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Enrichment of Bacillus subtilis DNA in the sequences for ribosomal RNA
466
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96361
E N R I C H M E N T OF B A C I L L U S
SUBTILIS
DNA IN T H E SEQUENCES F O R
RIBOSOMAL RNA V. SGARAMELLA*
Istituto di Genetica deU'Universita, 27100 Pavia (Italy) (Received July i4th, 1969)
SUMMARY
Bacillus subtilis DNA was heated at constant temperatures within the helixcoil transition interval. Slight increases in the temperature caused the denaturation of greater fractions of DNA. The native molecules were separated from the heatsensitive ones b y means of nitrocellulose chromatography. The fractions thus obtained were characterized for their base composition and ability to hybridize with ribosomal RNA (rRNA). As expected, heat resistance is inversely correlated with thymidine frequency and directly correlated with the content in sequences complementary to rRNA (rDNA).
INTRODUCTION
Most DNA preparations are composed of polynucleotide chains derived from mechanical and chemical fragmentation of the original molecules. These fragments m a y be heterogeneous in their base composition 1. Attempts to separate them make use of the resulting differences in their physical properties. Variations in the buoyant density have allowed the separation of fragments of ~ DNA ~, the enrichment for ribosomal RNA (rRNA) cistrons in completely denatured DNA from Escherichia coli a, and in native DNA from Xenopus laevis *,~A, as well as for specific genetic markers in pneumococcal DNA 5 and in Bacillus subtilis 6. The thermal resistance of DNA is known to depend linearly on its G + C contentT; controlled heating m a y lead to the melting of some molecules and not of others s. Various techniques have been used to separate the native from the denatured fraction: to mention some, density gradient centrifugation 6 and chromatography on various materials 9-12. One of the most versatile among them is nitrocellulose, which specifically interacts with denatured but not with native DNA is. This material has been used to prepare membranes 14 and to fill chromatographic columns, in which single-stranded DNA is selectively adsorbed 15-17. In this work the molecules of Bacillus subtilis DNA, which have not been denatured after fractional melting at given temperatures, have been separated from the Abbreviations: rRNA, ribosomal RNA; rDNA, the sequences of DNA complementary to rRNA; Tin, melting temperature. * Present address: Institute for Enzyme Research, University of Wisconsin, Madison, Wisc., U.S.A.
Biochim. Biophys. Acta, 195 (1969) 466-472
ENRICHMENT OF D N A IN SEQUENCES FOR r R N A
467
denatured ones. While the former are not retained by nitrocellulose columns, the latter are tightly adsorbed so that completely different conditions are required to elute them. In this way it has been possible to obtain fractions of DNA with increasing thermal stability. These fractions exhibit a thymidine content lower than unfractionated DNA and inversely correlated with their thermal resistance. The heatresistant fractions are considerably enriched in sequences complementary to rRNA.
MATERIALS AND METHODS
[3H]DNA preparation Cells of the thymine-requiring B. subtilis strain SB 556/1 were grown in a medium containing, (in g/l): glucose, 5.0; Casamino acids, 0.4; asparagine, I.O; tryptophan, o.oi; (NH4)2SO 4, I.O; K H , P 0 4 , 7.0; K2HP04, 2.0; MgSO 4, o.I; sodium citrate, o.i; unlabeled thymidine, 0.00175. The medium contained also 2.5 mC of [Me-3H] thymidine, 14 C/mmole, from Schwarz BioResearch (Orangeburg, N.Y.). After growth at 37 ° under alternative shaking, at the beginning of the stationary phase, the cells were centrifuged and washed. DNA was extracted following a previously described procedure and showed an s°20,~ of 35 S which, according to STUDIER TM, corresponds to an average molecular weight of 5" lOT-
32p-labeled rRNA preparation As previously described TM.
DNA denaturation For partial denaturations, 2-3-ml samples of DNA solutions at approx. 50/*g/ ml in 7.5 mM NaCl-o.75 mM sodium citrate (pH 7.8) were immersed for 15 min in a water bath at a temperature selected within the thermal transition interval. Temperature regulations to o.I ° were obtained with a Colora (Lorch, Germany) water bath. For complete denaturations, DNA was heated to IOO° for 15 min. Each sample, exposed to a given temperature, was then chilled b y transferring the tube into an ice-water mixture.
