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[105] Methods for characterization of nucleic acid
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found when DNA solutions are heated. The heat stability of DNA from various origins has been investigated by Zamenhof. 7 Light-scattering experiments have indicated that this decrease in viscosity brought about by heat treatment does not at first cause any decrease in the molecular weight. 8 Thus it appears that heat treatment is analogous to exposure to low pH values. Presumably the hydrogen bonds between the polynucleotide chains are broken by heat as well as by low pH. Conclusion. Although the measurement of the intrinsic viscosity in a manner which successfully eliminates the effects of both concentration and shear is difficult experimentally, its determination provides a sensitive measure of molecular size. Moreover, when such a sensitive measurement is combined with an independent molecular weight determination, the absolute value of the size can be obtained. On the other hand the molecular weight itself can be calculated by combination with the sedimentation constant. Viscosity measurements have proved especially valuable in investigating the effects of pH and heat on the DNA molecule. In both cases the molecular weight was found to remain constant whereas the size of the molecule, as reflected by the viscosity, undergoes substantial contraction. This contraction in size is thought to be the result of the destruction of hydrogen bonds between polynucleotide chains. 7 S. Zamenhof, H. E. Alexander, G. Leidy, J. Exp. Med. 98, 373-397 (1953). 8 Paul Doty and S. A. Rice, Biochim. et Biophys. Acta 16, 446-447 (1955).
[105] M e t h o d s for C h a r a c t e r i z a t i o n of N u c l e i c Acid 1
By ROLLIN D. HOTCHKISS I. Characterization of Nucleic Acids by Spectrophotometry
Principle. Both DNA and RNA absorb ultraviolet light strongly, with maximum absorption about 260 m~, falling off to a minimum about 230 to 240 m~ and almost disappearing at 310 m~. High-molecular DNA and RNA can undergo a denaturation change which results in an increase of ultraviolet absorption in saline solution of about 33 % at 260 m~ (and of similar magnitude along the whole absorption curve). The absorption increment from NaOH treatment has been used in the author's laboratory since 1948 to determine DNA in the presence of RNA (because the latter is usually not high molecular). 1 For colorimetric procedures for anatysi8 of nucleic acid constituents, see Vol. I I I [12, 13, 99].
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After Kunitz reported that pancreatic deoxyribonuclease (DNase) hydrolysis resulted in an increase in absorption, 2 it was quickly discovered that the alkali and enzymatic effects were equivalent and not additive. Enzymatically hydrolyzed DNA does not show further increase in ultraviolet absorption when made alkaline; likewise alkali- (or acid-) denatured DNA, reneutralized, no longer gives the increase on hydrolysis with enzyme. It was also shown at the same time (with M. McCarty) that high-molecular R N A from Pneumococcus would show a similar 30 to 33 % absorption increment when treated with ribonuclease (RNase) or alkali. A number of authors have observed some of these absorption shifts in the years since, particularly with DNA. It seems clear that some have come to look on the extinction coefficient in relation to phosphorus content as some sort of criterion of nativeness in DNA, 3 but the quantitative differences between different preparations and different modes of denaturation discouraged interpretation of the absorption increment of denaturation. Beaven el al. 4 have recently proposed the use of alkali denaturation, without phosphorus analysis, in much the same way as described here in judging the quality of a preparation. As stated, the method has been used by the author with increasing confidence during the past several years. It is important to make as clear as present knowledge permits what features of DNA or R N A structure can be correlated with a 33 % absorption increment. In operational terms, the increment is shown by bacterial DNA of high biological activity, tissue DNA prepared by a variety of gentle means, DNA isolated while still attached to histone 5 (on alkali denaturation), and highly viscous R N A preparations from pneum0coccus. The value of 33 % is believed to be close to the ideal maximum, although a number of DNA and RNA preparations have been described which gave absorption increments less than this on hydrolysis and had been considered native. The author believes such preparations to be nevertheless denatured and has been able to make similar ones by irreversibly damaging intact DNA by drying, by heating, or by suitably drastic shift of pH or ionic strength. His native material, like that of a number of other workers, gives an increment close to 33 %. Few R N A preparations of good 2 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 3 G. Frick, Biochim. et Biophys. Acta 8, 625 (1952); J. Shack, R. J. Jenkins, and J. M. Thompsett, J. Biol. Chem. 203, 373 (1953) ; R. Thomas, Biochim. et Biophys. Acta 14, 231 (1954); E. Chargaff, in "The Nucleic Acids" (Chargaff and Davidson, eds.), Vol. I, p. 336. Academic Press, New York, 1955. 4 G. H. Beaven, E. R. Holiday, and E. A. Johnson, in "The Nucleic Acids" (Chargaff and Davidson, eds.), Vol. I, p. 526. Academic Press, New York, 1955. 6 j. Shack and J. M. Thompsett, J. Biol. Chem. 197, 17 (1952) ; J. Natl. Cancer Inst. 13, 1425 (1953).
