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Template activity of DNA is restricted in chromatin
J. Mol. Biol. 16,242-244
LETTERS TO THE EDITOR
Template Activity of DNA is Restricted in Chromatin Many authors have assumed that the template activity of DNA in chromatin is restricted because the rate of transcription from chromatin is less than that from DNA in a cell-free incubation mixture. But this rate may be affected by many factors, especially the solubility of the components of the reaction. This was emphasized by Sonnenberg & Zubay (1965), who demonstrated that sonication increased the solubility of chromatin and at the same time enhanced its capacity to act as a template for RNA synthesis. In their work it was assumed that the effect of sonication was only to cause breaks in the chromatin, whereas it might be argued that it could also strip some of the protein from the underlying DNA, especially at points of breakage. The only certain way to obtain an answer to both questions is to determine whether RNA formed on a chromatin template is transcribed from all the DNA sequences present or from relatively few. This can be done by hybridizing RNA formed from chromatin with DNA derived from the same material. This letter reports such an experiment, which gives the unequivocal answer that the template activity of DNA in chromatin is restricted. Sonication permits transcription from more of the DNA. Chromatin was prepared from calf thymus. Nuclei were first isolated by homogenizing the chopped tissue in 10 volumes of 0·025 M-citric acid and repeatedly washing with citric acid until a clean preparation was obtained. The nuclei were then suspended in 0·2 M-phosphate buffer at pH 7. Mter centrifugation, the sediment was washed in 0·001 M-EDTA. The gel which formed was centrifuged at 10,000 g for 15 minutes. Finally, the resulting pellet was dissolved in distilled water and constituted chromatin. This was prepared fresh before each experiment. Crude DNA was first made by the method of Kay, Simmonds & Dounce (1952) and was then purified by digestion with DNase-free RNase and DNase-free pronase followed by repeated deproteinization with chloroform until no interphase formed. It was precipitated by adding two volumes of ethanol, redissolved in 1/100 SSC (SSC being 0·15 M-NaCl and 0·015 M-sodium citrate) and then reprecipitated by addition of isopropanol (Marmur, 1961). Sonication of DNA and chromatin was carried out at O°C for 90 seconds, using an M.S.E. ultrasonic disintegrator at 1·3 A. 10 ml. of either chromatin or DNA, containing 50 fLg DNA/ml. in 1/100 SSC, were treated at one time. RNA polymerase was prepared from M. lysodeikticus by the method of Nakamoto, Fox & Weiss (1964). Both this preparation and the chromatin were tested for ribonuclease activity by incubating with [14C]RNA for one hour at 30°C; this was not detected. For the preparation of RNA, incubation mixtures were set up containing the following to a total volume of 3 ml.: 150 fLmoles tris (pH 7·8); 7·5 fLmoles MnCI2 ; 60 Weiss units RNA polymerase; 2-4 fLmoles ATP; 2·4 fLmoles CTP; 2·4 fLmoles GTP; 2·4 fLmoles [3H]UTP (4 X 106 disintegrationsjminjumole UTP); 50 fLg DNA as whole DNA, sonicated DNA, whole chromatin or sonicated chromatin. 242
LETTERS TO THE EDITOR
243
The reaction mixtures were incubated at 30°C. Mter one hour, the DNA reactions were terminated by freezing; after six hours, the chromatin reactions were similarly terminated. RNA was extracted with phenol at room temperature. After the first extraction, DNase (10 fLgfml.) was added for 10 minutes and two further extractions were then performed. Mter removing the last traces of phenol (by extracting with ether and then removing it with nitrogen) the RNA was taken up in buffer and dialysed against 0·05 M-tris (pH 7,2), 0·001 M-MgCI 2 and 0·03% bentonite for 18 hours at O°C. In each case the amount of RNA obtained was about four times as great as the amount of DNA present in the mixture. Hybridization was carried out according to the method of Gillespie & Spiegelman (1965). DNA, denatured by treatment with alkali, was bound to nitrocellulose filters as described by these authors, using Ip,g DNA per filter. Varying amounts of each of the RNA preparations were used to permit the prediction of saturation values. The regressions obtained with the whole and sonicated DNA preparations were identical and the results were therefore combined to calculate the regression (y = 0·688 0'031x) in the double reciprocal plot (Fig. 1). It can be calculated that about
+
...
X.
