Volumne 7 Number 5 1979
Nucleic Acids Research
Structure of plant nuclear and ribosomal DNA containing chromatin
B.Leber and V.Hemleben Institut fiir Biologie II, Lehrstuhl f'ur Genetik, Universitat Tubingen, Auf der Morgenstelle 28, 7400 Tubingen, GFR Received 1 June 1979
ABSTRACT Digestion of plant chromatin from Brassica pekinensis and Matthiola incana with staphylococcus nuclease leads to a DNA repeat of 17518 and a core size of 140 base pairs. DNase I digestion results in multiples of 10 bases. Ribosomal RNA genes were studied as a model system for active plant chromatin because of their great redundancy and their high transcriptional activity in growing and differentiating tissues. The actively transcribed genes were identified by nascent RNA of ribosomal origin still attached to its matrix DNA. Hybridization techniques were used to demonstrate that even transcriptionally active gene sequences are present in nuclease generated chromatin subunits. Comparison of the DNase I kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicated that ribosomal RNA genes are digested, but not preferentially degraded by DNase I.
INTRODUCTION Since the basic organization of chromatin into nucleosomes has been proved for eukaryotic organisms, the main interest is focussed on the question, whether structural changes are nescessary to permit the process of transcription. Several approaches have been employed to characterize transcriptionally active chromatin in animal systems. The fact that cDNA complementary to various messenger RNAs hybridizes to nuclease generated chromatin subunits indicated, that coding sequences are organized into nucleosomes (1,2,3). The observation, however, that limited DNase I digestion results in a preferential degradation of those sequences which are transcribed into tissue specific messenger RNAs, demonstrated, that transcriptionally active nucleosomes have an altered conformation which renders
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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Nucleic Acids Research them particularly sensitive to DNase I (4,5). Studies on active plant chromatin are to date restricted to the fraction of the reiterated ribosomal RNA genes since in higher plants no abundant specific messenger RNAs are available for isolation. Contradicting the electron microscopic evidence (6,7,8) biochemical investigations show that ribosomal RNA genes have a similar nucleosomal organization as the bulk of chromatin (9,10,11). In Tetrahymena and Physarum preferential digestion of extrachromosomal DNA by DNase I was shown (9,12). In the present report we used hybridization techniques following staphylococcus nuclease or DNase I digestion of plant nuclei to study the structure of rDNA containing chromatin in higher plants.
MATERIALS AND METHODS Isolation of nuclei. Plant tissue - seedlings of Matthiola incana and Brassica pekinensis or flower petals of Matthiola was homogenized with an Ultra-Turrax at 40C in a medium containing 400 mM sucrose, 10 mM MgCl2, 50 mM Tris and 10 mM NaCl, pH 7.8. The homogenate was filtered through miracloth and Triton X-100 was added to lyse chloroplasts (0.1% final concentration). Nuclei were pelleted (5 min, 800 x g, 40C) and finally washed in isolation buffer. The isolated nuclei are morphologically intact and physiologically active as shown previously (13). Digestion of nuclei with staphylococcus nuclease and DNase I. To test the accessibility of nuclear DNA to staphylococcal endonuclease and DNase I and to identify newly synthesized RNA attached to it, nuclei were isolated from Matthiola and Brassica seedlings which were either unlabelled or had been grown for 20 hours in [6-3H] thymidine (27 Ci/mmol; 150 pCi/ml) or [5-3H] uridine (26 Ci/mmol; 150 pCi/ml). Nuclei were suspended in 2 ml of incubation buffer, either 10 mM Tris, 1 mM CaCl2 and 250 mM sucrose, pH 7.8 (for nuclease digestion) or 10 mM Tris, 2 mM MgCl2 and 250 mM sucrose, pH 7.5 (for DNase I digestion). The nuclear suspension was divided into fractions of 200 pl each. After preincubation (370C, 30 sec) 2 pl of staphylococcus nuclease (30 units) or 4 pl of DNase I (4 pg) were added and the nuclei were digested for the indicated times. Staphylococcus 1264
Nucleic Acids Research nuclease and DNase I were purchased from Boehringer. The reaction was terminated by addition of 20 p1 5 mM EDTA and 5 mM EGTA, pH 7.5 and chilling on ice. After centrifugation at 3000 x g for 5 min a first supernatant was recovered. The pellet of remaining nuclei was lysed by suspension in 500 p1 of 1 mM EDTA, pH 7.5, and recentrifuged. The resulting supernatant was suspended in 700 p1 of the above medium. The sediment and the combined supernatants were checked for radioactivity (dissolved in 6 ml Unisolve, Zinsser). For qualitative analysis unlabelled nuclei were incubated as described above. Nuclease digested chromatin was fractionated by two centrifugation steps and the first and second supernatant combined or separately were analyzed on isokinetic sucrose gradients. For analysis of the DNA aize distribution generated by staphylococcus nuclease or DNase I digestion both reactions were terminated by the addition of 1% SDS (final concentration) The DNA was isolated by chloroform-isoamylalcohol extraction and ethanol precipitation. Isokinetic sucrose gradients. The final nuclease digestion product (first or second supernatant) was layered on an isokinetic sucrose gradient (ct=0.5 M, cr=1.7 M and Vm=10 ml; 14) containing 1 mM EDTA, pH 7 and generally 100 mM NaCl. The gradients were centrifuged for 15 hours at 36000 rpm and 4°C in a Beckman SW 40 rotor. 6 drop-fractions were collected from the top. For subsequent studies of the DNA fractions of interest were pooled and treated with RNase (20 pg/ml) for 30 min. The DNA was isolated by chloroform-isoamylalcohol extraction and ethanol rreciDitation. Electrophoresis. The DNA fragments resulting from DNase I digestion were analyzed on 12% acrylamide slab gels containing 7 M urea according to Maniatis et al. (15). Running buffer was 0.09 M Tris-borat and 2.5 mM EDTA, pH 8.3. DNA samples were boiled for 10 min in 50% formamide and quickly chilled on ice. Electrophoresis was for 6 hours at 200 V. DNA fragments generated by staphylococcus endonuclease digestion were separated by slab gel electrophoresis on 2.5% agarose gels. The running buffer was 0.04 M Tris, 0.02 M sodium 1265
Nucleic Acids Research acetate, 1 mM EDTA, pH 7.8. Electrophoresis was for 6 hours at 140 V. Agarose and polyacrylamide gels were stained for 30 min with 1 pg/ml ethidium bromide dissolved in running buffer and photographed under UV illumination. Molecular weight markers on agarose gels were DNA fragments of 1686, 1320, 881, 535, 462, 357, 271, 230, 215, 180, 144, 133, 84, 38 and 34 base pairs of Hae III digested Advl DNA (kindly supported by Dr. R. Streeck). The repeat length was calculated by averaging the differences between adjacent multimer band sizes. Blotting and hybridization with 125I-rRNA. DNA fragments were transferred from the agarose gels to nitrocellulose filters according to Southern (16). The rRNA used for subsequent hybridization was iodinated in vitro. Hybridization was performed at 60°C for 16 - 18 hours with 125I-labelled 18S rRNA and 25S rRNA (0.2 pg/ml; specific radioactivity: 4.5 x 106 cpm/pg) in 6 x SSC containing 25% formamide. After the incubation, filters were washed three times with 2 x SSC (10 min), treated with RNase (20 p.g/ml; 45 min) washed again three times in 2 x SSC (10 min) and dried. Filters were exposed on a Kodak x-ray film (No Screen Film) for two weeks. Actinomycin-CsCl gradients. Total DNA of Matthiola incana isolated as described (17) was centrifuged in an actinomycincaesium chloride gradient according to Hemleben et al. (18) in order to separate ribosomal DNA from the bulk of DNA. DNA-RNA filter hybridization. DNA isolated from isokinetic sucrose gradient fractions dissolved in 1 ml SSC or fractions from actinomycin-CsCl gradients diluted with 1 ml of gradient buffer were denatured, neutralized and loaded onto nitrocellulose filters according to Hemleben et al. (18). Hybridization was carried out in 6 x SSC at 60 0C for 4 hours using either .purified 3H-uridine labelled 25S and 18S rRNA (specific radioactivity: 40,000 cpm/4g; 2 jg/ml) or 3H- uridine labelled RNA isolated from digested chromatin (specific radioactivity: 700 cpm/pg; 20 fjg/ml). Washing procedures, RNase treatment and DNA determination were performed as described (19). Radioactively labelled rRNA samples used for hybridization experiments were obtained by growing Matthiola seedlings in 1266
Nucleic Acids Research [5-3H] uridine (500 pCi/ml) for 3 days. RNA 18S and 25S rRNA resis (20).
