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The use of equilibrium centrifugation in actinomycin-caesium chloride for the purification of ribosomal DNA
Plant Science Letters, 9 (1977) 129--135 © Elsevier/North-Holland Scientific Publishers, Ltd.
129
THE USE OF EQUILIBRIUM CENTRIFUGATION IN ACTINOMYCINCAESIUM CHLORIDE FOR THE PURIFICATION OF RIBOSOMAL DNA
VERA HEMLEBEN*, DONALD GRIERSON** and HEIDI DERTMANN*
*Department of Biology II, University of TSbingen, A u f der MorgensteUe 28, 74 TiJbingen, W. Germany, and **Department of Physiology and Environmental Studies, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, LE12 5RD (Great Britain) (Received September 1st, 1976) (Accepted October 28th, 1976)
SUMMARY
The separation of main-band DNA and major satellite components from DNA coding for ribosomal RNA was studied using equilibrium centrifugation in CsC1 and actinomycin-CsC1. The results show that actinomycin-CsC1 gradients can be used for the purification of plant ribosomal DNA.
INTRODUCTION
A valuable initial step in the characterisation of the genome is to separate DNA into various components. This provides defined fractions which can be analysed by biochemical and biophysical techniques. Equilibrium centrifugation of animal and plant DNA in neutral CsC1 often reveals satellites which can be studied separately from the main-band DNA [1--3]. Centrifugation in Cs2SO4 gradients in the presence of heavy metals [4--6] has also shown additional DNA components not previously detected in CsC1 alone. Such studies have yielded information about the organisation, sequence complexity and chromosomal location of repeated DNA sequences [5,7]. However it is probable that many of these DNA satellites are not transcribed in vivo [ 1] and are therefore unsuitable as defined templates for in vitro studies on the regulation of RNA synthesis by purified RNA polymerases.Furthermore, there is evidence that in other cases prominent satellites detected in CsC1 gradients may represent organelle DNA [8]. We have investigated the use of actinomycin-CsC1 gradients [9] for the purification of ribosomal DNA from plants, free from main-band DNA and other satellite DNA.
130 MATERIALS AND METHODS Seeds of Matthiola incana, strain 17, were germinated and grown aseptically [10]. Seeds of Cucumis melo (melon) were separated from the fruit, which was bought locally and eaten, washed thoroughly, germinated and grown aseptically as described for Phaseolus aureus [11]. DNA was isolated and purified from seedlings grown for 5--7 days as described by Hemleben et al. [10] and used after reprecipitation several times from 0.1 M Tris pH 7.5 containing 5 mM EDTA, with 2 volumes of ethanol. Radioactive M. incana DNA was obtained by growing plants in [6-3H]thymidine (27 Ci/mmole) alone or together with [8-3H]adenine (21 Ci/mmole) (20 uCi/ml of each) for 7 days. Radioactive RNA was prepared by growing M. incana seedlings in [5-3H]uridine (26 Ci/mmole; 500 ~Ci/ml) for 3 days. R N A was extracted and purified as described by Grierson and Covey [ 12 ], fractionated by polyacrylamide gel electrophoresis and 25S and 18S, 5S and 4SRNA peaks eluted from gels and recovered [11]. 25S and 18S RNA purified in this way was used for hybridisation. • For b u o y a n t density centrifugation in CsCI, 8.3 g CsCl was dissolved in 6.65 ml Tris--EDTA--HC1 buffer, pH 7.5, containing 50--200 ~g of DNA. The density was adjusted to 1.70 g ml-' and the samples were centrifuged at 20°C for 60 h at 32 000 rpm in a 50 Ti (fixed angle) rotor of a Beckman model 2 L preparative ultracentrifuge. For actinomycin-CsCl gradients [9] 7.5 g CsCl was dissolved in 7.5 ml 0.025 M sodium tetraborate buffer, pH 9.2, containing 200--500 ~g DNA. The solution was cooled in an ice bath