Nitrocellulose chromatography Partially or totally denatured DNA solutions were adjusted to 0.3 M NaC10.03 M sodium citrate with appropriate additions of 3 M NaCl-o.3 M sodium citrate, They were then loaded on small columns (3.0 cm ×0.6 cm) filled with Nitrocel-S (Serva, Heidelberg) according to KLAMERTH15. Before loading, the columns were washed until no ultraviolet-absorbing material was eluted. After the sample had been adsorbed, the column was washed with three bed volumes of 0.3 M NaCl-o.o3 M sodium citrate. Native DNA was immediately released; its double-stranded structure was assessed with thermal hyperchromicity measurements. On the contrary, denatured molecules were tightly bound so that both low ionic strength (o.oi M NaC1) and alkaline conditions (pH up to 12) were necessary to elute them. Original DNA preparations, not submitted to any thermal treatment, were recovered from the columns in variable amounts, ranging between 60 and 9 ° ~/o, in different DNA preparations. If rechromatographed, the relessed material could be recovered almost quantitatively. Since the same batches exhibited similar retention, this effect seems ascribable to partially denatured molecules present in current DNA preparations le. Undenatured molecules were recovered by collecting 4-5 fractions of I.O ml. Biochim. Biophys. Acta, 195 (1969) 466-472
468
V. SGARAMELLA
Determination o] thymidine content o/ /ractionated DNA The specific radioactivity (as counts/rain per fg) in the fractions resistant to progressively higher temperatures was compared to that of the original, unfractionated DNA. The variation of this ratio was assumed to reflect a change in base composition. For radioactivity measurements, proportional aliquots of the DNA solutions, brought to the same final volume, were pipetted into discs of filter paper; these were counted in a liquid scintillation spectrometer using a toluene-2,5-diphenyloxazole-I,4-bis-(5-phenyloxazolyl-2)benzene system. DNA concentrations were determined by the diphenylamine test 2°.
Hybridization experiments Hybridizations were performed in solution, according to •YGAARD AND HALL13. Before annealing, native DNA and heat-resistant fractions were completely denatured by exposure to IOO° for 15 rain. Hybridized 32P-labeled rRNA was measured as previously described is.
RESULTS
Fractional denaturation and nitrocellulose adsorption First of all, it was considered necessary to study how the fraction of DNA adsorbed to nitrocellulose varied with increasing temperatures of preliminary heating, and the relationship between this variation and the process of thermal melting as followed by absorbance.
100I
~.,...-..~
1.30
.1.20
1.10
~o
%o 1.00 60 °
6 5 '-
70 °
75°
80 °
85 o
HEATING TEMPERATURE Fig. I. Influence of the t e m p e r a t u r e on a b s o r b a n c c at 260 m # and nitrocellulose adsorption. A s t o p p e r e d cuvette w i t h a i o - m m light p a t h was filled w i t h [SH]DNA (specific activity, 14 7o0 c o u n t s / m i n p e r fig) in 7.5 mM NaCl-o.75 mM s o d i u m citrate. A b s o r b a n c e at 26o m # was m e a s u r e d a g a i n s t the solvent in a B e c k m a n D U G-24oo S p e c t r o p h o t o m e t e r e q u i p p e d with h e a t i n g c h a m b e r . T e m p e r a t u r e was increased at a rate of o.3°/min and m e a s u r e d w i t h an i r o n - c o n s t a n t a n t h e r m o couple dipping in a s t o p p e r e d c u v e t t e filled w i t h vaseline oil. H y p e r c h r o m i c i t y is given as the ratio of the absorbance at the various t e m p e r a t u r e s on t h e a b s o r b a n c e at 25 °, w i t h o u t a n y volume correction. Nitrocellulose a d s o r p t i o n was m e a s u r e d in this way: 3-ml solutions of [SH]DNA, at 5 ° / z g / m l in 7.5 mM NaCl-o.75 mM s o d i u m citrate, were h e a t e d at the indicated t e m p e r a t u r e s for 15 rain and i m m e d i a t e l y a f t e r w a r d s were chilled in i c e - w a t e r m i x t u r e ; the solutions were t h e n b r o u g h t to 0. 3 M NaCl-o.o 3 M s o d i u m citrate w i t h the additions of 3 M NaCl-o. 3 M s o d i u m citrate a n d loaded on the nitrocellulose columns. Several I . o - m l fractions were collected until no more r a d i o a c t i v i t y was eluted. The difference b e t w e e n i n p u t and r e c o v e r y gave the adsorption of nitrocellulose columns.
Biochim. Biophys. Acta, 195 (1969) 466-472
ENRICHMENT OF D N A
469
IN SEQUENCES FOR r R N A
Fig. I shows that the amount of DNA adsorbed to nitrocellulose increases sharply following exposure to temperatures above 7 °° . The midpoint of the adsorption curve is near 73 °. Its sigmoid shape parallels the hyperchromicity curve, which is displaced towards the higher temperatures b y about 5 ° . Nitrocellulose adsorption can therefore be considered as an additional property of DNA affected b y its helix-coil transition. From a preparative point of view, it permits a simple separation of native from partially denatured molecules. The displacement between the two curves is discussed below.