were
was extracted and purified by polyacrylamide gel electro-
RESULTS Structure of nuclear chromatin. To obtain information on the chromatin organization of the used plant systems the accessibility of nuclear DNA to staphylococcus nuclease was studied. Nuclei of Matthiola seedlings were digested for 1 30 min with staphylococcal endonuclease. Resultant DNA fragments were analyzed on a 2.5% slab gel together with calibrated DNA fragments (Fig.1). The repeat length was calculated by averaging the differences between adjacent multimer sizes to be 175±8 base pairs. The core particle contains 140 base pairs of DNA as observed for all eukaryotic organisms studied. No difference in repeat length is obtained for Brassica and Matthiola or for seedlings and flower petals of Matthiola. Maintaining the DNA-protein interactions of chromatin staphylococcus nuclease generated chromatin subunits were separated on an isokinetic sucrose gradient containing 0.1 M NaCl (Fig.2A). As described previously (21) the separation is improved on gradients containing a salt concentration between 0.05 M and 0.1 M NaCl. The quality of separation can be inferred from the pattern of deproteinized DNA fragments on a 2.5% agarose gel, following the direction of sedimentation (Fig.2B). Digestion of chromatin with DNase I is reported to produce DNA fragments which are multiples of 10 bases if analyzed under denaturing conditions. Nuclei of Matthiola, labelled with [3H uridine or (3H] thymidine were digested for the indicated times with DNase I to investigate the kinetics of degradation. Fig.3 shows that initial degradation is high leading to about 50% of released 3H-thymidine labelled DNA after 2 min of digestion. After 10 min about 60% of degraded material is released from lysed nuclei. Release of 3H- uridine labelled RNA was not observed during the whole incubation period. The same experiment was performed with unlabelled nuclei to characterize the degradation products on a 12% acrylamide gel containing 7 M urea. -
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Figure 1. Nucleosomal DNA pattern of chromatin from Matthiola seedlings (slots 1-3), Matthiola flower petals (slots 4-6) and Brassica seedlings (slot 8) as obtained by electrophoresis on 2.5% agarose gels. Staphylococcal nuclease digest was for 3 min (slots I and 4), 6 mmn (slots 2 and 5), 10 mmn (slots 3 and 6) and 5 mmn (slot 8). Molecular weight markers were DNA fragments of 1686, 1320, 881, 535, 462, 357, 271, 230, 215, 180, 144, 133, 84, 38 and 34 base pairs of Rae III digested Xdvl DNA.
Fig.4 shows the banding pattern
of DNA fragments, which were isolated from DNase I digested nuclei. The lengths of visible bands are multiples of 10 bases. Structure of ribosomal liT'A genes. N~uclei of Brassica seedlings were digested for 7 mmn with staphylococcus nuclease. Resultant deproteinized DNA fragments were separated on a 2.5% 1268
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Figure 3. DNase I digestion of Matthiola nuclei. Nuclei of Matthiola seedlings labelled with J5Hj thymidine and[PH] uridine, respectively, were incubated for 10,20,30,40,50 sec and 1,2,5 and 10 min with DNase I (20 pg/ml). The percentage of DNA ) and 3H- uridine labelled RNA associated with degraded (it (C---O) was determined by pelleting the undegraded material of the lysed nuclei and measuring the release of radioactivity.
gel and transferred to nitrocellulose filters according to the method of Southern (16). Hybridization was performed with 125I-labelled 18S + 25S rRNA (Fig.5). The autoradiograph of hybridized filters (Fig.5B) shows a repeat pattern which coincides exactly with the electrophoretic pattern of the ethidium bromide stained DNA bands (Fig.5A). Ribosomal RNA genes must therefore generally be present within the histone protected fraction of nuclease digested chromatin. Quantitative information on the distribution of rDNA sequences among the subunit fractions was obtained by hybridization of DNA derived from monomeric and multimeric nucleosome particles to 3H-rRNA in excess. Nuclei of Brassica seedlings were digested with staphylococcal nuclease for 5 and 20 minutes. Following the fractionation procedure described in Materials agarose
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80 NT
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Figure 4. 12 percent polyacrylamide/7 M urea gel electrophoresis of DNase I digestion products. 20 pg of the extracted DNA were denatured and applied to each slot. a) 10 sec, b) 30 sec, c) 1 min, d) 5 min and e) 10 min digestion.