and 0.25 ml of actinomycin D (1 mg/ml stock solution} was added. The refractive index at 5°C was 1.3900. Samples were centrifuged at 3°C for 85 h at 30 000 rpm in a 50 Ti (fixed angle) rotor in a Beckman model 2 L ultracentrifuge. After centrifugation, tubes were punctured at the b o t t o m and 7-drop fractions collected. The refractive index of every tenth fraction was measured. Other fractions were diluted with 1 ml of buffer used to make up the gradients and the absorbance at 260 nm measured. For the experiment in Fig. 1, after measuring A260 am, the DNA of every remaining fraction was then precipitated by adding 1 ml carrier R N A (250 ug/ml) and 2 ml 10% TCA, trapped on nitrocellulose filters and counted in a toluene scintillator. For the experiment in Fig. 2 where [3H]adenine and [3H]thymidine-labelled M. incana DNA was fractionated in actinomycin-CsCl {Fig. 2A), gradient fractions were diluted with 1 ml buffer, 10 pl from each fraction was dried onto filter paper and counted in a toluene scintillator. The fractions indicated in Fig. 2A were then pooled, dialysed overnight at 4°C against 2 l of Tris--EDTA--HCI buffer, pH 7.5, with one change of buffer and recentrifuged on neutral CsC1 gradients together with unlabelled M. incana total DNA. A260 nm and radioactivity was then measured as described above. For subsequent hybridisation {Fig. 3) fractions from either C'sC1 or ac'cinomycin-CsCl gradients were diluted with 1 ml buffer for measurement of A260nm and then denatured with 1 ml 1 N NaOH; after 30 min fractions were
131
neutralised by adding 4 ml 1 N HCI; 1 M Tris, pH 8.0; 3 M NaCI (1:1:2) and the DNA loaded onto 13 mm diameter nitrocellulose filters (0.45 tam) [13]. Filters were dried, baked in a vacuum oven at 80°C for 2 h and used for hybridisation. In some experiments each filter was cut in half before hybridisation and each set of half-filters hybridised to different radioactive R N A samples. For hybridisation the general method of Birnstiel et al. [13] was followed. Hybridisation was in 2 × SSC, 70°C, for 2 h using 25S or 18S r R N A (specific activity 91 000 cpm/tag) at a concentration of 2 tag/ml. For all gradients used, both types of rRNA always hybridised to the same DNA fractions and therefore only results for 25S rRNA hybridisation are shown. The Tm of DNA-RNA hybrids was determined by incubating filters in 1 ml aliquots of 1 X SSC for 5 min at a given temperature (the solution was preheated to the desired temperature and checked with a thermometer), and measuring the a m o u n t of radioactive RNA released from the filter. The process was then repeated at successively higher temperatures. RESULTS
AND
DISCUSSION
In Fig. 1 DNA from Cucumis melo an~i Matthiola incana are compared by equilibrium centrifugation in CsC1. C. raelo main-band DNA has a b u o y a n t density of 1.692 g ml -~ and the prominent satellite, which represents approximately 25% of the total DNA, has a density of 1.706 g ml -~ [14]. M. incana, which like C. melo has a small genome (C. melo 1.9 pg per diploid nucleus [ 14] ; M. incana approximately 3 pg per diploid nucleus, our unpublished work) has a main band at 1.697 g ml -t with a small satellite DNA fraction at a b o u t 1.708 g ml -~ amounting to a b o u t 3% of the total DNA. Centrifugation of M. incana (Fig. 2A) or C. melo (Fig. 3C) DNA in actinomycin-CsC1 gradients produced quite different results. The apparent b u o y a n t "~:706 ""1-~1.697
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Fig. 1. Fractionation of M. incana and C. melo D N A in CsCI. 80 ug unlabelled C. melo D N A (A260, open circles)and 5 ug M. incana D N A (radioactivity, closed circles;specific activity 8.103 cprn/~g) were centrifuged together as described in M A T E R I A L S AND METHODS.