Thymidine content o//ractionated DNA A set of DNA fractions exhibiting increasing thermal stability b y their chromatographic behavior on nitrocellulose was prepared and its specific radioactivity determined as described in MATERIALS AND METHODS. Table I gives the results of TABLE
I
CORRELATION BETWEEN THERMAL STABILITY AND THYMIDINE CONTENT OF FRACTIONATED E3H]DNA F o r f r a c t i o n a l d e n a t u r a t i o n s a n d e v a l u a t i o n of D N A h e a t - r e s i s t a n t f r a c t i o n s see MATERIALS AND METHODS. The a p p a r e n t G + C c o n t e n t g i v e n b e t w e e n b r a c k e t s in t h e f i r s t r o w of t h e l a s t c o l u m n is t h e one o b t a i n e d f r o m t h e l i t e r a t u r e 5 a n d is t h e v a l u e to w h i c h t h e v a r i a t i o n s i n s pe c i fi c act i v i t y h a v e b e e n r e f e r r e d in o r d e r to c a l c u l a t e t h e a p p a r e n t G + C c o n t e n t of t h e h e a t - r e s i s t a n t fractions.
Heat-resistant [raction (%)
Specific activity (3Hcounts/rnin per I~g)
Decrease in [SH]thymidine content
Apparent G+ C content
Untreated 50 20
850 837 76o
-1.5 lO.6
(43) 43.8 48.9
(%)
(%)
such measurements. The correlation between beat resistance and specific activity seems consistent with the hypothesis of a decreasing thymidine content in the more thermally stable fractions. The inferred estimate of the G + C content of the various fractions (last column) based on the consideration that the DNA is all double-stranded, demonstrates that the most heat-resistant fraction (representing 20 % of the original untreated DNA) is almost 6 % richer in G + C than the unfractionated DNA.
Hybridization o//ractionated DNA with rRNA In B. subtilis the G + C content of DNA is 43 % (ref. 6), while that of r R N A is about 53 % (ref. 2I). This difference gave support to the hope that the most heatresistant fractions of DNA could contain a proportion of rRNA sequences greater than original DNA. To test this possibility, z2P-labeled r R N A was annealed with (a) original unfractionated DNA; (b) DNA heated at 73 ° and fractionated via nitrocellulose column. (About 40 % of input DNA was eluted from the column in the native fractions.); (c) DNA heated at 76.5 ° and isolated as above. (In this case, 97 % of DNA was withheld b y the column, only the 3 % being recovered in the native fractions.) Biochim. Biophys. Acta, 195 (1969) 466-472
470
V. SGARAMELLA
c~3000 ~. 2500
,~
.ooo 1
;2,~-~ed ~ - -
2 3 4 5 ~321~1 r R N A (/~g) _
J
ilI i ~0
37..
1 2 3 4 5 ~32pj r R N h ( p g )
Fig. 2. A b u n d a n c e of r D N A in t h e r m a l l y f r a c t i o n a t e d D N A . I n c r e a s i n g a m o u n t s of 32P-labeled r R N A , specific a c t i v i t y i 5 o ooo c o u n t s / m i n p e r / ~ g , were a n n e a l e d w i t h 4.o/zg of u n f f a c t i o n a t e d ESH]DNA, specific a c t i v i t y 14 7oo c o u n t s / m i n p e r # g , w i t h 1.6/zg of t h e f r a c t i o n o b t a i n e d a f t e r h e a t i n g a t 73 °, as d e s c r i b e d in MATERIALS AND METHODS, a n d w i t h o. 13/~g of t h e fraction o b t a i n e d a f t e r h e a t i n g a t 76.5 °. P a r t A g i v e s t h e t o t a l a m o u n t s of r a d i o a c t i v i t y s a t u r a t i n g t h e u n f r a c t i o n a t e d D N A ([]-Vq), t h e 4o % f r a c t i o n s t a b l e a t 73 ° ( O - O ) a n d t h e 3 %, fraction stable a t 76.5 ° ( / x - A ) . T h e filled m a r k s a t t h e b o t t o m are t h e r e s p e c t i v e c o n t r o l s for aspecific t r a p p i n g of s~p_ l a b e l e d r R N A b y t h e nitrocellulose m e m b r a n e ; t h e y r e p r e s e n t t h e r a d i o a c t i v i t y left on t h e m e m b r a n e a f t e r [SH]DNA a n d 32P-labeled r R N A , i n c u b a t e d u n d e r a n n e a l i n g c o n d i t i o n s b u t in s e p a r a t e t u b e s , h a v e b e e n m i x e d in t h e p r e s e n c e of r i b o n u c l e a s e ts, d i g e s t e d a n d finally filtered, as in MATERIALS AND METHODS. I n P a r t ]3 are s h o w n t h e relative a m o u n t s of r D N A c o n t a i n e d in t h e t h r e e D N A ' s of P a r t A, h e r e i n d i c a t e d w i t h t h e s a m e s y m b o l s .