and Methods the first and second supernatants were analyzed separately on isokinetic sucrose gradients (Fig.6). In the first supernatant there are no chromatin subunits which exceed monomer size (Fig.6A), whereas the second supernatant contains all higher order multimers (Fig.6C). After prolonged incubation multimers are reduced to monomer and core size (Fig.6D; second supernatant), monomers are reduced to cores and smaller fragments (Fig.6B; first supernatant). Hybridization of the corresponding 1271
Nucleic Acids Research Figure 5. Distribution of ribosomal DNA sequences in nuclease digested plant chromatin. Nuclei of Brassica pekinensis were digested for 7 min with staphylococcus nuclease (150 units/ml). DNA fragments were isolated, separated on a 2.5% agarose slab gel, stained with ethidium bromide and transferred to nitrocellulose filters according to the technique of Southern. Ribosomal DNA sequences were detected by hybridization using 125i-labelled 18S + 25S rRNA followed by autoradiography. A) photographic negative of the ethidium bromide stained DNA pattern,
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DNA fragments, which were loaded onto nitrocellulose filters, to purified H-uridine labelled rRNA does not suggest a preferential digestion of ribosomal sequences. The extent of hybridization in the first supernatant remains relatively constant over 20 min of digestion. The corresponding analysis of the second supernatant demonstrates that although the bulk of chromatin is already extensively degraded after short digestion time (Fig.6C) ribosomal genes are enriched in the region of subunit multimers comprising 7 and more nucleosomes. Only after a longer incubation period (Fig.6D) rDNA containing chromatin is reduced to monomer size. Compared to the corresponding unfractionated samples of undigested and nuclease digested plant 1272
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were analyzed on isokinetic sucrose gradients. The DNA the pooled fractions was isolated, denatured and fixed onto ni ~rocellulose filters. Filters were incubated for 4 hours at 3H- uridine labelled 18S 60 C in 6 x SS0 containing 2 j.Ig/ml 9% DNA h-ybridized Absorbance at 260 nxn; + 25S rRNA. 0--
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Nucleic Acids Research DNA fragments (no decrease of hybridizable rDNA sequences) we conclude that these studies on fractionated chromatin deal with a representative part of the ribosomal DNA sequences. 25S and 18S ribosomal RNA genes therefore appear to be arranged in a configuration which can be cleaved by staphylococcus nuclease attack into a similar subunit pattern as the bulk of chromatin. But the digestion seems to follow different kinetics. Evidence has to be presented that the ribosomal RNA genes studied are in the transcriptionally active state. To identify active genes by their attached nascent RNA chains, seedlings of Matthiola were labelled in vivo with [3H] uridine. Nuclei were isolated and treated for 3 min with staphylococcus nuclease. The digested chromatin was fractionated on an isokinetic sucrose gradient and the radioactivity of each fraction was measured (Fig.7A). The radioactive label is distributed over the complete repeat pattern and seems to coincide with the nucleosomal distribution. Isolated 3H- uridine labelled RNA from this gradient (fractions 20-40; Fig.7A) if recentrifuged on an isokinetic sucrose gradient together with digested unlabelled chromatin appears exclusively in the region corresponding to about 4-5S (Fig.7B). This result demonstrates that the sedimentation behaviour of the labelled RNA (Fig.7A) cannot be due to an unspecific aggregation between free RNA and chromatin. To demonstrate that ribosomal RNA sequences are present in this preparation the 3H- uridine labelled RNA derived from the nucleosomal fractions was hybridized to ribosomal DNA. The ribosomal DNA had first been separated from the bulk of DNA by centrifugation in a CsCl density gradient containing actinomycin (Fig.8). In this type of gradient ribosomal DNA sequences become located on the light side of the main band (18). The DNA of every single gradient fraction was denatured, fixed onto nitrocellulose filters and hybridized to the 3H- uridine labelled RNA described above. Fig.8 demonstrates that part of the radioactive RNA hybridizes to those fractions of the gradient which contain ribosomal DNA sequences. The remaining RNA hybridizes to main band DNA and is obviously of nonribosomal origin. Since the response of transcriptionally active sequences to digestion by DNase I was reported to be strikingly different 1274
Nucleic Acids Research
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Figure 7. Detection of newly transcribed RNA within fractionated chromatin subunits. Matthiola seedlings were incubated for 20 hours with [3H] uridinie invivo. Chromatin derived from staphylococcus nuclease Tigested nuclei of these seedlings was fractionated on an isokinetia sucrose gradient. RNA sequences were identified by their radioactive label (A). The size of the 3H- uridine labelled RNA isolated from the chromatin subunits (fractions 20-40) was determined by recentrifugation on an isokinetic sucrose gradient together with unlabelled digested chromatin (B). 3 Absorbance at 260 nm; X---x 3H-radioactivity v 1275
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Figure 6. Identification of ribosomal RNA sequences among the nascent 3H- uridine labelled RNA molecules attached to fractionated chromatin. Unlabelled total DNA from Matthiola incana was fractionated on an actinomycin-CsCl gradient. Each fraction was denatured, loaded onto filters and hybridized to 3H-labelled RNA isolated from chromatin subunits (fractions 20-40) of the sucrose gradient shown in Fig.7. - * Absorbance at 260 nm; X- --X 3H-radioactivity from that of inactive chromatin (4,5), DNase I degradation of nuclear chromatin was compared to degradation of ribosomal RNA genes. Nuclei of unlabelled Brassica seedlings were incubated up to 10 min with DNase I (Fig. 9 ). At the indicated times the DNA was isolated, fixed onto nitrocellulose filters and hybridized to excess 3H-labelled 18S + 25S rRNA. From the amount of ribosomal DNA hybridized the percentage of degraded DNA was calculated. In a parallel experiment the kinetics of DNase I was determined for 3H- thymidine labelled nuclear chromatin as described in Materials and Methods (Fig.9). Comparison of the kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicates that ribosomal genes are digested but not preferentially degraded. Initial degradation of nuclear DNA even appears to be higher than the degradation of ribosomal DNA 1276
Nucleic Acids Research
S
1
2
3
4 5 6 7 Digestion time (min)
8
9
10
Figure 9. Kinetics of DNase I digestion of ribosomal RNA genes as compared to digestion of nuclear chromatin. Nuclei of Brassica seedlings were incubated for 10,20,30 sec and 1,2,5 and 1-0 min with DNase I. DNA was isolated and hybridized for 4 hours to excess 3H- uridine labelled 18S + 253 rRNA. The percentage of rDNA degraded was calculated. The percentage of total DNA degraded was determined as described for Fig.3. *-* % ribosomal DNA degraded; X % total DNA degraded sequences. DISCUSSION
Staphylococcal nuclease and DNase I digestion of plant chromatin indicate a similar structure for animal and plant systems. Staphylococcal nuclease digestion of Brassica and Matthiola nuclei leads to a nucleosomal DNA repeat of 175±8 base pairs. This value is lower than the data reported for tobacco, barley (22) and rye (23) calculated by comparison to Hae III fragments of PM2 DNA and digested rat liver nuclei. Low values in the range of 150-175 base pairs have been described for Aspergillus, Saccharomyces, Neurospora and Physarum (for references see 24). Contrary to various animal systems no 1277