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quality have been described (they are often prepared with use of alkali). In particular, it should be noted that air or solvent drying of D N A usually leads to partial denaturation, in this sense. High specific viscosity is not a reliable criterion of integrity of DNA; the temperature dependence of viscosity (see Vol. III [103]) is probably a much better one. The ability to show an absorption increment, then, appears to depend on some not adequately understood feature of the spatial fitting or hydrogen bonding of the purine and pyrimidine bases, easily destroyed by denaturation. It may be that different nucleic acids of widely different base composition will show somewhat discrepant absorption increments. The methods which follow may therefore be somewhat liable to reinterpretation or quantitative correction as more is learned about the molecules involved.1 Differentiation of Nati~,e and Denatured DNA and RNA. Exactly 2.5 or 3.0 mh of the nucleic acid solution, at appropriate concentration (about 10 to 50 ~,/ml. in NaC1 of 0.1 M or stronger) is placed in a quartz cell having a 1-cm. light path. The optical density is determined at 320 m~ (as indication of extraneous scattering or absorption) and at 260 m~. Saline in the same volume serves as blank. Strong NaOH is added to each cell, 0.05 ml. of approximately 6 N NaOH per 3 ml., or an equivalent amount. The absorption of the alkaline solution, corrected back to the volume before dilution with alkali, less the initial absorption at 260 m~, gives the "absorption increment." This increment, multiplied by 3 (that is, 1/0.33), is taken as the 260-m~ absorption value for the native D N A and R N A initially present. An optical density of 1.0 at 260 m~ (1-era. light path) corresponds to about 45 5' of DNA or R N A per milliliter. Freshly made NaOH, kept in Pyrex, adds only a small absorption to the blank. The quantity used corresponds to a final concentration of 0.1 M, or about a threefold excess to allow for buffering by other constituents (final pH to be more than 11.5). Certain substances, such as phenols, or proteins in twentyfold excess over nucleic acid, might give sufficient alkali response to necessitate the before and after observation of absorption at a number of wavelengths, or other blank determinations. Specific Spectrophotometric Determination of High-Molecular DNA and R N A in Mixtures. In a similar way, small amounts of purified ribonuclease ~RNase) or deoxyribonuclease (DNase) may be added to neutral solutions of nucleic acid, and the absorption increment after enzymatic reaction is complete is a measure of the corresponding native nucleic acids. 1 It is convenient to add the enzymes in succession, RNase first, since it is more likely than DNase to be free from the other enzyme; it may also reduce inhibition which R N A can exert on certain preparations 1See Addendum, p. 715.
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of DNase. Magnesium salt is added at the beginning, since DNase will require Mg ++ as activator, and its addition may influence the absorption.
Reagents Mg-saline, 0.9% NaC1 containing 0.005 M MgC12 (or MgSO4). RNase, crystalline ribonuclease, 0.5 mg./ml, in H20. This preparation should be sufficiently free of DNase so as not to reduce the viscosity obviously when incubated 30 minutes with an equal volume (e.g., 0.3 ml. of 0.2%) of viscous calf thymus or other DNA. DNase, crystalline pancreatic deoxyribonuclease, 0.1 mg./ml, in 0.9 % NaC1. (Commercial streptococcal DNase will also serve.)