132
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Fig. 2. Comparison of banding position of M. incana DNA fractions in actinomycin-CsC1 and CsCI alone. In Fig. 2A 350 #g M. incana DNA (labelled with [ 3H]thymidine and [ 3H ]adenine; specific activity 1 • 105 cpm/#g) were fractionated in actinomycin-CsCl. 1% of each gradient fraction was counted for radioactivity (closed circles). Fractions I, II and III were collected, dialysed and recentrifuged in CsCI together with unlabelled M. incana DNA. Fig. 2B, fraction I; Fig. 2C, fraction II, Fig. 2D, fraction III. Open circles, A 26~; closed circles, radioactivity of DNA recovered from gradient 2A.
density of the DNA was lower, due to the binding of actinomycin. In general we observed a decrease in density ranging from 0.09 to 0.15 g m1-1 with mainband, satellite and ribosomal DNA. The density change varies for different DNA fractions and may also be governed by the experimental conditions. Fig. 2 provides evidence that the degree of density shift is correlated to some extent with the c o n t e n t of guanosine and cytosine. M. incana DNA labelled with [ 3H] thymidine and [ 3H] adenine was fractionated in actinomycin-CsCl. Fractions from the gradient (I, II, III; Fig. 2A) were recovered, dialysed and recentrifuged in normal CsC1 gradients with total unlabelled M. incana DNA as marker, in the absence of actinomycin D. The results show (Fig. 2B, C, D) that DNA which bands at a low density in actinomycin-CsCl behaves as high GC DNA in CsC1 alone {Fig. 2D). Conversely, DNA which bands at a relatively high density in actinomycin-CsC1 (fraction I) behaves as a low GC DNA in normaJ CsCl (Fig. 2B). This is consistent with the known selective binding of
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Fig. 3. Degree of separation of rRNA genes from main-band and satellite DNA in CsCI and actinomycin-CsC1. Unlabelled total DNA from M. incana (Fig. 3A, 3B) and C. melo (Fig. 3C, 3D) was fractionated in CsC1 (3A, 3C) or actinomycin-CsCl (3B, 3D). The position of the rRNA genes was then measured by hybridisation with radioactive 25S rRNA from M. incana. Open circles, A :s0 of unlabelled DNA; closed circles, radioactivity after hybridisation with 25S rRNA.
actinomycin to GC-rich DNA [15]. Thus a high GC DNA should bind relatively more actinomycin and therefore have a larger reduction of buoyant density in actinomycin-CsC1. In support of this, the prominent satellite of C. melo, which behaves as a high GC DNA in CsC1 alone (Fig. 1, Fig. 3C) appears to have a lower density than main-band DNA in actinomycin-CsCl (Fig. 3D). Finally, when labelled with [3H] adenine and [3H] thymidine M. incana DNA displays a shoulder in actinomycin-CsCl (fraction I in Fig. 1A). It is only detected when the adenosine and thymidine residues are selectively labelled. The relatively dense banding position in the presence of actinomycin is consistent with it being AT-rich DNA and this is confirmed by the low density in CsCl (Fig. 2B). In many plant species the genes coding for rRNA have a higher GC content and therefore higher density in CsC1, than main-band DNA [14,16]. Fig. 3A and 3C show that the ribosomal DNA has a buoyant density of 1.702 g ml-'
134 in M. incana and 1.71 g ml-' in C. melo. The rRNA genes were detected by hybridisation of each fraction from the gradient with radioactive 25 S rRNA from M. incana. Saturation hybridisation experiments with M. incana indicate that approximately 0.2% of the DNA is complementary to 25S plus 18S rRNA (data not shown} which corresponds to a b o u t 2000 genes per diploid nucleus. A similar number of genes has been calculated for C. melo [14]. The T m of the homologous DNA-rRNA hybrid in M. incana was 89°C in 1 X SSC, whereas in C. melo the heterologous hybrid showed a T m approximately 6°C lower (not shown}. This indicates a sequence divergence of