Nucleic Acids Research differences in repeat length have been observed for different plant species and tissues. The different physiological activity of growing seedlings compared to relatively inert, fully differentiated flower petals is only documented by an indefinite monomer band (Fig.1). The DNA content-of the core particle (140 base pairs) and the DNase I digestion pattern suggest, that the general organization of the DNA within the nucleosome is the same for;plants and animals. A striking difference in the release of 3H-radioactivity when 3H- thymidine labelled plant nuclei (label observed was in the DNA) and 3H- uridine labelled plant nuclei (label in the RNA) were digested with DNase I in parallel but equivalent experiments. Whereas a 60% release of radioactivity was obtained for the 3H- thymidine labelled chromatin no increase of released 3H- uridine labelled RNA occured during the whole incubation period suggesting that the in vivo labelled RNA is attached to an unsoluble chromatin fraction. This interpretation would conflict with results reported-by'several groups which demonstrate that transcriptionally active genes, to which nascent RNA chains are attached,are preferentially degraded by DNase I
(4,5,9,12). In our experiments ribosomal-RNA genes were used as a model system for active chromatin. No preferential digestion of rRNA genes was observed by staphylococcal nuclease (Fig.6) or DNase I (Fig.9). We cannot state with certainty that th1e rDNIA containing chromatin we examined contained actually avtive genes. A strong evidence, however, is given by the identification of nascent RNA chains in nuclease generated subunits. Unspecific, aggregation can be excluded (Fig.7B) and since the RNA molecules which are distributed over the whole nucleosomal repeat pattern exhibit a length of 4-5S if isolated and recentrifuged, their different sedimentation behaviour cannot be caused byassociation with proteins (RNP particles) but must be due to attachment to the transcriptional comp-exs. Evidence-that part of these newly synthesized RNA molecules are transeribed from ribosomal DNA arises from the hybridization experiment (Fig.8). Previous studies have shown tl,at 25S, 18S and 5S rRNA hybridize to GC-rich fractions which are.separated from the bulk of main 1278
Nucleic Acids Research band DNA on actinomycin/CsCl gradients. From the amount of radioactivity, however, it can be calculated that the nascent RNA molecules contain predominantly 18S and 25S rRNA sequences
(25). It is now generally accepted that ribosomal RNA genes are present in a nucleosomal structure (for references see 24). After staphylococcal nuclease digestion we identified ribosomal DNA in DNA fragments of all multimer lengths (Fig.5) and in particles sedimenting like nucleosomes (Fig.6). The distribution of the ribosomal DNA sequences, however, was found to be unequal among the various nucleosomal classes. After limited digestion, when the bulk of chromatin is already digested to monomer size, ribosomal genes are accumulated in multimers comprising 7 and more nucleosomes (Fig.6C). Extrachromosomal rDNA sequences of Physarum are reported to be primarily recovered in the monomer and so called peak A fraction (26). This difference may be due to a structural transition caused by the different salt concentrations used. Both results clearly agree on the different susceptibility to staphylococcal nuclease compared to the bulk of chromatin. Conflicting with nuclease digestion studies (9,10,11) electron microscopic observations agree on the absence of nucleosomes in actively transcribed ribosomal RNA genes (6,7,8). The facts, however, that actively transcribed DNA is still associated with basic proteins (27) and protected against staphylococcal nuclease (4,5,9,10,11) but not DNase I digestion (4,5,9,12) have suggested the conclusion that the tight binding between histones and DNA may be modulated in actively transcribed genes. Our results indicate that ribosomal RNA genes in the active and inactive state posess a nucleosomal periodicity. Ribosomal DNA sequences appear to be less susceptible to DNase I attack compared to the bulk of chromatin. Since a modified chromatin structure of ribosomal genes may result in a different solubilization behaviour the relation between ionic strength and structural transitions has to be further studied.
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Nucleic Acids Research ACKNOWLEDGEMENTS We are grateful to Miss Anne Roth for excellent technical assistance. Part of the results has been presented at the Symposium "Genome, Chromatin and Karyotype: Evolution and Function", held at Kaiserslautern, -GFR, October 13-15, 1978, and is going to be published in Plant Syst.Evol. Suppl.2, Springer, Wien, New York, 1979. The work has been supported by Deutsche Forschungsgemeinschaft.
REFERENCES 1 Axel, R., Cedar, H. and Felsenfeld, G.C. (1975) Biochemistry 14, 2489-2495 2 Lacy, E. and Axel,R. (1975) Proc. Nat. Acad. Sci. USA 72, 3978-3982 3 Kuo, T.M., Sahasrabuddhe, C.G. and Saunders, G.F. (1976) Proc. Nat. Acad. Sci. USA 73, 1572-1575 4 Weintraub,H. and Groudine, M. (1976) Science 193, 848-856 5 Garel, A. and Axel, R. (1976) Proc. Nat. Acad. Sci. USA 73 3966-3970 6 Foe, V.E., Wilkinson, L.E. and Laird, C.D. (1976) Cell 9,
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131-146 Laird, C.D., Wilkinson, L.E., Foe, V.E. and Chooi, W.Y. (1976) Chromosoma (Berl.) 58, 169-192 Scheer, U. (1978) Cell 13, 535-549 Mathis, D.J. and Gorovsky, M.A. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 773-778 Reeves, R. (1976) Science 194, 529-532 Reeves, R. and Jones, A. (1976) Nature 260, 495-500 Stalder, J., Seebeck, T. and Braun, R. (1978) Eur.J.Biochem.
90, 391-395
Leber, B. and Hemleben, V. (1979) Z. Pflanzenphysiol. 91, 305-316 14 Noll, H. (1967) Nature 215, 360-363 15 Maniatis, T., Jeffrey, A. and van de Sande, H. (1975) Biochemistry 14, 3787-3794 16 Southern, E.M. (1975) J.Mol.Biol. 98, 503-515 17 Hemleben, V., Ermisch, N., Kimmich, D., Leber, B. and Peter, G. (1975) Eur.J.Biochem. 56, 403-411 18 Hemleben, V., Grierson, D. and Dertmann, H. (1977) Plant Science Letters 9, 129-135 19 Spring, H., Grierson, D., Hemleben, V., Stohr, M., Krohne, G., Stadler, J. and Franke, W.W. (1978) Experimental Cell Research 114, 203-215 20 Grierson, D. and Hemleben, V. (1977) Biochim.Biophys.Acta 475, 424-436 21 Leber, B. and Hemleben, V. (1979) Plant Syst. Evol., Suppl.2 (in press) 22 Philipps, G. and Gigot, C. (1977) Nucl. Acids Res. 4, 3617-3626 23 Cheah, K.S.E. and Osborne, D.J. (1977) Bioch.J. 163, 141-144 13
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Thomas, J.0. (1978) in: International Review of Biochemistry, Biochemistry of Nucleic Acids II, Clark, B.F.C., Ed., Vol 17, pp.181-232, University Park Press Baltimore. Hemleben, V. and Grierson, D. (1978) Chromosoma (Berl.) 65,
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Allfrey, V.G., Johnson, E.M., Sun, I.Y.C., Littau, V.C., Matthews, H.R. and Bradbury, E.M. (1977) Cold Spring Harbor Symp. Quant.Biol. 42, 505-514 McKnight, S.L., Bustin, M. and Millar, O.L. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 741-754
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Structure of plant nuclear and ribosomal DNA containing chromatin
B.Leber and V.Hemleben Institut fiir Biologie II, Lehrstuhl f'ur Genetik, Universitat Tubingen, Auf der Morgenstelle 28, 7400 Tubingen, GFR Received 1 June 1979
ABSTRACT Digestion of plant chromatin from Brassica pekinensis and Matthiola incana with staphylococcus nuclease leads to a DNA repeat of 17518 and a core size of 140 base pairs. DNase I digestion results in multiples of 10 bases. Ribosomal RNA genes were studied as a model system for active plant chromatin because of their great redundancy and their high transcriptional activity in growing and differentiating tissues. The actively transcribed genes were identified by nascent RNA of ribosomal origin still attached to its matrix DNA. Hybridization techniques were used to demonstrate that even transcriptionally active gene sequences are present in nuclease generated chromatin subunits. Comparison of the DNase I kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicated that ribosomal RNA genes are digested, but not preferentially degraded by DNase I.
