c t i v e p r e c u r s o r . T h e labelled D N A was c h a r a c t e r i z e d b y d i f f e r e n t m e t h o d s . MATERIALS AND METHODS Dry seeds o f Pisum sativum L. were t h o r o u g h l y sterilized b y s h a k i n g in c h l o r a m i n e T (1%) and e x t e n s i v e washing with sterile water, b e f o r e and a f t e r i m b i b i t i o n (15 hr at 20°C). T h e seeds g e r m i n a t e d d u r i n g 5 or 6 d a y s in sterile Petri dishes, at 25 ° C, in t h e darkness. T h e seedlings were w o u n d e d b y r e m o v i n g t h e c o t y l e d o n s a n d the u p p e r p a r t o f the e p i c o t y l a n d p l a c e d with t h e i r r o o t s in an a q u e o u s s o l u t i o n o f [ 6 - 3 H ] t h y m i d i n e at 25°C. T h e w o u n d z o n e s were excised a f t e r one, t w o or t h r e e days.
140 The DNA was extracted following the m e t h o d of Stern ( 1 9 6 8 ) u s i n g deproteinization in 3 to 4 M NaC104 and furt her purified by preparative CsC1 gradient centrifugation (Flamm et al., 1966). Pea seedling DNA, when used in excess, was isolated from chromatin (Coucke et al., 1972). B u o y a n t densities were determined in CsC1 gradients in a Spinco Model E analytical centrifuge, using Micrococcus lysodeikticus DNA as a reference (Schildkraut et al., 1962). Renaturation of sonicated DNA was done in 0.12 M phosphate buffer (pH 6.8), 0.3 M NaC1 and 0.1% sodium lauryl sulphate, at 62 and 72°C, respectively 20 to 25°C under the Tm of pea and bacterial DNA in 1.0 SSC (0.15 M NaC1 -- 0.015 M Na citrate). The reaction mixture was analysed on a h y d r o x y l a p a t i t e (HAP)-column (McCallum et al., 1 9 6 7 ) a t different Cotvalues (Britten et al., 1966). The renaturated DNA was tested for thermal stability by using a HAP-column at 55 ° C, which was subsequently brought at higher temperatures. At each interval of 2.5°C the denatured DNA was eluted. All samples tested for radioactivity were diluted and c o u n t e d in Insta-gel scintillator (Packard), using the Packard Tri-Carb 3380 scintillation counter. The sterility of the medium and the wounded plants were tested by incubation o f diluted aliquots on agar plates enriched with general bacterial and yeast media. Bacterial DNA was isolated following the m e t h o d of Marmur
(1961). RESULTS The preparative CsC1 gradient profiles from several DNA preparations of l-day-old wound tissues were not exactly reproducible. In most cases a labelled satellite DNA peak (5 = 1.712--1.716 g/cm 3) was found at the heavy side o f the weakly labelled main-band DNA (Fig. la). The characteristics of r en atu r atio n at 62 ° (Table Ia) and 72°C (Table Ib) of the total DNA, isolated f r o m w o u n d e d tissues (OD260 measurement) and of the labelled satellite DNA (radioactivity counting) were different. When renaturation of labelled w o u n d tissue satellite DNA was studied by renaturation--hybridization at 62°C in the presence of a 100- to 1000-fold excess of pea seedling DNA (Table Ic) we could n o t d e t e c t any significant hybridization between w o un d tissue satellite DNA and the excess pea seedling DNA. These negative results, together with some observations by other authors, led to a more critical study. Obviously, a small a m o u n t of c o n t a m i n a t e d seedlings at the beginning of the incubation period were responsible for the clear c o n t a m i n a t i o n at the end of the incubation period. Mostly, 2 or 3 contaminating bacteria were isolated, sometimes only one contaminating bacterium was found. This organism most closely resembled an Erwinia, as established in the L a b o r a t o r y for Microbiology, Faculty of Sciences, State University of Ghent. From these contaminating bacteria, mixed with carrier pea seedlings, the total DNA was isolated. The CsC1 gradient profile and
141
0D260 0,360
cpm
\ o
/.00
03,-0
,
300 0400I 200 (~360[
0320 0.300
100
/
0320[%0"0.0. °-O.o
0,28(~
0-28012'0
OD260
I
0-~/.01
2/,
•
"°'° "°'o-.o /o...... j /,
8
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u
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c.
12000-
0650
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i
60003000-
0~50 °°
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2z.