the order of only 5--9 bases between M. incana r R N A and C. melo ribosomal DNA [17] and justifies the use of a heterologous hybridisation probe for the detection of rRNA genes. Although CsC1 centrifugation does achieve a partial purification of DNA coding for rRNA, considerable contamination with either main-band or satellite DNA remains (Fig. 3A and C). This is due in the case of M. incana to the small density difference between main-band DNA and the genes for rRNA, and in C. melo to the near-coincidence in b u o y a n t density between the large amount of satellite DNA and the small a m o u n t of DNA complementary to rRNA [14]. Centrifugation of DNA in the presence of actinomycin D, however, results in a much better separation of the r R N A genes. The inclusion of actinomycin seems to accentuate the density difference between the r R N A genes and main-band DNA in M. incana (Fig. 3B). This also occurs in the case of C. melo b u t here in addition actinomycin brings a b o u t the complete separation of the prominent satellite DNA from the DNA complementary to r R N A {Fig. 3D). The separation is greater than one might expect on the basis of b u o y a n t density in CsC1 and this implies that some additional factor such as sequence organisation may effect binding of actinomycin. For comparison we also used Ag÷-Cs2SO4 gradients for the fractionation of DNA complementary to r R N A in M. incana. The best results {average density 1.5 g ml -~, pH 7.0; DNA-P: Ag ÷ ratio 0.4) gave a qualitatively similar pattern to that shown in Fig. 3A for normal CsC1, with only a marginally better separation. Therefore we preferred the actinomycin-CsC1 method on the basis of resolving power. In addition, it proved to be much easier to standardise and reproduce than the Ag÷-Cs2SO4 gradients. It has previously been argued that there is no direct relationship between satellite DNA a m o u n t and rRNA gene number and that small amounts of rDNA satellite are therefore often masked in CsCI by large amounts of unrelated satellite DNA [14]. Our results show that these fractions can be resolved by using actinomycin. The saturation hybridisation values would lead us to expect 0.4--0.6% of the double-stranded DNA to code for rRNA. This value might be doubled to take into account non-conserved sequences transcribed in the rRNA precursor, or untranscribed spacer DNA. Therefore we might expect pure ribosomal R N A genes to represent approximately 1% of total DNA. Our preliminary calculations are that less than 3% of the total DNA is recovered from the region of actinomycin-CsCl gradients which contains sequences complementary to rRNA. The molecular weight of the DNA prepara-
135 tions used was a p p r o x i m a t e l y 5- 106 daltons. It is n o t necessary t o shear the D N A t o smaller size. On the c o n t r a r y , since the r R N A genes are p r o b a b l y clustered as t a n d e m repeats, f r a c t i o n a t i o n s h o u l d still o c c u r with D N A o f m u c h higher m o l e c u l a r weight. T h e p r e s e n t results indicate a substantial e n r i c h m e n t for r R N A genes, b u t f u r t h e r w o r k will be necessary t o establish the degree o f p u r i t y . This s h o u l d provide interesting i n f o r m a t i o n a b o u t the" s e q u e n c e organisation o f p l a n t r R N A genes. In a d d i t i o n , p r e p a r a t i o n s o f p l a n t R N A p o l y m e r a s e I, w h i c h is k n o w n t o be the e n z y m e responsible f o r synthesising r R N A in vivo, are n o w available [ 1 8 - - 2 4 ] . T h e r e f o r e studies on the regulation o f R N A synthesis in vitro using a d e f i n e d h o m o l o g o u s t e m p l a t e and e n z y m e m a y be possible. ACKNOWLEDGEMENT D.G. was s u p p o r t e d b y an E.M.B.O. l o n g - t e r m fellowship. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