10
o
-:
• •
10
o2 x 1'9 1RNA
1
FIG. 1. Kinetics of hybridization to thymus DNA of [3H]RNA prepared from thymus DNA and chromatin with M.lysodeikticUB RNA polymerase: double reciprocal plot of RNA concentration in annealing mixture and RNA hybridizing with DNA (in disintegrationsjrnin). -X-X-, Unsonicated DNA; - 0 - 0 - , sonicated DNA; -e-e-, whole chromatin; -L-L-, sonicated chromatin.
0·4 to 0·5 p,g of RNA would be bound to 1 p,g of DNA at complete saturation. On the other hand, RNA transcribed from whole chromatin reached saturation at a level which corresponded to about 10% of the saturation level for RNA transcribed from DNA (i.e. at about 5% of the whole DNA). RNA prepared from sonicated chromatin saturated about twice as much of the DNA. Hence the amount of DNA available as a template for transcription by RNA polymerase in our chromatin preparation corresponds to no more than about 5 to 10% of the total DNA. Because of the criteria used, one can conclude that the same 90to 95% of each set of DNA molecules is "masked". This may imply that regulation of the transcription of some genes in animal cells is not readily reversible and may, therefore,
244
J. PAUL AND R. S. GILMOUR
involve mechanisms other than those postulated for the control of transcription in Escherichia coli. It was also observed that sonication increased the amount of DNA available for transcription. Because of the criteria used, it again seems likely that specific sections of DNA were thereby made available for transcription and that the effect was not due to random stripping of protein from some portions of DNA. This research was supported by Grant CA-05855 from the National Cancer Institute, U.S. Public Health Services. Department of Biochemistry University of Glasgow Glasgow, Scotland
JOHN PAUL
R.
STEWART GILMOUR
Received 10 December, 1965 REFERENCES Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Kay, E. R. M., Simmonds, N. S. & Dounce, A. L. (1952). J. Amer. Chem, Soc. 74, 1724. Marmur, J. (1961). J. Mol. Biol. 3, 208. Nakamoto, T., Fox, F. C. & Weiss, S. B. (1964). J. Biol. Chem, 239, 167. Sonnenberg, B. P. & Zubay, G. (1965). Proc, Nat. Acad. Sci., Wash. 54, 415.
LETTERS TO THE EDITOR
Template Activity of DNA is Restricted in Chromatin Many authors have assumed that the template activity of DNA in chromatin is restricted because the rate of transcription from chromatin is less than that from DNA in a cell-free incubation mixture. But this rate may be affected by many factors, especially the solubility of the components of the reaction. This was emphasized by Sonnenberg & Zubay (1965), who demonstrated that sonication increased the solubility of chromatin and at the same time enhanced its capacity to act as a template for RNA synthesis. In their work it was assumed that the effect of sonication was only to cause breaks in the chromatin, whereas it might be argued that it could also strip some of the protein from the underlying DNA, especially at points of breakage. The only certain way to obtain an answer to both questions is to determine whether RNA formed on a chromatin template is transcribed from all the DNA sequences present or from relatively few. This can be done by hybridizing RNA formed from chromatin with DNA derived from the same material. This letter reports such an experiment, which gives the unequivocal answer that the template activity of DNA in chromatin is restricted. Sonication permits transcription from more of the DNA. Chromatin was prepared from calf thymus. Nuclei were first isolated by homogenizing the chopped tissue in 10 volumes of 0·025 M-citric acid and repeatedly washing with citric acid until a clean preparation was obtained. The nuclei were then suspended in 0·2 M-phosphate buffer at pH 7. Mter centrifugation, the sediment was washed in 0·001 M-EDTA. The gel which formed was centrifuged at 10,000 g for 15 minutes. Finally, the resulting pellet was dissolved in distilled water and constituted chromatin. This was prepared fresh before each experiment. Crude DNA was first made by the method of Kay, Simmonds & Dounce (1952) and was then purified by digestion with DNase-free RNase and DNase-free pronase followed by repeated deproteinization with chloroform until no interphase formed. It was precipitated by adding two volumes of ethanol, redissolved in 1/100 SSC (SSC being 0·15 M-NaCl and 0·015 M-sodium citrate) and