Procedure. The nucleic acid is diluted in Mg-saline to an approximate total concentration of 10 to 50 -y/ml. and brought to pH between 6 and 7.5. A known 2.5- or 3.0-ml. amount is placed in a 1-cm. quartz cell, and the optical density measured at 260 m~ (and at 320 mu to estimate extraneous scattering or absorption) against a Mg-saline control. A known volume of RNase (0.01 ml. or the equivalent, with micropipet) is added to the blank and the unknown with mixing. Hydrolysis requires less than 10 minutes at room temperature when active enzyme is used. When the absorption at 260 mu becomes stable, the optical density, corrected to the volume before RNase addition, less the initial value, gives the absorption increment due to ribonucleic acid hydrolysis. The increment, times 3, is taken as the original absorption of native RNA. Mter the absorption at 260 mu is constant, a known volume of DNase (0.06 ml. of above solution, or the equivalent) is added to the blank and the unknown. The corrected absorption increment is calculated exactly as for RNA; the onset of change usually comes only after some delay, since the early stages of hydrolysis do not result in optical density change. The increment, times 3, is taken as the original absorption of native D N A (45 -y/ml. giving optical density of 1.0 per centimeter). The initial absorption at 260 m~, less the sum of native R N A and D N A determined as above, gives a measure of the denatured or degraded nucleic acids, plus other constituents absorbing at this wavelength. Remarks. The absorption increment is a property the meaning of which is not yet fully clear. As indicated, it may for the moment be considered an empirical property (like the inevitable proportionality constant in a colorimetric analysis), one which seems to be closely correlated with biological and physical integrity. The method outlined above may need some quantitative revision as more information on nucleic acid structure becomes known. For economy, the small correction due to the enzymes added can be
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separately determined for each nuclease and deducted, instead of eliminated by incorporation in the blank as above. Either RNase or DNase may be used alone; if the DNase preparation has RNase activity, however, it will be necessary to add pure RNase before using theDNase. Since high-molecular R N A is not usually available, the DNase preparation should be tested for RNase in some other way, e.g., by observing or measuring whether it decreases the acid precipitability of an ordinary R N A preparation.
II. Characterization of DNA by Biological Activity (Bacterial Transformation) Principle. D N A acts as the bearer of specific genetic properties in some bacteria, and perhaps in most cells. By introducing DNA coming from one strain into a suitable, related but different, strain of certain bacteria, heritable properties characteristic of the donor cells can be transferred to the recipient line. s,7 The proportion of the recipient cells "transformed" is a measure of a specific biological activity of the material. This proportion is determined quantitatively by exposing a measured sample of cells from the transformed culture to a selective environment which will permit growth of only the modified cells, and counting the number of cells which have produced colonies after a suitable time. 7,s The response of a pneumococcal population to suitable DNA is dependent on (1) appropriate pH and concentrations of calcium and serum albumin, (2) the physiological state of the culture, (3) randomness or synchrony in the division state 8 of individual cells, (4) the total DNA concentration, and (5) the proportion of this DNA which carries the biological activity being measured. Factor 1 is provided by choosing suitable media; factors 2 and 3 have to be controlled in each experiment by comparison against a standard transforming DNA. Increasing concentration of DNA (factor 4) gives a dose-response curve showing transformants increasing linearly with DNA at low concentrations (up to about 0.2 7/ml.), leveling off to a saturation plateau at high concentrations (0.2 to 5 ~,/ml.). D N A from a species not suitable for transformation can be tested by incorporating it as an inhibitory substance in known ratios to active DNA. The inhibitory activity of the DNA from a heterologous source is a test of its own biological intactness, since various kinds of denatured D N A do not inhibit transformation. Materials. TRANSFORMABLEBACTERIA. A number of strains of Pneumococcus have been transformed, especially several derived from strain 60. T. Avery, C. M. MacLeod, and M. McCarty, J. Exptl. Med. 79, 137 (1944). R. D. Hotehkiss, Cold Spring Harbor Symposia Quant. Biol. 16, 457 (1951). a R. D. Hotchkiss, Proc. Natl. Acad. Sci. U.S. 40, 49 (1954).