W.G. Flamm, Int. Rev. Cytol., 32 (1972) 1. J. Ingle, G.G. Pearson and J. Sinclair, Nature New Biol., 242 (1973) 193. Y. Coudray, F. Quetier and E. Guille, Biochim. Biophys. Acta, 217 (1970) 259. G, Corneo, E. Ginelli and E. Polli, Biochemistry, 9 (1970) 1565. J. Sinclair, R. Wells, B. Deumling and J. Ingle, Biochem. J., 149 (1975) 31. J. Retel and H. van Keulen, Eur. J. Biochem., 58 (1975) 51. J.N. Timmis, B. Deumling and J. Ingle, Nature, 257 (1975) 155. A. Kadouri, D. Atsmon and M. Edelman, Proc. Natl. Acad. Sci. USA, 72 (1975) 2260. M. Birnstiel, J. Telford, E. Weinberg and D. Stafford, Proc. Natl. Acad. Sci. USA, 71 (1974) 2900. V. Hemleben, N. Ermisch, D. Kimmich, B. Leber and G. Peter, Eur. J. Biochem., 56 (1975) 403. D. Grierson, Eur. J. Biochem., 44 (1974) 509. D. Grierson and S. Covey, Planta (Berl.), 127 (1975) 77. M. Birnstiel, J. Speirs, I. Purdom, K. Jones and U.E. Loening, Nature, 219 (1968) 454. J. Ingle, J.N. Timmis and J. Sinclair, Plant Physiol., 55 (1975) 496. W. Miiller and D.M. Crothers, J. Mol. Biol., 35 (1968) 251. K. Matsuda and A. Siegel, Proc. Natl. Acad. Sci. USA, 58 (1967) 673. R.J. Britten, D.E. Graham and B.R. Neufeld, Analysis of repeating DNA sequences by reassociation, in L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 29, Part E, Academic Press, New York, 1974, p. 363. P.H. Horgen and J.L. Key, Biochim. Biophys. Acta, 294 (1973) 227. H. Mondal, R.K. Mandal and B. Biswas, Eur. J. Biochem., 25 (1972) 463. T.J. Guilfoyle, C.Y. Lin, Y.M. Chen, R.T. Nagoa and J.L. Key, Proc. Natl. Acad. Sci. USA, 72 (1975) 69. C.Y. Lin, T.J. Guilfoyle, Y.M. Chen, R.T. Nagoa and J.L. Key, Biochem. Biophys. Res. Commun., 60 (1974) 498. P.J. Rizzo, J.A. Cherry, K. Pederson and V.L. Dunham, Plant Physiol., 54 (1974) 349. Y. Sasaki, R. Sasaki, T. Hashizume and Y. Yamada, Biochem. Biophys. Res. Commun., 50 (1973) 785. G.C. Strain, K.P. Mullinix and L. Bogorad, Proc. Natl. Acad. Sci. USA, 68 (1971) 2647.
129
THE USE OF EQUILIBRIUM CENTRIFUGATION IN ACTINOMYCINCAESIUM CHLORIDE FOR THE PURIFICATION OF RIBOSOMAL DNA
VERA HEMLEBEN*, DONALD GRIERSON** and HEIDI DERTMANN*
*Department of Biology II, University of TSbingen, A u f der MorgensteUe 28, 74 TiJbingen, W. Germany, and **Department of Physiology and Environmental Studies, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, LE12 5RD (Great Britain) (Received September 1st, 1976) (Accepted October 28th, 1976)
SUMMARY
The separation of main-band DNA and major satellite components from DNA coding for ribosomal RNA was studied using equilibrium centrifugation in CsC1 and actinomycin-CsC1. The results show that actinomycin-CsC1 gradients can be used for the purification of plant ribosomal DNA.
INTRODUCTION
A valuable initial step in the characterisation of the genome is to separate DNA into various components. This provides defined fractions which can be analysed by biochemical and biophysical techniques. Equilibrium centrifugation of animal and plant DNA in neutral CsC1 often reveals satellites which can be studied separately from the main-band DNA [1--3]. Centrifugation in Cs2SO4 gradients in the presence of heavy metals [4--6] has also shown additional DNA components not previously detected in CsC1 alone. Such studies have yielded information about the organisation, sequence complexity and chromosomal location of repeated DNA sequences [5,7]. However it is probable that many of these DNA satellites are not transcribed in vivo [ 1] and are therefore unsuitable as defined templates for in vitro studies on the regulation of RNA synthesis by purified RNA polymerases.Furthermore, there is evidence that in other cases prominent satellites detected in CsC1 gradients may represent organelle DNA [8]. We have investigated the use of actinomycin-CsC1 gradients [9] for the purification of ribosomal DNA from plants, free from main-band DNA and other satellite DNA.
