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[92] Preparation of tritium-labeled DNA from Bacillus subtilis and Escherichia coli
[92]
SH-DNA
693
Hot Acid Extraction o] DNA. Quantitative extraction of DNA by hot acid appears to be difficult, if not impossible, for some tissues. Hutchison et al. 1" concluded that the efficiency of extraction of DNA by hot acid, free of non-DNA material, is a complicated function of acid concentration, of temperature, and of the type of biological material undergoing analysis. As an aside, estimations based on deoxyribose color reactions may give low values because of the destruction of deoxyribose by the more vigorous hot acid extractions. 2 While the above procedures appear to be satisfactory for bacterial studies, they may have to be altered for estimations of DNA radioactivity in some plant and animal tissues. Of course, in cases in which the label is not present in the protein fraction, the hot acid extraction can be omitted, and the radioactive DNA then counted as the acid precipitate. The separation of DNA from RNA by alkaline treatment appears to be quite satisfactory for all types of biological material. 2 Polysaccharides. Cold TCA can be used to extract polysaccharides from gram-positive bacteria, 2° and it is likely that in certain biological material, at least part of the polysaccharide component would be removed in the initial cold acid extractions. Also, in some cells it represents a minor fraction of the dry weight, e.g., less than 3% of the dry weight of E. coll. 1,~° However, in other cells it might be a more significant fraction and a troublesome component to remove. Therefore, if labeled by the precursor, special modifications might be necessary to extract it. 1, W. C. Hutchison, E. D. Downie, and H. N. Munro, Biochim. Biophys. Aeta 55, 561 (1962). M. R. J. Salton, "The Bacterial Cell Wall." Elsevier, Amsterdam, 1964.
[92 ] P r e p a r a t i o n of T r i t i u m - L a b e l e d D N A f r o m B a c i l l u s s u b t i l i s a n d E s c h e r i c h i a coli
By INGA MAHLER The following procedure is applicable to the isolation of bacterial DNA from selected gram positive and gram negative organisms. The use of the labeled nucleoside, thymidine-methyl-SH is recommended since the uptake of thymine is negligible in strains of E. coli and B. subtilis. The uptake of thymidine is also limited since cultures of these organisms have been found to convert the nucleoside rapidly to thymine?, 2 1W. F. Bodmer and C. Schildkraut, Anal. Biochem. 8, 229 (1964). M. Rachmeler, G. Gerhart, and J. Nosher, Biochim. Biophys. Aeta 49, 222 (1961).
694
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[92]
Growth Medium Bacillus subtilis dipotassium phosphate monopotassium phosphate ammonium sulfate sodium citrate-2 H20 magnesium sulfate. 7 H20 distilled water After autoclaving supplement with: 10~ glucose acid casein hydrolyzate (N.B.C.)
14 g 6g 2g lg 0.2 g 1 liter 50 ml 10 ml
Escherichia coli ammonium chloride sodium chloride magnesium sulfate monopotassium phosphate disodium phosphate distilled water After autoclaving, supplement with: 10% glucose
lg 0.5 g 2g 3g 6g 1 liter 50 ml
Boyce and Setlow3 have reported that the addition of certain nucleosides and deoxynucleosides at concentrations of 250 ~g/ml to the growth medium of prototrophic strains of E. coli significantly enhances the uptake of exogenous thymidine. Growth of Cells A 250-ml flask containing 50 ml of growth medium is inoculated and shaken overnight at 37 °. The overnight culture is added as inoculum to 450 ml of growth medium in a 2-1iter flask, and the incubation is continued for approximately 2 hours (cell density 2 X 108). The labeled nucleoside is then added. Thymidine-methyl-SH is supplied in sterile aqueous solution by New England Nuclear Corporation, and can be added directly to the medium. The cells are harvested after at least one doubling of cell concentration has occurred. In strains of E. coli B and C, and in strains of B. subtilis, maximal incorporation of the isotope has been found to take place within 30-60 minutes2 ,4 Uptake of label into whole cells may be assayed by filtering 0.1 ml R. P. Boyce and R. B. Setlow, Biochim. Biophys. Acta 61, 618 (1962). ' W. F. Bodmer and S. Grether, J. Bacteriol. 89, 1011 (1965).