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R36A. 6 Hemophilus 9 and meningococcal :° strains have been used similarly. It is likely that other transformable strains and species will become available from time to time; this expectation is one reason for attempting to illustrate here a general method of bioassay. D N A PREPARATION. In the example given, the quantitative marker introduced will be streptomycin resistance of Pneumococcus. The DNA used comes from one-step, high-level streptomycin resistant pneumococci. (Hemophilus or meningococcal, etc., mutants could be used, corresponding to the transformable species of bacteria available.) Preparation is carried out as described in Vol. III [102] the DNA being precipitated with alcohol to sterilize it, but never dried to a water-free state. Dilutions are made in sterile 0.9% NaC1, standard and unknown being prepared in comparable dilutions. DNAsE. Commercial pancreatic deoxyribonuclease is used in a working dilution of 0.03 mg./ml, in sterile growth medium, or saline, stored in the refrigerator. SERUM ALBUMIN.Bovine serum albumin (Armour Plasma Fraction V) is dissolved at 4 g. per 100 ml. in distilled H20, adjusted to pH 7.5, and sterilized by filtration. Other purified albumin preparations from various species suffice, crude serum and other proteins do not. GROWTH MEDIUM. This should be suited to the organism used. For pneumococcus, the medium may be 1% neopeptone (Difco), 5% fresh meat infusion (lean beef ground into an equal weight of distilled water, strained, heated to 85 °, and filtered)l and 5% yeast extract (baker's yeast cake slurried into an equal weight of boiling water, filtered). When brought to pH 7.6, to 0.3% concentration of NaC1, clarified, and autoclaved, this is the basal growth medium. Additional requirements for transformation are supplied by adding K2HPO4 to a final concentration of M/40, glucose to 0.03 %, serum albumin to 0.2 %, and CaCl~ to 0.003 % (all in per cent weight per 100-ml. volume). For Hemophilus and other organisms, serum albumin has not proved necessary. Other media, such as one based on casein hydrolyzate and vitamins,:: may be used, with appropriate addition of serum albumin. SELECT:VE MEDIUM. In this example, streptomycin is the selective agent. It has also been used for Hemophilus; 9 other quantitative selection principles are described elsewhere 7,:2 for Pneumococcus. In the author's laboratory, quantitation is customarily carried out in liquid selective media containing antiserum globulin which causes single cells to grow as 9 H. E. Alexander and G. Leidy, J. Exptl. Med. 97, 17 (1953). lo H. E. Alexander and W. Redman, J. Exptl. Med. 97, 797 (1953). 11 M. H. Adams and A. S. Roe, J. Bacteriol. 49, 401 (1945). 1~R. D. Hotchkiss and J. Marmur, Proc. Natl. Acad. Sci. U.S. 40, 55 (1954); J. Marmur and R. D. Hotchklss, J. Biol. Chem. 214, 383 (1955).
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colonies. 8 Since this method has not been worked out for other bacteria as yet, the more adaptable streptomycin-blood-agar plate scoring is described here. For many organisms nutrient agar without blood would suffice. The basal neopeptone medium described is brought to 100 °, and 15 g. of agar per liter dissolved in it. After cooling to 45 °, 0.1% serum albumin, 2% sterile defibrinated whole rabbit or human blood, and streptomycin (150 -~/ml.) are added, and standard plates of about 20 ml. are poured. Such plates, made without the streptomycin, should support growth of surface colonies from single cells of the sensitive recipient strain to be used; with the drug present, only the resistant transformants or mutants should grow.