130 MATERIALS AND METHODS Seeds of Matthiola incana, strain 17, were germinated and grown aseptically [10]. Seeds of Cucumis melo (melon) were separated from the fruit, which was bought locally and eaten, washed thoroughly, germinated and grown aseptically as described for Phaseolus aureus [11]. DNA was isolated and purified from seedlings grown for 5--7 days as described by Hemleben et al. [10] and used after reprecipitation several times from 0.1 M Tris pH 7.5 containing 5 mM EDTA, with 2 volumes of ethanol. Radioactive M. incana DNA was obtained by growing plants in [6-3H]thymidine (27 Ci/mmole) alone or together with [8-3H]adenine (21 Ci/mmole) (20 uCi/ml of each) for 7 days. Radioactive RNA was prepared by growing M. incana seedlings in [5-3H]uridine (26 Ci/mmole; 500 ~Ci/ml) for 3 days. R N A was extracted and purified as described by Grierson and Covey [ 12 ], fractionated by polyacrylamide gel electrophoresis and 25S and 18S, 5S and 4SRNA peaks eluted from gels and recovered [11]. 25S and 18S RNA purified in this way was used for hybridisation. • For b u o y a n t density centrifugation in CsCI, 8.3 g CsCl was dissolved in 6.65 ml Tris--EDTA--HC1 buffer, pH 7.5, containing 50--200 ~g of DNA. The density was adjusted to 1.70 g ml-' and the samples were centrifuged at 20°C for 60 h at 32 000 rpm in a 50 Ti (fixed angle) rotor of a Beckman model 2 L preparative ultracentrifuge. For actinomycin-CsCl gradients [9] 7.5 g CsCl was dissolved in 7.5 ml 0.025 M sodium tetraborate buffer, pH 9.2, containing 200--500 ~g DNA. The solution was cooled in an ice bath and 0.25 ml of actinomycin D (1 mg/ml stock solution} was added. The refractive index at 5°C was 1.3900. Samples were centrifuged at 3°C for 85 h at 30 000 rpm in a 50 Ti (fixed angle) rotor in a Beckman model 2 L ultracentrifuge. After centrifugation, tubes were punctured at the b o t t o m and 7-drop fractions collected. The refractive index of every tenth fraction was measured. Other fractions were diluted with 1 ml of buffer used to make up the gradients and the absorbance at 260 nm measured. For the experiment in Fig. 1, after measuring A260 am, the DNA of every remaining fraction was then precipitated by adding 1 ml carrier R N A (250 ug/ml) and 2 ml 10% TCA, trapped on nitrocellulose filters and counted in a toluene scintillator. For the experiment in Fig. 2 where [3H]adenine and [3H]thymidine-labelled M. incana DNA was fractionated in actinomycin-CsCl {Fig. 2A), gradient fractions were diluted with 1 ml buffer, 10 pl from each fraction was dried onto filter paper and counted in a toluene scintillator. The fractions indicated in Fig. 2A were then pooled, dialysed overnight at 4°C against 2 l of Tris--EDTA--HCI buffer, pH 7.5, with one change of buffer and recentrifuged on neutral CsC1 gradients together with unlabelled M. incana total DNA. A260 nm and radioactivity was then measured as described above. For subsequent hybridisation {Fig. 3) fractions from either C'sC1 or ac'cinomycin-CsCl gradients were diluted with 1 ml buffer for measurement of A260nm and then denatured with 1 ml 1 N NaOH; after 30 min fractions were
131
neutralised by adding 4 ml 1 N HCI; 1 M Tris, pH 8.0; 3 M NaCI (1:1:2) and the DNA loaded onto 13 mm diameter nitrocellulose filters (0.45 tam) [13]. Filters were dried, baked in a vacuum oven at 80°C for 2 h and used for hybridisation. In some experiments each filter was cut in half before hybridisation and each set of half-filters hybridised to different radioactive R N A samples. For hybridisation the general method of Birnstiel et al. [13] was followed. Hybridisation was in 2 × SSC, 70°C, for 2 h using 25S or 18S r R N A (specific activity 91 000 cpm/tag) at a concentration of 2 tag/ml. For all gradients used, both types of rRNA always hybridised to the same DNA fractions and therefore only results for 25S rRNA hybridisation are shown. The Tm of DNA-RNA hybrids was determined by incubating filters in 1 ml aliquots of 1 X SSC for 5 min at a given temperature (the solution was preheated to the desired temperature and checked with a thermometer), and measuring the a m o u n t of radioactive RNA released from the filter. The process was then repeated at successively higher temperatures. RESULTS
AND
DISCUSSION
In Fig. 1 DNA from Cucumis melo an~i Matthiola incana are compared by equilibrium centrifugation in CsC1. C. raelo main-band DNA has a b u o y a n t density of 1.692 g ml -~ and the prominent satellite, which represents approximately 25% of the total DNA, has a density of 1.706 g ml -~ [14]. M. incana, which like C. melo has a small genome (C. melo 1.9 pg per diploid nucleus [ 14] ; M. incana approximately 3 pg per diploid nucleus, our unpublished work) has a main band at 1.697 g ml -t with a small satellite DNA fraction at a b o u t 1.708 g ml -~ amounting to a b o u t 3% of the total DNA. Centrifugation of M. incana (Fig. 2A) or C. melo (Fig. 3C) DNA in actinomycin-CsC1 gradients produced quite different results. The apparent b u o y a n t "~:706 ""1-~1.697
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Fig. 1. Fractionation of M. incana and C. melo D N A in CsCI. 80 ug unlabelled C. melo D N A (A260, open circles)and 5 ug M. incana D N A (radioactivity, closed circles;specific activity 8.103 cprn/~g) were centrifuged together as described in M A T E R I A L S AND METHODS.