INTRODUCTION Since the basic organization of chromatin into nucleosomes has been proved for eukaryotic organisms, the main interest is focussed on the question, whether structural changes are nescessary to permit the process of transcription. Several approaches have been employed to characterize transcriptionally active chromatin in animal systems. The fact that cDNA complementary to various messenger RNAs hybridizes to nuclease generated chromatin subunits indicated, that coding sequences are organized into nucleosomes (1,2,3). The observation, however, that limited DNase I digestion results in a preferential degradation of those sequences which are transcribed into tissue specific messenger RNAs, demonstrated, that transcriptionally active nucleosomes have an altered conformation which renders
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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Nucleic Acids Research them particularly sensitive to DNase I (4,5). Studies on active plant chromatin are to date restricted to the fraction of the reiterated ribosomal RNA genes since in higher plants no abundant specific messenger RNAs are available for isolation. Contradicting the electron microscopic evidence (6,7,8) biochemical investigations show that ribosomal RNA genes have a similar nucleosomal organization as the bulk of chromatin (9,10,11). In Tetrahymena and Physarum preferential digestion of extrachromosomal DNA by DNase I was shown (9,12). In the present report we used hybridization techniques following staphylococcus nuclease or DNase I digestion of plant nuclei to study the structure of rDNA containing chromatin in higher plants.
MATERIALS AND METHODS Isolation of nuclei. Plant tissue - seedlings of Matthiola incana and Brassica pekinensis or flower petals of Matthiola was homogenized with an Ultra-Turrax at 40C in a medium containing 400 mM sucrose, 10 mM MgCl2, 50 mM Tris and 10 mM NaCl, pH 7.8. The homogenate was filtered through miracloth and Triton X-100 was added to lyse chloroplasts (0.1% final concentration). Nuclei were pelleted (5 min, 800 x g, 40C) and finally washed in isolation buffer. The isolated nuclei are morphologically intact and physiologically active as shown previously (13). Digestion of nuclei with staphylococcus nuclease and DNase I. To test the accessibility of nuclear DNA to staphylococcal endonuclease and DNase I and to identify newly synthesized RNA attached to it, nuclei were isolated from Matthiola and Brassica seedlings which were either unlabelled or had been grown for 20 hours in [6-3H] thymidine (27 Ci/mmol; 150 pCi/ml) or [5-3H] uridine (26 Ci/mmol; 150 pCi/ml). Nuclei were suspended in 2 ml of incubation buffer, either 10 mM Tris, 1 mM CaCl2 and 250 mM sucrose, pH 7.8 (for nuclease digestion) or 10 mM Tris, 2 mM MgCl2 and 250 mM sucrose, pH 7.5 (for DNase I digestion). The nuclear suspension was divided into fractions of 200 pl each. After preincubation (370C, 30 sec) 2 pl of staphylococcus nuclease (30 units) or 4 pl of DNase I (4 pg) were added and the nuclei were digested for the indicated times. Staphylococcus 1264
Nucleic Acids Research nuclease and DNase I were purchased from Boehringer. The reaction was terminated by addition of 20 p1 5 mM EDTA and 5 mM EGTA, pH 7.5 and chilling on ice. After centrifugation at 3000 x g for 5 min a first supernatant was recovered. The pellet of remaining nuclei was lysed by suspension in 500 p1 of 1 mM EDTA, pH 7.5, and recentrifuged. The resulting supernatant was suspended in 700 p1 of the above medium. The sediment and the combined supernatants were checked for radioactivity (dissolved in 6 ml Unisolve, Zinsser). For qualitative analysis unlabelled nuclei were incubated as described above. Nuclease digested chromatin was fractionated by two centrifugation steps and the first and second supernatant combined or separately were analyzed on isokinetic sucrose gradients. For analysis of the DNA aize distribution generated by staphylococcus nuclease or DNase I digestion both reactions were terminated by the addition of 1% SDS (final concentration) The DNA was isolated by chloroform-isoamylalcohol extraction and ethanol precipitation. Isokinetic sucrose gradients. The final nuclease digestion product (first or second supernatant) was layered on an isokinetic sucrose gradient (ct=0.5 M, cr=1.7 M and Vm=10 ml; 14) containing 1 mM EDTA, pH 7 and generally 100 mM NaCl. The gradients were centrifuged for 15 hours at 36000 rpm and 4°C in a Beckman SW 40 rotor. 6 drop-fractions were collected from the top. For subsequent studies of the DNA fractions of interest were pooled and treated with RNase (20 pg/ml) for 30 min. The DNA was isolated by chloroform-isoamylalcohol extraction and ethanol rreciDitation. Electrophoresis. The DNA fragments resulting from DNase I digestion were analyzed on 12% acrylamide slab gels containing 7 M urea according to Maniatis et al. (15). Running buffer was 0.09 M Tris-borat and 2.5 mM EDTA, pH 8.3. DNA samples were boiled for 10 min in 50% formamide and quickly chilled on ice. Electrophoresis was for 6 hours at 200 V. DNA fragments generated by staphylococcus endonuclease digestion were separated by slab gel electrophoresis on 2.5% agarose gels. The running buffer was 0.04 M Tris, 0.02 M sodium 1265