Procedure for Quantitative Transformation. Albumin-containing medium is inoculated with approximately 5000 cells/ml. (10-5 dilution of a fresh full-grown culture of transformable strain), and divided into 1.5-ml. portions in small culture tubes. After 4 hours of undisturbed incubation (in a water bath at 37 ° for accurate definition of time intervals), standard and unknown DNA's are added in a series of saline dilutions to separate cultures. Appropriate amounts are from about 1 to 2 ~,/ml. (saturation level) down to 0.01 .~,/ml. or less, in any desired dilution steps. Exactly 5 (or 10) minutes after addition of DNA, a drop of DNase solution is added to each culture to stop the formation of further transformants, and incubation is continued for 75 minutes. At the end of this time, the cultures are chilled in ice and diluted as soon as possible for streaking on selective medium. There should be a countable number of transformant colonies from 0.10 ml. evenly spread on the surface of the selective medium, using 1 : 20 dilution (at high DNA) or undiluted culture (for low DNA concentrations). The yield of colonies, in relation to those from a parallel culture treated with a standard D N A bearing the same marker, is a reliable measure of the transforming actiwity of a preparation. The slope of the linear region of the response curve indicates the active DNA concentration. The number of transformants at saturation concentration expresses the activity, or quality, level of the D N A preparation (active/total, modified by affinity constants, degree of degradation, etc.). D N A from heterologous sources can be evaluated by its ability, in known admixtures, to interfere with transformation by active specific material. The absolute yield is meaningful only when the condition and amount of inoculum, the time of growth, and the medium have been closely controlled. A larger inoculum gives a larger population, sensitive to D N A considerably earlier than the 4 hours used here, but the period of sensitivity is brief and must be rather closely outlined or it may be entirely missed.
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Alternative assay methods, in which DNA is diluted to the end point at which transformation ceases to show up, ~,9.13 are possible but are quantitatively less reliable, since both statistical and physiological uncertainties produce much scatter at the dilution end point. 1~ S. Zamenhof, H. E. Alexander, and G. Leidy, J. Exptl. Med. 98, 373 (1953); A. W. Ravin, Exptl. Cell Research 7, 58 (1954). ADDENDUM The increasing evidence that many high-molecular R N A preparations tend to depolymerize spontaneously in saline suggests (May, 1956) that one should if possible determine for each R N A the denaturation increment (more or less than 33%) with reference to an arbitrarily defined native state.
[106] M e t h o d s f o r C h a r a c t e r i z a t i o n of N u c l e i c A c i d s b y Base 1 Composition
By AARON BENDICH I. Methods of Hydrolysis
Principle. The liberation of bases from nucleic acids depends on the cleavage of acid-labile glycosidic bonds. Except for the T-even bacteriophages of Escherichia coli, the DNA's of which contain the base 5-hydroxymethylcytosine 2 (which is largely destroyed during hydrolysis with HC104), hydrolysis by means of HC10~ is the method of choice for both R N A and DNA. For DNA's in general, and for the above phage DNA's in particular, hydrolysis with formic acid is also valuable. Reagents 70 to 72% HC10~ (ca. 12 N). A good analytical reagent grade is supplied by the Mallinckrodt Chemical Works (catalog No. 2766). 88% formic acid reagent grade supplied by Merck & Co.
Procedure. (a) (HC104). 3 A weighed nucleic acid preparation (dry) is intimately mixed with 70 to 72% HC104 (5 mg. per 0.1 ml. of HC104) 4 in a small glass-stoppered test tube or centrifuge tube and is heated at 100 ° for 60 minutes with occasional agitation. 5 The mixture is cooled and 1 The term " b a s e " refers to the purines and pyrimidines isolable from nucleic acids, despite the lack of demonstrable basic properties in uracil and thymine. G. R. W y a t t and S. S. Cohen, Biochem. J. 55, 774 (1953). Based mainly on A. Marshak and H. J. Vogel, J. Biol. Chem. 189, 597 (1951). 4 A 2.5% loss of thymine from insect virus D N A has been found by G. R. Wyatt [J. Gen. Physiol. 36, 201 (1952)] with this proportion of D N A to HC104. He advises the use of half this quantity of HC10~. 5 At somewhat lower temperatures, incomplete liberation of cytosine from R N A is observed together with the appearance of a cytidylic acid [P. M. Roll and I. Welicky, J. Biol. Chem. 213, 509 (1955)]. It is imperative that a temperature of 100 ° be maintained.