Fraction number
Fig. 1. P r e p a r a t i v e CsCl g r a d i e n t c e n t r i f u g a t i o n of a) o n e - d a y - o l d w o u n d tissue D N A isolated f r o m c o n t a m i n a t e d seedlings; b) r a d i o a c t i v e bacterial D N A isolated in the prese n c e of carrier p l a n t tissues; c) o n e - d a y - o l d w o u n d tissue D N A isolated f r o m asceptic seedlings. ~3 --~J, U V - m e a s u r e m e n t at 260 nm. v. e, r a d i o a c t i v i t y m e a s u r e m e n t .
density of this labelled bacterial DNA was identical to the profile and density of w o u n d tissue satellite DNA. The isolated bacterial species was cultured on large scale. Fr om these cultures a sample was transferred to a liquid medium, enriched with [6-3 H] thymidine. Labelled bacterial DNA was again isolated in the presence of carrier plant tissues; the density of this 'satellite' DNA (Fig. l b ) was identical to the density of w o u n d tissue satellite DNA. Thermally denatured, labelled satellite DNA from w ound tissues and a 100- to 1000-fold excess of single-stranded bacterial DNA wery incubated together: the excess bacterial DNA favoured the renat urat i on at 72°C of the w o u n d tissue satellite DNA (Table Id). In the control experiment, labelled bacterial DNA incubated in the presence of excess unlabelled bacterial DNA r e n atu r ed roughly to the same e x t e n t as the unlabelled DNA (Table Ie). The thermal stability and the melting t e m p e r a t u r e (Tm) of the di fferent DNAs
142 TABLE I Renaturation of total DNA isolated from wounded tissues, and of labelled wound tissue satellite DNA and bacterial DNA in the presence of excess unlabelled DNA of different origin (HAP-chromatography).
Cot
0.12 M fraction
0.16 M fraction
0.30 M fraction
OD26o (%)
cpm (%)
OD260 (%)
cpm (%)
OD26o (%)
cpm (%)
a) 1.10 -I 1.10 ° 1.10 J 7.7.101
63.3 33.0 26.8 21.1
90.1 75.9 59.7 52.7
12.5 19.9 15.7 14.9
5.9 8.5 13.4 14.5
24.2 47.1 57.5 64.0
4.0 i5.6 26.9 32.8
b) 1.10 -1 1.10 o 1.101 7.7.101
71.6 41.8 32.8 28.9
90.7 77.5 60.2 54.2
11.5 14.5 12.3 17.4
5.6 7.9 14.2 9.9
16.9 43.7 54.9 61.7
3.7 14.6 25.6 35.9
c) 1.101 1.102 1.103 *
36.6 28.5 21.7
88.6 79.8 65.7 72.5
11.9 I 1.0 7.1 --
4.3 8.5 10.1 6.3
51.5 60.5 71.2 --
7.1 11.7 24.2 21.1
d) 5.102 *
1.5 --
3.4 77.5
0.5 --
0.2 10.2
98.0 --
96.4 12.3
e) 4.5.102 *
4.6 --
8.9 96.0
0.8 --
0.6 1.0
94.6 --
90.5 3.0
Fractionation of the reaction mixture was done following the method of McCallum et al. (1967); the OD260 values obtained for the single-stranded DNA (0.12 M fraction) were corrected for hyperchromicity (--30%); partially renatured DNA was eluted with 0.16 M phosphate buffer; the 0.30 M fraction contains the renatured DNA. * Control reaction in which single-stranded labelled DNA was incubated in the same concentration and for the same time, as for the incubation in the presence of excess DNA. were c o m p a r e d and f o u n d to be closely related: 9 3 . 0 0 ° C for bacterial D N A and 92.50°C for wound tissue satellite DNA; 88.75°C for the renatured d u p l e x s t r u c t u r e s at Cot 500 w h e n w o u n d tissue satellite D N A or labelled b a c t e r i a l D N A r e n a t u r e d in t h e p r e s e n c e o f e x c e s s b a c t e r i a l D N A . By a d d i n g the f o l l o w i n g a n t i b i o t i c s to the r a d i o a c t i v e i n c u b a t i o n m e d i u m : penicillin (100 U/ml), streptomycin (100 pg/ml) and chloramphenicol (50 p g / m l ) ( S k o o d e t al., 1 9 7 2 ) , o n l y a c c i d e n t a l b a c t e r i a l c o n t a m i n a t i o n t o a s m a l l e x t e n t c o u l d be d e m o n s t r a t e d . N o s a t e l l i t e D N A was p r e s e n t in lab e l l e d D N A p r e p a r a t i o n s o f 1, 2 o r 3 - d a y - o l d w o u n d t i s s u e s ( F i g . l c ) . T h e labelling pattern coincided with the UV-absorbing fractions of the main-band D N A in p r e p a r a t i v e CsC1 g r a d i e n t c e n t r i f u g a t i o n . M i c r o d e n s i t o m e t e r t r a c i n g s of U V - a b s o r p t i o n p h o t o g r a p h s o f D N A isolated f r o m w o u n d tissues, after a n a l y t i c a l CsC1 g r a d i e n t a n a l y s i s r e v e a l s o n e s y m m e t r i c a l D N A b a n d
143
Q.