Before hybridization, the isolated native material had to be completely denatured, as in MATERIALS AND METHODS. Fig. 2 reports the results of experiments in which these DNA preparations were saturated with rRNA. The absolute quantity of 8zP-labeled r R N A hybridized with the two fractions of DNA is lower than with original DNA, but the 4 ° % fraction of DNA undenatured after exposure to 73 ° seems to contain more than 80 % of the total sequences hybridizable with rRNA, while the 3 % of DNA that is undenatured after treatment at 76.5 ° contains around 25 % of those sequences. In summary, 80 % of the DNA sequences hybridizable with rRNA seem to be present in the fraction left native at 73.0 °, and about 25 % of them are present in the fraction native at 76.5 °. The missing ribosomal DNA (rDNA) sequences have been trapped by the column, probably because they are contained in prevalently denatured molecules. Indeed, it has been found that without any fractionation and further heating, these partially denatured mixtures could, as such, form hybrids with rRNA to an extent very close to their content in denatured rDNA sequences.
DISCUSSION
The main requirements of any DNA fractionation technique are (a) possibility to process large amounts, (b) mildness of the treatments involved, (c) simplicity and rapidity, (d) reproducibility. The fractional denaturation and nitrocellulose adsorpJBiochim. Biophys. Acta, 195 (1969) 466-472
ENRICHMENT OF D N A
IN SEQUENCES FOR r R N A
471
tion, as described in this paper, reasonably fulfill all these requirements. It is likely that controlled fragmentation of DNA may improve the resolution power of this technique. The fact that original DNA is withheld in a variable proportion TM probably means that it is strictly required that the DNA is in double-stranded structure in order to pass freely through nitrocellulose. This would seem to indicate that ordinary bacterial DNA preparations present partially denatured regions. The fact that nitrocellulose chromatography discriminates according to the secondary structure is proved by the observation that the released material, when rechromatographed, can be recovered quantitatively. The strict requirements of integrity in the secondary structure may explain also the shifting of the adsorption curve towards the lower temperatures with respect to the hyperchromicity one (Fig. I); those molecules which underwent only partial denaturation when the solutions were heated at the various temperatures, contributed to the hyperchromic effect only with their denatured fractions. When the solutions are chilled to stop the denaturation process, they are probably "frozen" in their partially denatured structure. Since chromatography is performed immediately afterwards and takes only a few minutes, they may be unable to completely renature and are therefore retained in the columns together with totally denatured molecules, while fully double-stranded molecules are completely released. This might help to explain why only 25 9/0 of rDNA sequences were found in the fraction of DNA (3 ~/o) undenatured at 76.5 °, since it is known that the various rDNA cistrons are intermingled with other sequences of different base composition4,~2, 24. Nevertheless, such a fraction contains over 7 times more rDNA per #g of DNA than does total DNA. Probably, more interesting and useful results can be obtained using, instead of rRNA, purified specific tRNA's to anneal with DNA fractionated according to this technique, assuming that the sequences from which they are transcribed are considerably richer in G + C than the total DNA, as it appears to be at least in the E. coli tRNA's sequenced so far 23.
ACKNOWLEDGMENTS
The author was supported by a fellowship of Euratom, Brussels. Thanks are given to Prof. A. Falaschi for invaluable suggestions and criticisms and to Prof. L. L. Cavalli-Sforza and O. Ciferri. Dr. U. Bertazzoni, Euratom, Ispra, Italy pointed out the usefulness of nitrocellulose and Mr. G. Mazza, undergraduate student of the Istituto di Genetica dell'UniversitA di Pavia, Italy, helped in many experiments. The work was supported by financial help from the Consigiio Nazionale delle Ricerche, Rome.
REFERENCES I 2 3 4 5 6 7 8
N. SUEOKA, J. Mol. Biol., 3 (1961) 31. A. SKALKA, E. BURGI AND A. D. HERSHEY, J. Mol. Biol., 34 (I968) I. P- F. DAVlSON, Science, 152 (1966) 509 . D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 661. R. ROLFE AND H. EPHRUSSI-TAYLOR, Proc. Natl. Acad. Sci. U.S., 47 (1961) 145°. A. T. GANESAN AND J. LEDERBERG, J. Mol. Biol., 9 (1964) 683. J. MARMUR AND P. DOTY, J. Mol. Biol., 5 (1962) lO9. M. ROGER AND R. D. HOTCHKISS, Proc. Natl. Acad, Sci. U.S., 47 (1961) 653.