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found when DNA solutions are heated. The heat stability of DNA from various origins has been investigated by Zamenhof. 7 Light-scattering experiments have indicated that this decrease in viscosity brought about by heat treatment does not at first cause any decrease in the molecular weight. 8 Thus it appears that heat treatment is analogous to exposure to low pH values. Presumably the hydrogen bonds between the polynucleotide chains are broken by heat as well as by low pH. Conclusion. Although the measurement of the intrinsic viscosity in a manner which successfully eliminates the effects of both concentration and shear is difficult experimentally, its determination provides a sensitive measure of molecular size. Moreover, when such a sensitive measurement is combined with an independent molecular weight determination, the absolute value of the size can be obtained. On the other hand the molecular weight itself can be calculated by combination with the sedimentation constant. Viscosity measurements have proved especially valuable in investigating the effects of pH and heat on the DNA molecule. In both cases the molecular weight was found to remain constant whereas the size of the molecule, as reflected by the viscosity, undergoes substantial contraction. This contraction in size is thought to be the result of the destruction of hydrogen bonds between polynucleotide chains. 7 S. Zamenhof, H. E. Alexander, G. Leidy, J. Exp. Med. 98, 373-397 (1953). 8 Paul Doty and S. A. Rice, Biochim. et Biophys. Acta 16, 446-447 (1955).
[105] M e t h o d s for C h a r a c t e r i z a t i o n of N u c l e i c Acid 1
By ROLLIN D. HOTCHKISS I. Characterization of Nucleic Acids by Spectrophotometry
Principle. Both DNA and RNA absorb ultraviolet light strongly, with maximum absorption about 260 m~, falling off to a minimum about 230 to 240 m~ and almost disappearing at 310 m~. High-molecular DNA and RNA can undergo a denaturation change which results in an increase of ultraviolet absorption in saline solution of about 33 % at 260 m~ (and of similar magnitude along the whole absorption curve). The absorption increment from NaOH treatment has been used in the author's laboratory since 1948 to determine DNA in the presence of RNA (because the latter is usually not high molecular). 1 For colorimetric procedures for anatysi8 of nucleic acid constituents, see Vol. I I I [12, 13, 99].
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After Kunitz reported that pancreatic deoxyribonuclease (DNase) hydrolysis resulted in an increase in absorption, 2 it was quickly discovered that the alkali and enzymatic effects were equivalent and not additive. Enzymatically hydrolyzed DNA does not show further increase in ultraviolet absorption when made alkaline; likewise alkali- (or acid-) denatured DNA, reneutralized, no longer gives the increase on hydrolysis with enzyme. It was also shown at the same time (with M. McCarty) that high-molecular R N A from Pneumococcus would show a similar 30 to 33 % absorption increment when treated with ribonuclease (RNase) or alkali. A number of authors have observed some of these absorption shifts in the years since, particularly with DNA. It seems clear that some have come to look on the extinction coefficient in relation to phosphorus content as some sort of criterion of nativeness in DNA, 3 but the quantitative differences between different preparations and different modes of denaturation discouraged interpretation of the absorption increment of denaturation. Beaven el al. 4 have recently proposed the use of alkali denaturation, without phosphorus analysis, in much the same way as described here in judging the quality of a preparation. As stated, the method has been used by the author with increasing confidence during the past several years. It is important to make as clear as present knowledge permits what features of DNA or R N A structure can be correlated with a 33 % absorption increment. In operational terms, the increment is shown by bacterial DNA of high biological activity, tissue DNA prepared by a variety of gentle means, DNA isolated while still attached to histone 5 (on alkali denaturation), and highly viscous R N A preparations from pneum0coccus. The value of 33 % is believed to be close to the ideal maximum, although a number of DNA and RNA preparations have been described which gave absorption increments less than this on hydrolysis and had been considered native. The author believes such preparations to be nevertheless denatured and has been able to make similar ones by irreversibly damaging intact DNA by drying, by heating, or by suitably drastic shift of pH or ionic strength. His native material, like that of a number of other workers, gives an increment close to 33 %. Few R N A preparations of good 2 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 3 G. Frick, Biochim. et Biophys. Acta 8, 625 (1952); J. Shack, R. J. Jenkins, and J. M. Thompsett, J. Biol. Chem. 203, 373 (1953) ; R. Thomas, Biochim. et Biophys. Acta 14, 231 (1954); E. Chargaff, in "The Nucleic Acids" (Chargaff and Davidson, eds.), Vol. I, p. 336. Academic Press, New York, 1955. 4 G. H. Beaven, E. R. Holiday, and E. A. Johnson, in "The Nucleic Acids" (Chargaff and Davidson, eds.), Vol. I, p. 526. Academic Press, New York, 1955. 6 j. Shack and J. M. Thompsett, J. Biol. Chem. 197, 17 (1952) ; J. Natl. Cancer Inst. 13, 1425 (1953).