Nucleic Acids Research acetate, 1 mM EDTA, pH 7.8. Electrophoresis was for 6 hours at 140 V. Agarose and polyacrylamide gels were stained for 30 min with 1 pg/ml ethidium bromide dissolved in running buffer and photographed under UV illumination. Molecular weight markers on agarose gels were DNA fragments of 1686, 1320, 881, 535, 462, 357, 271, 230, 215, 180, 144, 133, 84, 38 and 34 base pairs of Hae III digested Advl DNA (kindly supported by Dr. R. Streeck). The repeat length was calculated by averaging the differences between adjacent multimer band sizes. Blotting and hybridization with 125I-rRNA. DNA fragments were transferred from the agarose gels to nitrocellulose filters according to Southern (16). The rRNA used for subsequent hybridization was iodinated in vitro. Hybridization was performed at 60°C for 16 - 18 hours with 125I-labelled 18S rRNA and 25S rRNA (0.2 pg/ml; specific radioactivity: 4.5 x 106 cpm/pg) in 6 x SSC containing 25% formamide. After the incubation, filters were washed three times with 2 x SSC (10 min), treated with RNase (20 p.g/ml; 45 min) washed again three times in 2 x SSC (10 min) and dried. Filters were exposed on a Kodak x-ray film (No Screen Film) for two weeks. Actinomycin-CsCl gradients. Total DNA of Matthiola incana isolated as described (17) was centrifuged in an actinomycincaesium chloride gradient according to Hemleben et al. (18) in order to separate ribosomal DNA from the bulk of DNA. DNA-RNA filter hybridization. DNA isolated from isokinetic sucrose gradient fractions dissolved in 1 ml SSC or fractions from actinomycin-CsCl gradients diluted with 1 ml of gradient buffer were denatured, neutralized and loaded onto nitrocellulose filters according to Hemleben et al. (18). Hybridization was carried out in 6 x SSC at 60 0C for 4 hours using either .purified 3H-uridine labelled 25S and 18S rRNA (specific radioactivity: 40,000 cpm/4g; 2 jg/ml) or 3H- uridine labelled RNA isolated from digested chromatin (specific radioactivity: 700 cpm/pg; 20 fjg/ml). Washing procedures, RNase treatment and DNA determination were performed as described (19). Radioactively labelled rRNA samples used for hybridization experiments were obtained by growing Matthiola seedlings in 1266
Nucleic Acids Research [5-3H] uridine (500 pCi/ml) for 3 days. RNA 18S and 25S rRNA resis (20).
were
was extracted and purified by polyacrylamide gel electro-
RESULTS Structure of nuclear chromatin. To obtain information on the chromatin organization of the used plant systems the accessibility of nuclear DNA to staphylococcus nuclease was studied. Nuclei of Matthiola seedlings were digested for 1 30 min with staphylococcal endonuclease. Resultant DNA fragments were analyzed on a 2.5% slab gel together with calibrated DNA fragments (Fig.1). The repeat length was calculated by averaging the differences between adjacent multimer sizes to be 175±8 base pairs. The core particle contains 140 base pairs of DNA as observed for all eukaryotic organisms studied. No difference in repeat length is obtained for Brassica and Matthiola or for seedlings and flower petals of Matthiola. Maintaining the DNA-protein interactions of chromatin staphylococcus nuclease generated chromatin subunits were separated on an isokinetic sucrose gradient containing 0.1 M NaCl (Fig.2A). As described previously (21) the separation is improved on gradients containing a salt concentration between 0.05 M and 0.1 M NaCl. The quality of separation can be inferred from the pattern of deproteinized DNA fragments on a 2.5% agarose gel, following the direction of sedimentation (Fig.2B). Digestion of chromatin with DNase I is reported to produce DNA fragments which are multiples of 10 bases if analyzed under denaturing conditions. Nuclei of Matthiola, labelled with [3H uridine or (3H] thymidine were digested for the indicated times with DNase I to investigate the kinetics of degradation. Fig.3 shows that initial degradation is high leading to about 50% of released 3H-thymidine labelled DNA after 2 min of digestion. After 10 min about 60% of degraded material is released from lysed nuclei. Release of 3H- uridine labelled RNA was not observed during the whole incubation period. The same experiment was performed with unlabelled nuclei to characterize the degradation products on a 12% acrylamide gel containing 7 M urea. -
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Figure 1. Nucleosomal DNA pattern of chromatin from Matthiola seedlings (slots 1-3), Matthiola flower petals (slots 4-6) and Brassica seedlings (slot 8) as obtained by electrophoresis on 2.5% agarose gels. Staphylococcal nuclease digest was for 3 min (slots I and 4), 6 mmn (slots 2 and 5), 10 mmn (slots 3 and 6) and 5 mmn (slot 8). Molecular weight markers were DNA fragments of 1686, 1320, 881, 535, 462, 357, 271, 230, 215, 180, 144, 133, 84, 38 and 34 base pairs of Rae III digested Xdvl DNA.
Fig.4 shows the banding pattern
of DNA fragments, which were isolated from DNase I digested nuclei. The lengths of visible bands are multiples of 10 bases. Structure of ribosomal liT'A genes. N~uclei of Brassica seedlings were digested for 7 mmn with staphylococcus nuclease. Resultant deproteinized DNA fragments were separated on a 2.5% 1268
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Figure 3. DNase I digestion of Matthiola nuclei. Nuclei of Matthiola seedlings labelled with J5Hj thymidine and[PH] uridine, respectively, were incubated for 10,20,30,40,50 sec and 1,2,5 and 10 min with DNase I (20 pg/ml). The percentage of DNA ) and 3H- uridine labelled RNA associated with degraded (it (C---O) was determined by pelleting the undegraded material of the lysed nuclei and measuring the release of radioactivity.
gel and transferred to nitrocellulose filters according to the method of Southern (16). Hybridization was performed with 125I-labelled 18S + 25S rRNA (Fig.5). The autoradiograph of hybridized filters (Fig.5B) shows a repeat pattern which coincides exactly with the electrophoretic pattern of the ethidium bromide stained DNA bands (Fig.5A). Ribosomal RNA genes must therefore generally be present within the histone protected fraction of nuclease digested chromatin. Quantitative information on the distribution of rDNA sequences among the subunit fractions was obtained by hybridization of DNA derived from monomeric and multimeric nucleosome particles to 3H-rRNA in excess. Nuclei of Brassica seedlings were digested with staphylococcal nuclease for 5 and 20 minutes. Following the fractionation procedure described in Materials agarose
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80 NT
40
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Figure 4. 12 percent polyacrylamide/7 M urea gel electrophoresis of DNase I digestion products. 20 pg of the extracted DNA were denatured and applied to each slot. a) 10 sec, b) 30 sec, c) 1 min, d) 5 min and e) 10 min digestion.