[93]
15N-DEUTERATED DNA
695
of the culture, before cell lysis, through Millipore filters (HA; pore size, 0.45). Incorporation into acid-insoluble material may be determined by extracting 0.1 ml of culture with 0.9 ml of 0.5 N cold perchloric acid followed by filtration through an HA Millipore filter. The amount of 3H which is taken up into acid-insoluble material depends to some extent on growth conditions. Although proportions as high as 25% have been reported, the usual uptake ranges from 10 to 12% of the added 3H. Labeled DNA of high molecular weight may be obtained by the method described by Marmur. 5 ~J. Marmur, ,l. Mol. Biol. 3, 208 (1961).
[93] T h e P r e p a r a t i o n a n d P r o p e r t i e s of 1 5 N - D e u t e r a t e d DNA from Bacteria By C. L, SCHILDKRAUT In studying the interactions of nucleic acids, as well as their genetic fate and function using the technique of cesium chloride density gradient centrifugation,1-3 it may be desirable to prepare DNA as well as RNA of high density by homogeneous labeling. The heavy isotopes lSN, lsC, and 2H have been used successfully for this purpose. 4-6 The combination of 15N and 2H has been found convenient to use for the heavy labeling of DNA from bacteria and bacteriophage2 -8 The incorporation of SH as a radioisotope together with these two heavy isotopes is easily accomplished? Growth Medium Two different media containing ~SN and deuterium are presented here. The constituents (listed in Table I) of the first are relatively easy to obtain. This medium contains 15NH4C1 as the sole source of nitrogen and two nondeuterated carbon sources: glycerol and lactate. The second (listed in Table II) is a richer medium and provides the advantage of a greatly 1M. Meselson, F. W. Stahl, and J. Vinograd, Proc. Natl. Acad. Sci. U~. 43, 581 (1957). *See Volume VI [120]. SSee Vol. XII, Part B [108], in preparation. "M. Meselson and F. W. Stahl, Proc. Natl. Acad. Sci. U$. 44, 671 (1958). uC. I. Davern and M. Meselson, J. Mol. Biol. 2, 153 (1960). ej. Marmur and C. L. Schildkraut, Nature 189, 636 (1961). TH. Crespi, J. Marmur, and J. J. Katz, J. Amer. Chem. Soc. 84, 3489 (1962). s W. Bodmer and C. Schildkraut, Anal. Biochem. 8, 229 (1964).
SH-DNA
693
Hot Acid Extraction o] DNA. Quantitative extraction of DNA by hot acid appears to be difficult, if not impossible, for some tissues. Hutchison et al. 1" concluded that the efficiency of extraction of DNA by hot acid, free of non-DNA material, is a complicated function of acid concentration, of temperature, and of the type of biological material undergoing analysis. As an aside, estimations based on deoxyribose color reactions may give low values because of the destruction of deoxyribose by the more vigorous hot acid extractions. 2 While the above procedures appear to be satisfactory for bacterial studies, they may have to be altered for estimations of DNA radioactivity in some plant and animal tissues. Of course, in cases in which the label is not present in the protein fraction, the hot acid extraction can be omitted, and the radioactive DNA then counted as the acid precipitate. The separation of DNA from RNA by alkaline treatment appears to be quite satisfactory for all types of biological material. 2 Polysaccharides. Cold TCA can be used to extract polysaccharides from gram-positive bacteria, 2° and it is likely that in certain biological material, at least part of the polysaccharide component would be removed in the initial cold acid extractions. Also, in some cells it represents a minor fraction of the dry weight, e.g., less than 3% of the dry weight of E. coll. 1,~° However, in other cells it might be a more significant fraction and a troublesome component to remove. Therefore, if labeled by the precursor, special modifications might be necessary to extract it. 1, W. C. Hutchison, E. D. Downie, and H. N. Munro, Biochim. Biophys. Aeta 55, 561 (1962). M. R. J. Salton, "The Bacterial Cell Wall." Elsevier, Amsterdam, 1964.
[92 ] P r e p a r a t i o n of T r i t i u m - L a b e l e d D N A f r o m B a c i l l u s s u b t i l i s a n d E s c h e r i c h i a coli
By INGA MAHLER The following procedure is applicable to the isolation of bacterial DNA from selected gram positive and gram negative organisms. The use of the labeled nucleoside, thymidine-methyl-SH is recommended since the uptake of thymine is negligible in strains of E. coli and B. subtilis. The uptake of thymidine is also limited since cultures of these organisms have been found to convert the nucleoside rapidly to thymine?, 2 1W. F. Bodmer and C. Schildkraut, Anal. Biochem. 8, 229 (1964). M. Rachmeler, G. Gerhart, and J. Nosher, Biochim. Biophys. Aeta 49, 222 (1961).