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quality have been described (they are often prepared with use of alkali). In particular, it should be noted that air or solvent drying of D N A usually leads to partial denaturation, in this sense. High specific viscosity is not a reliable criterion of integrity of DNA; the temperature dependence of viscosity (see Vol. III [103]) is probably a much better one. The ability to show an absorption increment, then, appears to depend on some not adequately understood feature of the spatial fitting or hydrogen bonding of the purine and pyrimidine bases, easily destroyed by denaturation. It may be that different nucleic acids of widely different base composition will show somewhat discrepant absorption increments. The methods which follow may therefore be somewhat liable to reinterpretation or quantitative correction as more is learned about the molecules involved.1 Differentiation of Nati~,e and Denatured DNA and RNA. Exactly 2.5 or 3.0 mh of the nucleic acid solution, at appropriate concentration (about 10 to 50 ~,/ml. in NaC1 of 0.1 M or stronger) is placed in a quartz cell having a 1-cm. light path. The optical density is determined at 320 m~ (as indication of extraneous scattering or absorption) and at 260 m~. Saline in the same volume serves as blank. Strong NaOH is added to each cell, 0.05 ml. of approximately 6 N NaOH per 3 ml., or an equivalent amount. The absorption of the alkaline solution, corrected back to the volume before dilution with alkali, less the initial absorption at 260 m~, gives the "absorption increment." This increment, multiplied by 3 (that is, 1/0.33), is taken as the 260-m~ absorption value for the native D N A and R N A initially present. An optical density of 1.0 at 260 m~ (1-era. light path) corresponds to about 45 5' of DNA or R N A per milliliter. Freshly made NaOH, kept in Pyrex, adds only a small absorption to the blank. The quantity used corresponds to a final concentration of 0.1 M, or about a threefold excess to allow for buffering by other constituents (final pH to be more than 11.5). Certain substances, such as phenols, or proteins in twentyfold excess over nucleic acid, might give sufficient alkali response to necessitate the before and after observation of absorption at a number of wavelengths, or other blank determinations. Specific Spectrophotometric Determination of High-Molecular DNA and R N A in Mixtures. In a similar way, small amounts of purified ribonuclease ~RNase) or deoxyribonuclease (DNase) may be added to neutral solutions of nucleic acid, and the absorption increment after enzymatic reaction is complete is a measure of the corresponding native nucleic acids. 1 It is convenient to add the enzymes in succession, RNase first, since it is more likely than DNase to be free from the other enzyme; it may also reduce inhibition which R N A can exert on certain preparations 1See Addendum, p. 715.
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of DNase. Magnesium salt is added at the beginning, since DNase will require Mg ++ as activator, and its addition may influence the absorption.
Reagents Mg-saline, 0.9% NaC1 containing 0.005 M MgC12 (or MgSO4). RNase, crystalline ribonuclease, 0.5 mg./ml, in H20. This preparation should be sufficiently free of DNase so as not to reduce the viscosity obviously when incubated 30 minutes with an equal volume (e.g., 0.3 ml. of 0.2%) of viscous calf thymus or other DNA. DNase, crystalline pancreatic deoxyribonuclease, 0.1 mg./ml, in 0.9 % NaC1. (Commercial streptococcal DNase will also serve.)