132
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Fig. 2. Comparison of banding position of M. incana DNA fractions in actinomycin-CsC1 and CsCI alone. In Fig. 2A 350 #g M. incana DNA (labelled with [ 3H]thymidine and [ 3H ]adenine; specific activity 1 • 105 cpm/#g) were fractionated in actinomycin-CsCl. 1% of each gradient fraction was counted for radioactivity (closed circles). Fractions I, II and III were collected, dialysed and recentrifuged in CsCI together with unlabelled M. incana DNA. Fig. 2B, fraction I; Fig. 2C, fraction II, Fig. 2D, fraction III. Open circles, A 26~; closed circles, radioactivity of DNA recovered from gradient 2A.
density of the DNA was lower, due to the binding of actinomycin. In general we observed a decrease in density ranging from 0.09 to 0.15 g m1-1 with mainband, satellite and ribosomal DNA. The density change varies for different DNA fractions and may also be governed by the experimental conditions. Fig. 2 provides evidence that the degree of density shift is correlated to some extent with the c o n t e n t of guanosine and cytosine. M. incana DNA labelled with [ 3H] thymidine and [ 3H] adenine was fractionated in actinomycin-CsCl. Fractions from the gradient (I, II, III; Fig. 2A) were recovered, dialysed and recentrifuged in normal CsC1 gradients with total unlabelled M. incana DNA as marker, in the absence of actinomycin D. The results show (Fig. 2B, C, D) that DNA which bands at a low density in actinomycin-CsCl behaves as high GC DNA in CsC1 alone {Fig. 2D). Conversely, DNA which bands at a relatively high density in actinomycin-CsC1 (fraction I) behaves as a low GC DNA in normaJ CsCl (Fig. 2B). This is consistent with the known selective binding of
133
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FRACTION NO,
Fig. 3. Degree of separation of rRNA genes from main-band and satellite DNA in CsCI and actinomycin-CsC1. Unlabelled total DNA from M. incana (Fig. 3A, 3B) and C. melo (Fig. 3C, 3D) was fractionated in CsC1 (3A, 3C) or actinomycin-CsCl (3B, 3D). The position of the rRNA genes was then measured by hybridisation with radioactive 25S rRNA from M. incana. Open circles, A :s0 of unlabelled DNA; closed circles, radioactivity after hybridisation with 25S rRNA.