694
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[92]
Growth Medium Bacillus subtilis dipotassium phosphate monopotassium phosphate ammonium sulfate sodium citrate-2 H20 magnesium sulfate. 7 H20 distilled water After autoclaving supplement with: 10~ glucose acid casein hydrolyzate (N.B.C.)
14 g 6g 2g lg 0.2 g 1 liter 50 ml 10 ml
Escherichia coli ammonium chloride sodium chloride magnesium sulfate monopotassium phosphate disodium phosphate distilled water After autoclaving, supplement with: 10% glucose
lg 0.5 g 2g 3g 6g 1 liter 50 ml
Boyce and Setlow3 have reported that the addition of certain nucleosides and deoxynucleosides at concentrations of 250 ~g/ml to the growth medium of prototrophic strains of E. coli significantly enhances the uptake of exogenous thymidine. Growth of Cells A 250-ml flask containing 50 ml of growth medium is inoculated and shaken overnight at 37 °. The overnight culture is added as inoculum to 450 ml of growth medium in a 2-1iter flask, and the incubation is continued for approximately 2 hours (cell density 2 X 108). The labeled nucleoside is then added. Thymidine-methyl-SH is supplied in sterile aqueous solution by New England Nuclear Corporation, and can be added directly to the medium. The cells are harvested after at least one doubling of cell concentration has occurred. In strains of E. coli B and C, and in strains of B. subtilis, maximal incorporation of the isotope has been found to take place within 30-60 minutes2 ,4 Uptake of label into whole cells may be assayed by filtering 0.1 ml R. P. Boyce and R. B. Setlow, Biochim. Biophys. Acta 61, 618 (1962). ' W. F. Bodmer and S. Grether, J. Bacteriol. 89, 1011 (1965).
[93]
15N-DEUTERATED DNA
695
of the culture, before cell lysis, through Millipore filters (HA; pore size, 0.45). Incorporation into acid-insoluble material may be determined by extracting 0.1 ml of culture with 0.9 ml of 0.5 N cold perchloric acid followed by filtration through an HA Millipore filter. The amount of 3H which is taken up into acid-insoluble material depends to some extent on growth conditions. Although proportions as high as 25% have been reported, the usual uptake ranges from 10 to 12% of the added 3H. Labeled DNA of high molecular weight may be obtained by the method described by Marmur. 5 ~J. Marmur, ,l. Mol. Biol. 3, 208 (1961).
[93] T h e P r e p a r a t i o n a n d P r o p e r t i e s of 1 5 N - D e u t e r a t e d DNA from Bacteria By C. L, SCHILDKRAUT In studying the interactions of nucleic acids, as well as their genetic fate and function using the technique of cesium chloride density gradient centrifugation,1-3 it may be desirable to prepare DNA as well as RNA of high density by homogeneous labeling. The heavy isotopes lSN, lsC, and 2H have been used successfully for this purpose. 4-6 The combination of 15N and 2H has been found convenient to use for the heavy labeling of DNA from bacteria and bacteriophage2 -8 The incorporation of SH as a radioisotope together with these two heavy isotopes is easily accomplished? Growth Medium Two different media containing ~SN and deuterium are presented here. The constituents (listed in Table I) of the first are relatively easy to obtain. This medium contains 15NH4C1 as the sole source of nitrogen and two nondeuterated carbon sources: glycerol and lactate. The second (listed in Table II) is a richer medium and provides the advantage of a greatly 1M. Meselson, F. W. Stahl, and J. Vinograd, Proc. Natl. Acad. Sci. U~. 43, 581 (1957). *See Volume VI [120]. SSee Vol. XII, Part B [108], in preparation. "M. Meselson and F. W. Stahl, Proc. Natl. Acad. Sci. U$. 44, 671 (1958). uC. I. Davern and M. Meselson, J. Mol. Biol. 2, 153 (1960). ej. Marmur and C. L. Schildkraut, Nature 189, 636 (1961). TH. Crespi, J. Marmur, and J. J. Katz, J. Amer. Chem. Soc. 84, 3489 (1962). s W. Bodmer and C. Schildkraut, Anal. Biochem. 8, 229 (1964).