and Methods the first and second supernatants were analyzed separately on isokinetic sucrose gradients (Fig.6). In the first supernatant there are no chromatin subunits which exceed monomer size (Fig.6A), whereas the second supernatant contains all higher order multimers (Fig.6C). After prolonged incubation multimers are reduced to monomer and core size (Fig.6D; second supernatant), monomers are reduced to cores and smaller fragments (Fig.6B; first supernatant). Hybridization of the corresponding 1271
Nucleic Acids Research Figure 5. Distribution of ribosomal DNA sequences in nuclease digested plant chromatin. Nuclei of Brassica pekinensis were digested for 7 min with staphylococcus nuclease (150 units/ml). DNA fragments were isolated, separated on a 2.5% agarose slab gel, stained with ethidium bromide and transferred to nitrocellulose filters according to the technique of Southern. Ribosomal DNA sequences were detected by hybridization using 125i-labelled 18S + 25S rRNA followed by autoradiography. A) photographic negative of the ethidium bromide stained DNA pattern,
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DNA fragments, which were loaded onto nitrocellulose filters, to purified H-uridine labelled rRNA does not suggest a preferential digestion of ribosomal sequences. The extent of hybridization in the first supernatant remains relatively constant over 20 min of digestion. The corresponding analysis of the second supernatant demonstrates that although the bulk of chromatin is already extensively degraded after short digestion time (Fig.6C) ribosomal genes are enriched in the region of subunit multimers comprising 7 and more nucleosomes. Only after a longer incubation period (Fig.6D) rDNA containing chromatin is reduced to monomer size. Compared to the corresponding unfractionated samples of undigested and nuclease digested plant 1272
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were analyzed on isokinetic sucrose gradients. The DNA the pooled fractions was isolated, denatured and fixed onto ni ~rocellulose filters. Filters were incubated for 4 hours at 3H- uridine labelled 18S 60 C in 6 x SS0 containing 2 j.Ig/ml 9% DNA h-ybridized Absorbance at 260 nxn; + 25S rRNA. 0--
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Nucleic Acids Research DNA fragments (no decrease of hybridizable rDNA sequences) we conclude that these studies on fractionated chromatin deal with a representative part of the ribosomal DNA sequences. 25S and 18S ribosomal RNA genes therefore appear to be arranged in a configuration which can be cleaved by staphylococcus nuclease attack into a similar subunit pattern as the bulk of chromatin. But the digestion seems to follow different kinetics. Evidence has to be presented that the ribosomal RNA genes studied are in the transcriptionally active state. To identify active genes by their attached nascent RNA chains, seedlings of Matthiola were labelled in vivo with [3H] uridine. Nuclei were isolated and treated for 3 min with staphylococcus nuclease. The digested chromatin was fractionated on an isokinetic sucrose gradient and the radioactivity of each fraction was measured (Fig.7A). The radioactive label is distributed over the complete repeat pattern and seems to coincide with the nucleosomal distribution. Isolated 3H- uridine labelled RNA from this gradient (fractions 20-40; Fig.7A) if recentrifuged on an isokinetic sucrose gradient together with digested unlabelled chromatin appears exclusively in the region corresponding to about 4-5S (Fig.7B). This result demonstrates that the sedimentation behaviour of the labelled RNA (Fig.7A) cannot be due to an unspecific aggregation between free RNA and chromatin. To demonstrate that ribosomal RNA sequences are present in this preparation the 3H- uridine labelled RNA derived from the nucleosomal fractions was hybridized to ribosomal DNA. The ribosomal DNA had first been separated from the bulk of DNA by centrifugation in a CsCl density gradient containing actinomycin (Fig.8). In this type of gradient ribosomal DNA sequences become located on the light side of the main band (18). The DNA of every single gradient fraction was denatured, fixed onto nitrocellulose filters and hybridized to the 3H- uridine labelled RNA described above. Fig.8 demonstrates that part of the radioactive RNA hybridizes to those fractions of the gradient which contain ribosomal DNA sequences. The remaining RNA hybridizes to main band DNA and is obviously of nonribosomal origin. Since the response of transcriptionally active sequences to digestion by DNase I was reported to be strikingly different 1274
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Figure 7. Detection of newly transcribed RNA within fractionated chromatin subunits. Matthiola seedlings were incubated for 20 hours with [3H] uridinie invivo. Chromatin derived from staphylococcus nuclease Tigested nuclei of these seedlings was fractionated on an isokinetia sucrose gradient. RNA sequences were identified by their radioactive label (A). The size of the 3H- uridine labelled RNA isolated from the chromatin subunits (fractions 20-40) was determined by recentrifugation on an isokinetic sucrose gradient together with unlabelled digested chromatin (B). 3 Absorbance at 260 nm; X---x 3H-radioactivity v 1275
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Figure 6. Identification of ribosomal RNA sequences among the nascent 3H- uridine labelled RNA molecules attached to fractionated chromatin. Unlabelled total DNA from Matthiola incana was fractionated on an actinomycin-CsCl gradient. Each fraction was denatured, loaded onto filters and hybridized to 3H-labelled RNA isolated from chromatin subunits (fractions 20-40) of the sucrose gradient shown in Fig.7. - * Absorbance at 260 nm; X- --X 3H-radioactivity from that of inactive chromatin (4,5), DNase I degradation of nuclear chromatin was compared to degradation of ribosomal RNA genes. Nuclei of unlabelled Brassica seedlings were incubated up to 10 min with DNase I (Fig. 9 ). At the indicated times the DNA was isolated, fixed onto nitrocellulose filters and hybridized to excess 3H-labelled 18S + 25S rRNA. From the amount of ribosomal DNA hybridized the percentage of degraded DNA was calculated. In a parallel experiment the kinetics of DNase I was determined for 3H- thymidine labelled nuclear chromatin as described in Materials and Methods (Fig.9). Comparison of the kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicates that ribosomal genes are digested but not preferentially degraded. Initial degradation of nuclear DNA even appears to be higher than the degradation of ribosomal DNA 1276
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S
1
2
3
4 5 6 7 Digestion time (min)
8
9
10
Figure 9. Kinetics of DNase I digestion of ribosomal RNA genes as compared to digestion of nuclear chromatin. Nuclei of Brassica seedlings were incubated for 10,20,30 sec and 1,2,5 and 1-0 min with DNase I. DNA was isolated and hybridized for 4 hours to excess 3H- uridine labelled 18S + 253 rRNA. The percentage of rDNA degraded was calculated. The percentage of total DNA degraded was determined as described for Fig.3. *-* % ribosomal DNA degraded; X % total DNA degraded sequences. DISCUSSION