Biochim. Biophys. Acta, 195 (1969) 466-472
472 9 IO II 12 13 14 15 16 17 I8 19 20 21 22 23 24
V. SGARAMELLA
G. BERNARDI, Biochem. J., 83 (1962) 32P. ]~. T. BOLTON AND B. J. McCARTHY, J. Mol. Biol., 8 (1964) 2Ol. Y. ]V~IYAZAWAAND C. A. THOMAS, JR., J . Mol. Biol., I I (1965) 223. M. OISHI, Proc. Natl. Acad. Sci. U.S., 6o (1968) 329 . A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 12 (1963) 98. D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. O. KLAMERTH, Nature, 208 (1965) 1318. J. A. BOEZI AND R. L. ARMSTRONG, in L. GROSSMANN AND K. ~V[OLDAVE,Methods in Enzymology, Voh X I I A , Academic Press, N e w York, 1967, p. 684. A. J. MAZAITIS AND E. K. F. BAUTZ, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1633. V. SGARAMELLA AND M. POLSlNELLI, Bioehim. Biophys. Acta, 149 (1967) 496. F. W. STUDIER, J. Mol. Biol., i i (1965) 373. W. C. SCHNEIDER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, N e w York, 1957, P. 680. J. E. M. MIDGLEY, Biochim. Biophys. Acta, 61 (1962) 513 . M. BIRNSTIEL, J. SPEIRS, I. PURDOM, K. JONES AND U. E. LOENING, Nature, 219 (1968) 454. Atlas ot Protein Sequence and Structure, 4, Natl. Biomed. Res. F o u n d a t i o n , Silver Spring, Md., 1969, pp. D-229 and D-23o. D. D. BROWN AND C. S. WEBER, J. Mol. Biol., 34 (1968) 681.
Biochim. Biophys. dcta, 195 {I969) 466 472
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96361
E N R I C H M E N T OF B A C I L L U S
SUBTILIS
DNA IN T H E SEQUENCES F O R
RIBOSOMAL RNA V. SGARAMELLA*
Istituto di Genetica deU'Universita, 27100 Pavia (Italy) (Received July i4th, 1969)
SUMMARY
Bacillus subtilis DNA was heated at constant temperatures within the helixcoil transition interval. Slight increases in the temperature caused the denaturation of greater fractions of DNA. The native molecules were separated from the heatsensitive ones b y means of nitrocellulose chromatography. The fractions thus obtained were characterized for their base composition and ability to hybridize with ribosomal RNA (rRNA). As expected, heat resistance is inversely correlated with thymidine frequency and directly correlated with the content in sequences complementary to rRNA (rDNA).
INTRODUCTION
Most DNA preparations are composed of polynucleotide chains derived from mechanical and chemical fragmentation of the original molecules. These fragments m a y be heterogeneous in their base composition 1. Attempts to separate them make use of the resulting differences in their physical properties. Variations in the buoyant density have allowed the separation of fragments of ~ DNA ~, the enrichment for ribosomal RNA (rRNA) cistrons in completely denatured DNA from Escherichia coli a, and in native DNA from Xenopus laevis *,~A, as well as for specific genetic markers in pneumococcal DNA 5 and in Bacillus subtilis 6. The thermal resistance of DNA is known to depend linearly on its G + C contentT; controlled heating m a y lead to the melting of some molecules and not of others s. Various techniques have been used to separate the native from the denatured fraction: to mention some, density gradient centrifugation 6 and chromatography on various materials 9-12. One of the most versatile among them is nitrocellulose, which specifically interacts with denatured but not with native DNA is. This material has been used to prepare membranes 14 and to fill chromatographic columns, in which single-stranded DNA is selectively adsorbed 15-17. In this work the molecules of Bacillus subtilis DNA, which have not been denatured after fractional melting at given temperatures, have been separated from the Abbreviations: rRNA, ribosomal RNA; rDNA, the sequences of DNA complementary to rRNA; Tin, melting temperature. * Present address: Institute for Enzyme Research, University of Wisconsin, Madison, Wisc., U.S.A.
Biochim. Biophys. Acta, 195 (1969) 466-472
ENRICHMENT OF D N A IN SEQUENCES FOR r R N A
467
denatured ones. While the former are not retained by nitrocellulose columns, the latter are tightly adsorbed so that completely different conditions are required to elute them. In this way it has been possible to obtain fractions of DNA with increasing thermal stability. These fractions exhibit a thymidine content lower than unfractionated DNA and inversely correlated with their thermal resistance. The heatresistant fractions are considerably enriched in sequences complementary to rRNA.
MATERIALS AND METHODS
[3H]DNA preparation Cells of the thymine-requiring B. subtilis strain SB 556/1 were grown in a medium containing, (in g/l): glucose, 5.0; Casamino acids, 0.4; asparagine, I.O; tryptophan, o.oi; (NH4)2SO 4, I.O; K H , P 0 4 , 7.0; K2HP04, 2.0; MgSO 4, o.I; sodium citrate, o.i; unlabeled thymidine, 0.00175. The medium contained also 2.5 mC of [Me-3H] thymidine, 14 C/mmole, from Schwarz BioResearch (Orangeburg, N.Y.). After growth at 37 ° under alternative shaking, at the beginning of the stationary phase, the cells were centrifuged and washed. DNA was extracted following a previously described procedure and showed an s°20,~ of 35 S which, according to STUDIER TM, corresponds to an average molecular weight of 5" lOT-
32p-labeled rRNA preparation As previously described TM.