Ref.
\A
Fig. 2. M i e r o d e n s i t o m e t e r tracings of U V - a b s o r p t i o n p h o t o g r a p h s of 3 t o 4 /.tg pea D N A a n d i pg r e f e r e n c e D N A ( M i c r o c o c c u s l y s o d e i k t i c u s , 5 = 1.731 g / c m 3 ) in CsCI ( S u p r a p u r - - Merck). a) D N A isolated f r o m c o n t r o l 12-day-old u n w o u n d e d e p i c o t y l tissues; b) D N A i s o l a t e d f r o m 1-day-old w o u n d tissues; c) D N A i s o l a t e d f r o m 2-day-old w o u n d tissues; d) D N A isolated f r o m 3-day-old w o u n d tissue; M = meniscus.
144 (5 = 1.695 g/cm 3) in all preparations (Fig. 2). The main-band DNA, synthesized after wounding is actually under investigation. DISCUSSION Our data concerning DNA synthesis in wounded peas clearly show the bacterial origin of the satellite DNA and emphasize the essentiality of efficient precautions in labelling experiments. No satellite DNA could be demonstrated using asceptic conditions. Contaminating bacteria which poorly contribute to UV-absorption of nucleic acids are of great importance in studies with radioactive precursors and could lead to extremely different interpretations (e.g. Lonberg-Holm, 1967; Bendich, 1972). Several authors {e.g. Green et al., 1966; Meyer et al., 1967; Sobota et al., 1968; Pearson et al., 1972; Sarrouy-Balat et al., 1973; Delsemy, 1974) recall different results in the field of satellite DNA and the so-called metabolic DNA, because of the objection cited above. The DNA synthesized in wounded pea seedlings is undoubtedly characterized as main-band DNA. Preparative CsC1 gradient centrifugation of aseptically cultured wounded pea seedlings demonstrates only main-band DNA synthesis; analytical CsC1 centrifugation reveals a symmetrical DNA band with a density of 1.695 g/cm 3 as observed by Beridze et al. (1967) and Ingle et al. (1973). The satellite DNA (6 = 1.707 g/cm 3) observed by Beridze et al. {1967) is probably of mitochondrial origin (Kolodner et al., 1972a). Also pea chloroplast DNA bands as a satellite peak (5 = 1.698 g/cm 3) (Kolodner et al., 1972b). Both organelle DNAs are not present in our preparations, or at least masked in the main-band DNA. Up till now, pea nuclear satellite DNA is u n k n o w n . These results differ from the results of Guill6 et al. (1968) who developed a model for the integration of bacterial DNA into the host plant genome (Guill6 et al., 1970a, b), based on the synthesis of a nuclear GC-rich DNA, partly homologous to Agrobacterium tumefaciens DNA (Qu6tier et al., 1969). This model implies the presence of A. tumefaciens DNA in the gehome of transformed cells, an assumption lacking convincing evidences since recent attempts to demonstrate this were unsuccessful (Chilton et al., 1974; Drlica et al., 1974; Eden et al., 1974). The proposed model is not only premature but certainly not a general one. The synthesis of a nuclear GC-rich DNA is probably not an obligatory step in Crown gall induction. We suppose that wounding plants rather induces DNA synthesis of the same type as during the onset of the germination, instead of a specific type of DNA synthesis. ACKNOWLEDGEMENTS We wish to thank Prof. Dr. J. De Ley and Mr. R. Tytgat (Laboratory of Microbiology, Faculty of Sciences, State University of Ghent, Belgium) for the analytical CsCl gradient
145 experiments and for fruitful discussions; Dr. P. Coucke for aid and advice in chromatin isolation.
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