Staphylococcal nuclease and DNase I digestion of plant chromatin indicate a similar structure for animal and plant systems. Staphylococcal nuclease digestion of Brassica and Matthiola nuclei leads to a nucleosomal DNA repeat of 175±8 base pairs. This value is lower than the data reported for tobacco, barley (22) and rye (23) calculated by comparison to Hae III fragments of PM2 DNA and digested rat liver nuclei. Low values in the range of 150-175 base pairs have been described for Aspergillus, Saccharomyces, Neurospora and Physarum (for references see 24). Contrary to various animal systems no 1277
Nucleic Acids Research differences in repeat length have been observed for different plant species and tissues. The different physiological activity of growing seedlings compared to relatively inert, fully differentiated flower petals is only documented by an indefinite monomer band (Fig.1). The DNA content-of the core particle (140 base pairs) and the DNase I digestion pattern suggest, that the general organization of the DNA within the nucleosome is the same for;plants and animals. A striking difference in the release of 3H-radioactivity when 3H- thymidine labelled plant nuclei (label observed was in the DNA) and 3H- uridine labelled plant nuclei (label in the RNA) were digested with DNase I in parallel but equivalent experiments. Whereas a 60% release of radioactivity was obtained for the 3H- thymidine labelled chromatin no increase of released 3H- uridine labelled RNA occured during the whole incubation period suggesting that the in vivo labelled RNA is attached to an unsoluble chromatin fraction. This interpretation would conflict with results reported-by'several groups which demonstrate that transcriptionally active genes, to which nascent RNA chains are attached,are preferentially degraded by DNase I
(4,5,9,12). In our experiments ribosomal-RNA genes were used as a model system for active chromatin. No preferential digestion of rRNA genes was observed by staphylococcal nuclease (Fig.6) or DNase I (Fig.9). We cannot state with certainty that th1e rDNIA containing chromatin we examined contained actually avtive genes. A strong evidence, however, is given by the identification of nascent RNA chains in nuclease generated subunits. Unspecific, aggregation can be excluded (Fig.7B) and since the RNA molecules which are distributed over the whole nucleosomal repeat pattern exhibit a length of 4-5S if isolated and recentrifuged, their different sedimentation behaviour cannot be caused byassociation with proteins (RNP particles) but must be due to attachment to the transcriptional comp-exs. Evidence-that part of these newly synthesized RNA molecules are transeribed from ribosomal DNA arises from the hybridization experiment (Fig.8). Previous studies have shown tl,at 25S, 18S and 5S rRNA hybridize to GC-rich fractions which are.separated from the bulk of main 1278
Nucleic Acids Research band DNA on actinomycin/CsCl gradients. From the amount of radioactivity, however, it can be calculated that the nascent RNA molecules contain predominantly 18S and 25S rRNA sequences
(25). It is now generally accepted that ribosomal RNA genes are present in a nucleosomal structure (for references see 24). After staphylococcal nuclease digestion we identified ribosomal DNA in DNA fragments of all multimer lengths (Fig.5) and in particles sedimenting like nucleosomes (Fig.6). The distribution of the ribosomal DNA sequences, however, was found to be unequal among the various nucleosomal classes. After limited digestion, when the bulk of chromatin is already digested to monomer size, ribosomal genes are accumulated in multimers comprising 7 and more nucleosomes (Fig.6C). Extrachromosomal rDNA sequences of Physarum are reported to be primarily recovered in the monomer and so called peak A fraction (26). This difference may be due to a structural transition caused by the different salt concentrations used. Both results clearly agree on the different susceptibility to staphylococcal nuclease compared to the bulk of chromatin. Conflicting with nuclease digestion studies (9,10,11) electron microscopic observations agree on the absence of nucleosomes in actively transcribed ribosomal RNA genes (6,7,8). The facts, however, that actively transcribed DNA is still associated with basic proteins (27) and protected against staphylococcal nuclease (4,5,9,10,11) but not DNase I digestion (4,5,9,12) have suggested the conclusion that the tight binding between histones and DNA may be modulated in actively transcribed genes. Our results indicate that ribosomal RNA genes in the active and inactive state posess a nucleosomal periodicity. Ribosomal DNA sequences appear to be less susceptible to DNase I attack compared to the bulk of chromatin. Since a modified chromatin structure of ribosomal genes may result in a different solubilization behaviour the relation between ionic strength and structural transitions has to be further studied.
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Nucleic Acids Research ACKNOWLEDGEMENTS We are grateful to Miss Anne Roth for excellent technical assistance. Part of the results has been presented at the Symposium "Genome, Chromatin and Karyotype: Evolution and Function", held at Kaiserslautern, -GFR, October 13-15, 1978, and is going to be published in Plant Syst.Evol. Suppl.2, Springer, Wien, New York, 1979. The work has been supported by Deutsche Forschungsgemeinschaft.
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131-146 Laird, C.D., Wilkinson, L.E., Foe, V.E. and Chooi, W.Y. (1976) Chromosoma (Berl.) 58, 169-192 Scheer, U. (1978) Cell 13, 535-549 Mathis, D.J. and Gorovsky, M.A. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 773-778 Reeves, R. (1976) Science 194, 529-532 Reeves, R. and Jones, A. (1976) Nature 260, 495-500 Stalder, J., Seebeck, T. and Braun, R. (1978) Eur.J.Biochem.
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Leber, B. and Hemleben, V. (1979) Z. Pflanzenphysiol. 91, 305-316 14 Noll, H. (1967) Nature 215, 360-363 15 Maniatis, T., Jeffrey, A. and van de Sande, H. (1975) Biochemistry 14, 3787-3794 16 Southern, E.M. (1975) J.Mol.Biol. 98, 503-515 17 Hemleben, V., Ermisch, N., Kimmich, D., Leber, B. and Peter, G. (1975) Eur.J.Biochem. 56, 403-411 18 Hemleben, V., Grierson, D. and Dertmann, H. (1977) Plant Science Letters 9, 129-135 19 Spring, H., Grierson, D., Hemleben, V., Stohr, M., Krohne, G., Stadler, J. and Franke, W.W. (1978) Experimental Cell Research 114, 203-215 20 Grierson, D. and Hemleben, V. (1977) Biochim.Biophys.Acta 475, 424-436 21 Leber, B. and Hemleben, V. (1979) Plant Syst. Evol., Suppl.2 (in press) 22 Philipps, G. and Gigot, C. (1977) Nucl. Acids Res. 4, 3617-3626 23 Cheah, K.S.E. and Osborne, D.J. (1977) Bioch.J. 163, 141-144 13
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Thomas, J.0. (1978) in: International Review of Biochemistry, Biochemistry of Nucleic Acids II, Clark, B.F.C., Ed., Vol 17, pp.181-232, University Park Press Baltimore. Hemleben, V. and Grierson, D. (1978) Chromosoma (Berl.) 65,
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Allfrey, V.G., Johnson, E.M., Sun, I.Y.C., Littau, V.C., Matthews, H.R. and Bradbury, E.M. (1977) Cold Spring Harbor Symp. Quant.Biol. 42, 505-514 McKnight, S.L., Bustin, M. and Millar, O.L. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 741-754
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