then reprecipitated by addition of isopropanol (Marmur, 1961). Sonication of DNA and chromatin was carried out at O°C for 90 seconds, using an M.S.E. ultrasonic disintegrator at 1·3 A. 10 ml. of either chromatin or DNA, containing 50 fLg DNA/ml. in 1/100 SSC, were treated at one time. RNA polymerase was prepared from M. lysodeikticus by the method of Nakamoto, Fox & Weiss (1964). Both this preparation and the chromatin were tested for ribonuclease activity by incubating with [14C]RNA for one hour at 30°C; this was not detected. For the preparation of RNA, incubation mixtures were set up containing the following to a total volume of 3 ml.: 150 fLmoles tris (pH 7·8); 7·5 fLmoles MnCI2 ; 60 Weiss units RNA polymerase; 2-4 fLmoles ATP; 2·4 fLmoles CTP; 2·4 fLmoles GTP; 2·4 fLmoles [3H]UTP (4 X 106 disintegrationsjminjumole UTP); 50 fLg DNA as whole DNA, sonicated DNA, whole chromatin or sonicated chromatin. 242
LETTERS TO THE EDITOR
243
The reaction mixtures were incubated at 30°C. Mter one hour, the DNA reactions were terminated by freezing; after six hours, the chromatin reactions were similarly terminated. RNA was extracted with phenol at room temperature. After the first extraction, DNase (10 fLgfml.) was added for 10 minutes and two further extractions were then performed. Mter removing the last traces of phenol (by extracting with ether and then removing it with nitrogen) the RNA was taken up in buffer and dialysed against 0·05 M-tris (pH 7,2), 0·001 M-MgCI 2 and 0·03% bentonite for 18 hours at O°C. In each case the amount of RNA obtained was about four times as great as the amount of DNA present in the mixture. Hybridization was carried out according to the method of Gillespie & Spiegelman (1965). DNA, denatured by treatment with alkali, was bound to nitrocellulose filters as described by these authors, using Ip,g DNA per filter. Varying amounts of each of the RNA preparations were used to permit the prediction of saturation values. The regressions obtained with the whole and sonicated DNA preparations were identical and the results were therefore combined to calculate the regression (y = 0·688 0'031x) in the double reciprocal plot (Fig. 1). It can be calculated that about
+
...
X.
10
o
-:
• •
10
o2 x 1'9 1RNA
1
FIG. 1. Kinetics of hybridization to thymus DNA of [3H]RNA prepared from thymus DNA and chromatin with M.lysodeikticUB RNA polymerase: double reciprocal plot of RNA concentration in annealing mixture and RNA hybridizing with DNA (in disintegrationsjrnin). -X-X-, Unsonicated DNA; - 0 - 0 - , sonicated DNA; -e-e-, whole chromatin; -L-L-, sonicated chromatin.
0·4 to 0·5 p,g of RNA would be bound to 1 p,g of DNA at complete saturation. On the other hand, RNA transcribed from whole chromatin reached saturation at a level which corresponded to about 10% of the saturation level for RNA transcribed from DNA (i.e. at about 5% of the whole DNA). RNA prepared from sonicated chromatin saturated about twice as much of the DNA. Hence the amount of DNA available as a template for transcription by RNA polymerase in our chromatin preparation corresponds to no more than about 5 to 10% of the total DNA. Because of the criteria used, one can conclude that the same 90to 95% of each set of DNA molecules is "masked". This may imply that regulation of the transcription of some genes in animal cells is not readily reversible and may, therefore,
244
J. PAUL AND R. S. GILMOUR
involve mechanisms other than those postulated for the control of transcription in Escherichia coli. It was also observed that sonication increased the amount of DNA available for transcription. Because of the criteria used, it again seems likely that specific sections of DNA were thereby made available for transcription and that the effect was not due to random stripping of protein from some portions of DNA. This research was supported by Grant CA-05855 from the National Cancer Institute, U.S. Public Health Services. Department of Biochemistry University of Glasgow Glasgow, Scotland
JOHN PAUL
R.
STEWART GILMOUR
Received 10 December, 1965 REFERENCES Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Kay, E. R. M., Simmonds, N. S. & Dounce, A. L. (1952). J. Amer. Chem, Soc. 74, 1724. Marmur, J. (1961). J. Mol. Biol. 3, 208. Nakamoto, T., Fox, F. C. & Weiss, S. B. (1964). J. Biol. Chem, 239, 167. Sonnenberg, B. P. & Zubay, G. (1965). Proc, Nat. Acad. Sci., Wash. 54, 415.