DNA denaturation For partial denaturations, 2-3-ml samples of DNA solutions at approx. 50/*g/ ml in 7.5 mM NaCl-o.75 mM sodium citrate (pH 7.8) were immersed for 15 min in a water bath at a temperature selected within the thermal transition interval. Temperature regulations to o.I ° were obtained with a Colora (Lorch, Germany) water bath. For complete denaturations, DNA was heated to IOO° for 15 min. Each sample, exposed to a given temperature, was then chilled b y transferring the tube into an ice-water mixture.
Nitrocellulose chromatography Partially or totally denatured DNA solutions were adjusted to 0.3 M NaC10.03 M sodium citrate with appropriate additions of 3 M NaCl-o.3 M sodium citrate, They were then loaded on small columns (3.0 cm ×0.6 cm) filled with Nitrocel-S (Serva, Heidelberg) according to KLAMERTH15. Before loading, the columns were washed until no ultraviolet-absorbing material was eluted. After the sample had been adsorbed, the column was washed with three bed volumes of 0.3 M NaCl-o.o3 M sodium citrate. Native DNA was immediately released; its double-stranded structure was assessed with thermal hyperchromicity measurements. On the contrary, denatured molecules were tightly bound so that both low ionic strength (o.oi M NaC1) and alkaline conditions (pH up to 12) were necessary to elute them. Original DNA preparations, not submitted to any thermal treatment, were recovered from the columns in variable amounts, ranging between 60 and 9 ° ~/o, in different DNA preparations. If rechromatographed, the relessed material could be recovered almost quantitatively. Since the same batches exhibited similar retention, this effect seems ascribable to partially denatured molecules present in current DNA preparations le. Undenatured molecules were recovered by collecting 4-5 fractions of I.O ml. Biochim. Biophys. Acta, 195 (1969) 466-472
468
V. SGARAMELLA
Determination o] thymidine content o/ /ractionated DNA The specific radioactivity (as counts/rain per fg) in the fractions resistant to progressively higher temperatures was compared to that of the original, unfractionated DNA. The variation of this ratio was assumed to reflect a change in base composition. For radioactivity measurements, proportional aliquots of the DNA solutions, brought to the same final volume, were pipetted into discs of filter paper; these were counted in a liquid scintillation spectrometer using a toluene-2,5-diphenyloxazole-I,4-bis-(5-phenyloxazolyl-2)benzene system. DNA concentrations were determined by the diphenylamine test 2°.
Hybridization experiments Hybridizations were performed in solution, according to •YGAARD AND HALL13. Before annealing, native DNA and heat-resistant fractions were completely denatured by exposure to IOO° for 15 rain. Hybridized 32P-labeled rRNA was measured as previously described is.
RESULTS
Fractional denaturation and nitrocellulose adsorption First of all, it was considered necessary to study how the fraction of DNA adsorbed to nitrocellulose varied with increasing temperatures of preliminary heating, and the relationship between this variation and the process of thermal melting as followed by absorbance.
100I
~.,...-..~
1.30
.1.20
1.10
~o
%o 1.00 60 °
6 5 '-
70 °
75°
80 °
85 o
HEATING TEMPERATURE Fig. I. Influence of the t e m p e r a t u r e on a b s o r b a n c c at 260 m # and nitrocellulose adsorption. A s t o p p e r e d cuvette w i t h a i o - m m light p a t h was filled w i t h [SH]DNA (specific activity, 14 7o0 c o u n t s / m i n p e r fig) in 7.5 mM NaCl-o.75 mM s o d i u m citrate. A b s o r b a n c e at 26o m # was m e a s u r e d a g a i n s t the solvent in a B e c k m a n D U G-24oo S p e c t r o p h o t o m e t e r e q u i p p e d with h e a t i n g c h a m b e r . T e m p e r a t u r e was increased at a rate of o.3°/min and m e a s u r e d w i t h an i r o n - c o n s t a n t a n t h e r m o couple dipping in a s t o p p e r e d c u v e t t e filled w i t h vaseline oil. H y p e r c h r o m i c i t y is given as the ratio of the absorbance at the various t e m p e r a t u r e s on t h e a b s o r b a n c e at 25 °, w i t h o u t a n y volume correction. Nitrocellulose a d s o r p t i o n was m e a s u r e d in this way: 3-ml solutions of [SH]DNA, at 5 ° / z g / m l in 7.5 mM NaCl-o.75 mM s o d i u m citrate, were h e a t e d at the indicated t e m p e r a t u r e s for 15 rain and i m m e d i a t e l y a f t e r w a r d s were chilled in i c e - w a t e r m i x t u r e ; the solutions were t h e n b r o u g h t to 0. 3 M NaCl-o.o 3 M s o d i u m citrate w i t h the additions of 3 M NaCl-o. 3 M s o d i u m citrate a n d loaded on the nitrocellulose columns. Several I . o - m l fractions were collected until no more r a d i o a c t i v i t y was eluted. The difference b e t w e e n i n p u t and r e c o v e r y gave the adsorption of nitrocellulose columns.