actinomycin to GC-rich DNA [15]. Thus a high GC DNA should bind relatively more actinomycin and therefore have a larger reduction of buoyant density in actinomycin-CsC1. In support of this, the prominent satellite of C. melo, which behaves as a high GC DNA in CsC1 alone (Fig. 1, Fig. 3C) appears to have a lower density than main-band DNA in actinomycin-CsCl (Fig. 3D). Finally, when labelled with [3H] adenine and [3H] thymidine M. incana DNA displays a shoulder in actinomycin-CsCl (fraction I in Fig. 1A). It is only detected when the adenosine and thymidine residues are selectively labelled. The relatively dense banding position in the presence of actinomycin is consistent with it being AT-rich DNA and this is confirmed by the low density in CsCl (Fig. 2B). In many plant species the genes coding for rRNA have a higher GC content and therefore higher density in CsC1, than main-band DNA [14,16]. Fig. 3A and 3C show that the ribosomal DNA has a buoyant density of 1.702 g ml-'
134 in M. incana and 1.71 g ml-' in C. melo. The rRNA genes were detected by hybridisation of each fraction from the gradient with radioactive 25 S rRNA from M. incana. Saturation hybridisation experiments with M. incana indicate that approximately 0.2% of the DNA is complementary to 25S plus 18S rRNA (data not shown} which corresponds to a b o u t 2000 genes per diploid nucleus. A similar number of genes has been calculated for C. melo [14]. The T m of the homologous DNA-rRNA hybrid in M. incana was 89°C in 1 X SSC, whereas in C. melo the heterologous hybrid showed a T m approximately 6°C lower (not shown}. This indicates a sequence divergence of the order of only 5--9 bases between M. incana r R N A and C. melo ribosomal DNA [17] and justifies the use of a heterologous hybridisation probe for the detection of rRNA genes. Although CsC1 centrifugation does achieve a partial purification of DNA coding for rRNA, considerable contamination with either main-band or satellite DNA remains (Fig. 3A and C). This is due in the case of M. incana to the small density difference between main-band DNA and the genes for rRNA, and in C. melo to the near-coincidence in b u o y a n t density between the large amount of satellite DNA and the small a m o u n t of DNA complementary to rRNA [14]. Centrifugation of DNA in the presence of actinomycin D, however, results in a much better separation of the r R N A genes. The inclusion of actinomycin seems to accentuate the density difference between the r R N A genes and main-band DNA in M. incana (Fig. 3B). This also occurs in the case of C. melo b u t here in addition actinomycin brings a b o u t the complete separation of the prominent satellite DNA from the DNA complementary to r R N A {Fig. 3D). The separation is greater than one might expect on the basis of b u o y a n t density in CsC1 and this implies that some additional factor such as sequence organisation may effect binding of actinomycin. For comparison we also used Ag÷-Cs2SO4 gradients for the fractionation of DNA complementary to r R N A in M. incana. The best results {average density 1.5 g ml -~, pH 7.0; DNA-P: Ag ÷ ratio 0.4) gave a qualitatively similar pattern to that shown in Fig. 3A for normal CsC1, with only a marginally better separation. Therefore we preferred the actinomycin-CsC1 method on the basis of resolving power. In addition, it proved to be much easier to standardise and reproduce than the Ag÷-Cs2SO4 gradients. It has previously been argued that there is no direct relationship between satellite DNA a m o u n t and rRNA gene number and that small amounts of rDNA satellite are therefore often masked in CsCI by large amounts of unrelated satellite DNA [14]. Our results show that these fractions can be resolved by using actinomycin. The saturation hybridisation values would lead us to expect 0.4--0.6% of the double-stranded DNA to code for rRNA. This value might be doubled to take into account non-conserved sequences transcribed in the rRNA precursor, or untranscribed spacer DNA. Therefore we might expect pure ribosomal R N A genes to represent approximately 1% of total DNA. Our preliminary calculations are that less than 3% of the total DNA is recovered from the region of actinomycin-CsCl gradients which contains sequences complementary to rRNA. The molecular weight of the DNA prepara-
135 tions used was a p p r o x i m a t e l y 5- 106 daltons. It is n o t necessary t o shear the D N A t o smaller size. On the c o n t r a r y , since the r R N A genes are p r o b a b l y clustered as t a n d e m repeats, f r a c t i o n a t i o n s h o u l d still o c c u r with D N A o f m u c h higher m o l e c u l a r weight. T h e p r e s e n t results indicate a substantial e n r i c h m e n t for r R N A genes, b u t f u r t h e r w o r k will be necessary t o establish the degree o f p u r i t y . This s h o u l d provide interesting i n f o r m a t i o n a b o u t the" s e q u e n c e organisation o f p l a n t r R N A genes. In a d d i t i o n , p r e p a r a t i o n s o f p l a n t R N A p o l y m e r a s e I, w h i c h is k n o w n t o be the e n z y m e responsible f o r synthesising r R N A in vivo, are n o w available [ 1 8 - - 2 4 ] . T h e r e f o r e studies on the regulation o f R N A synthesis in vitro using a d e f i n e d h o m o l o g o u s t e m p l a t e and e n z y m e m a y be possible. ACKNOWLEDGEMENT D.G. was s u p p o r t e d b y an E.M.B.O. l o n g - t e r m fellowship. REFERENCES
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