Biochim. Biophys. Acta, 195 (1969) 466-472
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Fig. I shows that the amount of DNA adsorbed to nitrocellulose increases sharply following exposure to temperatures above 7 °° . The midpoint of the adsorption curve is near 73 °. Its sigmoid shape parallels the hyperchromicity curve, which is displaced towards the higher temperatures b y about 5 ° . Nitrocellulose adsorption can therefore be considered as an additional property of DNA affected b y its helix-coil transition. From a preparative point of view, it permits a simple separation of native from partially denatured molecules. The displacement between the two curves is discussed below.
Thymidine content o//ractionated DNA A set of DNA fractions exhibiting increasing thermal stability b y their chromatographic behavior on nitrocellulose was prepared and its specific radioactivity determined as described in MATERIALS AND METHODS. Table I gives the results of TABLE
I
CORRELATION BETWEEN THERMAL STABILITY AND THYMIDINE CONTENT OF FRACTIONATED E3H]DNA F o r f r a c t i o n a l d e n a t u r a t i o n s a n d e v a l u a t i o n of D N A h e a t - r e s i s t a n t f r a c t i o n s see MATERIALS AND METHODS. The a p p a r e n t G + C c o n t e n t g i v e n b e t w e e n b r a c k e t s in t h e f i r s t r o w of t h e l a s t c o l u m n is t h e one o b t a i n e d f r o m t h e l i t e r a t u r e 5 a n d is t h e v a l u e to w h i c h t h e v a r i a t i o n s i n s pe c i fi c act i v i t y h a v e b e e n r e f e r r e d in o r d e r to c a l c u l a t e t h e a p p a r e n t G + C c o n t e n t of t h e h e a t - r e s i s t a n t fractions.
Heat-resistant [raction (%)
Specific activity (3Hcounts/rnin per I~g)
Decrease in [SH]thymidine content
Apparent G+ C content
Untreated 50 20
850 837 76o
-1.5 lO.6
(43) 43.8 48.9
(%)
(%)
such measurements. The correlation between beat resistance and specific activity seems consistent with the hypothesis of a decreasing thymidine content in the more thermally stable fractions. The inferred estimate of the G + C content of the various fractions (last column) based on the consideration that the DNA is all double-stranded, demonstrates that the most heat-resistant fraction (representing 20 % of the original untreated DNA) is almost 6 % richer in G + C than the unfractionated DNA.
Hybridization o//ractionated DNA with rRNA In B. subtilis the G + C content of DNA is 43 % (ref. 6), while that of r R N A is about 53 % (ref. 2I). This difference gave support to the hope that the most heatresistant fractions of DNA could contain a proportion of rRNA sequences greater than original DNA. To test this possibility, z2P-labeled r R N A was annealed with (a) original unfractionated DNA; (b) DNA heated at 73 ° and fractionated via nitrocellulose column. (About 40 % of input DNA was eluted from the column in the native fractions.); (c) DNA heated at 76.5 ° and isolated as above. (In this case, 97 % of DNA was withheld b y the column, only the 3 % being recovered in the native fractions.) Biochim. Biophys. Acta, 195 (1969) 466-472
470
V. SGARAMELLA
c~3000 ~. 2500
,~
.ooo 1
;2,~-~ed ~ - -
2 3 4 5 ~321~1 r R N A (/~g) _
J
ilI i ~0
37..
1 2 3 4 5 ~32pj r R N h ( p g )
Fig. 2. A b u n d a n c e of r D N A in t h e r m a l l y f r a c t i o n a t e d D N A . I n c r e a s i n g a m o u n t s of 32P-labeled r R N A , specific a c t i v i t y i 5 o ooo c o u n t s / m i n p e r / ~ g , were a n n e a l e d w i t h 4.o/zg of u n f f a c t i o n a t e d ESH]DNA, specific a c t i v i t y 14 7oo c o u n t s / m i n p e r # g , w i t h 1.6/zg of t h e f r a c t i o n o b t a i n e d a f t e r h e a t i n g a t 73 °, as d e s c r i b e d in MATERIALS AND METHODS, a n d w i t h o. 13/~g of t h e fraction o b t a i n e d a f t e r h e a t i n g a t 76.5 °. P a r t A g i v e s t h e t o t a l a m o u n t s of r a d i o a c t i v i t y s a t u r a t i n g t h e u n f r a c t i o n a t e d D N A ([]-Vq), t h e 4o % f r a c t i o n s t a b l e a t 73 ° ( O - O ) a n d t h e 3 %, fraction stable a t 76.5 ° ( / x - A ) . T h e filled m a r k s a t t h e b o t t o m are t h e r e s p e c t i v e c o n t r o l s for aspecific t r a p p i n g of s~p_ l a b e l e d r R N A b y t h e nitrocellulose m e m b r a n e ; t h e y r e p r e s e n t t h e r a d i o a c t i v i t y left on t h e m e m b r a n e a f t e r [SH]DNA a n d 32P-labeled r R N A , i n c u b a t e d u n d e r a n n e a l i n g c o n d i t i o n s b u t in s e p a r a t e t u b e s , h a v e b e e n m i x e d in t h e p r e s e n c e of r i b o n u c l e a s e ts, d i g e s t e d a n d finally filtered, as in MATERIALS AND METHODS. I n P a r t ]3 are s h o w n t h e relative a m o u n t s of r D N A c o n t a i n e d in t h e t h r e e D N A ' s of P a r t A, h e r e i n d i c a t e d w i t h t h e s a m e s y m b o l s .
Before hybridization, the isolated native material had to be completely denatured, as in MATERIALS AND METHODS. Fig. 2 reports the results of experiments in which these DNA preparations were saturated with rRNA. The absolute quantity of 8zP-labeled r R N A hybridized with the two fractions of DNA is lower than with original DNA, but the 4 ° % fraction of DNA undenatured after exposure to 73 ° seems to contain more than 80 % of the total sequences hybridizable with rRNA, while the 3 % of DNA that is undenatured after treatment at 76.5 ° contains around 25 % of those sequences. In summary, 80 % of the DNA sequences hybridizable with rRNA seem to be present in the fraction left native at 73.0 °, and about 25 % of them are present in the fraction native at 76.5 °. The missing ribosomal DNA (rDNA) sequences have been trapped by the column, probably because they are contained in prevalently denatured molecules. Indeed, it has been found that without any fractionation and further heating, these partially denatured mixtures could, as such, form hybrids with rRNA to an extent very close to their content in denatured rDNA sequences.
DISCUSSION
The main requirements of any DNA fractionation technique are (a) possibility to process large amounts, (b) mildness of the treatments involved, (c) simplicity and rapidity, (d) reproducibility. The fractional denaturation and nitrocellulose adsorpJBiochim. Biophys. Acta, 195 (1969) 466-472
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tion, as described in this paper, reasonably fulfill all these requirements. It is likely that controlled fragmentation of DNA may improve the resolution power of this technique. The fact that original DNA is withheld in a variable proportion TM probably means that it is strictly required that the DNA is in double-stranded structure in order to pass freely through nitrocellulose. This would seem to indicate that ordinary bacterial DNA preparations present partially denatured regions. The fact that nitrocellulose chromatography discriminates according to the secondary structure is proved by the observation that the released material, when rechromatographed, can be recovered quantitatively. The strict requirements of integrity in the secondary structure may explain also the shifting of the adsorption curve towards the lower temperatures with respect to the hyperchromicity one (Fig. I); those molecules which underwent only partial denaturation when the solutions were heated at the various temperatures, contributed to the hyperchromic effect only with their denatured fractions. When the solutions are chilled to stop the denaturation process, they are probably "frozen" in their partially denatured structure. Since chromatography is performed immediately afterwards and takes only a few minutes, they may be unable to completely renature and are therefore retained in the columns together with totally denatured molecules, while fully double-stranded molecules are completely released. This might help to explain why only 25 9/0 of rDNA sequences were found in the fraction of DNA (3 ~/o) undenatured at 76.5 °, since it is known that the various rDNA cistrons are intermingled with other sequences of different base composition4,~2, 24. Nevertheless, such a fraction contains over 7 times more rDNA per #g of DNA than does total DNA. Probably, more interesting and useful results can be obtained using, instead of rRNA, purified specific tRNA's to anneal with DNA fractionated according to this technique, assuming that the sequences from which they are transcribed are considerably richer in G + C than the total DNA, as it appears to be at least in the E. coli tRNA's sequenced so far 23.
ACKNOWLEDGMENTS
The author was supported by a fellowship of Euratom, Brussels. Thanks are given to Prof. A. Falaschi for invaluable suggestions and criticisms and to Prof. L. L. Cavalli-Sforza and O. Ciferri. Dr. U. Bertazzoni, Euratom, Ispra, Italy pointed out the usefulness of nitrocellulose and Mr. G. Mazza, undergraduate student of the Istituto di Genetica dell'UniversitA di Pavia, Italy, helped in many experiments. The work was supported by financial help from the Consigiio Nazionale delle Ricerche, Rome.
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