Procedure. The nucleic acid is diluted in Mg-saline to an approximate total concentration of 10 to 50 -y/ml. and brought to pH between 6 and 7.5. A known 2.5- or 3.0-ml. amount is placed in a 1-cm. quartz cell, and the optical density measured at 260 m~ (and at 320 mu to estimate extraneous scattering or absorption) against a Mg-saline control. A known volume of RNase (0.01 ml. or the equivalent, with micropipet) is added to the blank and the unknown with mixing. Hydrolysis requires less than 10 minutes at room temperature when active enzyme is used. When the absorption at 260 mu becomes stable, the optical density, corrected to the volume before RNase addition, less the initial value, gives the absorption increment due to ribonucleic acid hydrolysis. The increment, times 3, is taken as the original absorption of native RNA. Mter the absorption at 260 mu is constant, a known volume of DNase (0.06 ml. of above solution, or the equivalent) is added to the blank and the unknown. The corrected absorption increment is calculated exactly as for RNA; the onset of change usually comes only after some delay, since the early stages of hydrolysis do not result in optical density change. The increment, times 3, is taken as the original absorption of native D N A (45 -y/ml. giving optical density of 1.0 per centimeter). The initial absorption at 260 m~, less the sum of native R N A and D N A determined as above, gives a measure of the denatured or degraded nucleic acids, plus other constituents absorbing at this wavelength. Remarks. The absorption increment is a property the meaning of which is not yet fully clear. As indicated, it may for the moment be considered an empirical property (like the inevitable proportionality constant in a colorimetric analysis), one which seems to be closely correlated with biological and physical integrity. The method outlined above may need some quantitative revision as more information on nucleic acid structure becomes known. For economy, the small correction due to the enzymes added can be
712
NUCLEIC ACIDS AND DERIVATIVES
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separately determined for each nuclease and deducted, instead of eliminated by incorporation in the blank as above. Either RNase or DNase may be used alone; if the DNase preparation has RNase activity, however, it will be necessary to add pure RNase before using theDNase. Since high-molecular R N A is not usually available, the DNase preparation should be tested for RNase in some other way, e.g., by observing or measuring whether it decreases the acid precipitability of an ordinary R N A preparation.
II. Characterization of DNA by Biological Activity (Bacterial Transformation) Principle. D N A acts as the bearer of specific genetic properties in some bacteria, and perhaps in most cells. By introducing DNA coming from one strain into a suitable, related but different, strain of certain bacteria, heritable properties characteristic of the donor cells can be transferred to the recipient line. s,7 The proportion of the recipient cells "transformed" is a measure of a specific biological activity of the material. This proportion is determined quantitatively by exposing a measured sample of cells from the transformed culture to a selective environment which will permit growth of only the modified cells, and counting the number of cells which have produced colonies after a suitable time. 7,s The response of a pneumococcal population to suitable DNA is dependent on (1) appropriate pH and concentrations of calcium and serum albumin, (2) the physiological state of the culture, (3) randomness or synchrony in the division state 8 of individual cells, (4) the total DNA concentration, and (5) the proportion of this DNA which carries the biological activity being measured. Factor 1 is provided by choosing suitable media; factors 2 and 3 have to be controlled in each experiment by comparison against a standard transforming DNA. Increasing concentration of DNA (factor 4) gives a dose-response curve showing transformants increasing linearly with DNA at low concentrations (up to about 0.2 7/ml.), leveling off to a saturation plateau at high concentrations (0.2 to 5 ~,/ml.). D N A from a species not suitable for transformation can be tested by incorporating it as an inhibitory substance in known ratios to active DNA. The inhibitory activity of the DNA from a heterologous source is a test of its own biological intactness, since various kinds of denatured D N A do not inhibit transformation. Materials. TRANSFORMABLEBACTERIA. A number of strains of Pneumococcus have been transformed, especially several derived from strain 60. T. Avery, C. M. MacLeod, and M. McCarty, J. Exptl. Med. 79, 137 (1944). R. D. Hotehkiss, Cold Spring Harbor Symposia Quant. Biol. 16, 457 (1951). a R. D. Hotchkiss, Proc. Natl. Acad. Sci. U.S. 40, 49 (1954).
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NUCLEIC ACID CHARACTERIZATION
713
R36A. 6 Hemophilus 9 and meningococcal :° strains have been used similarly. It is likely that other transformable strains and species will become available from time to time; this expectation is one reason for attempting to illustrate here a general method of bioassay. D N A PREPARATION. In the example given, the quantitative marker introduced will be streptomycin resistance of Pneumococcus. The DNA used comes from one-step, high-level streptomycin resistant pneumococci. (Hemophilus or meningococcal, etc., mutants could be used, corresponding to the transformable species of bacteria available.) Preparation is carried out as described in Vol. III [102] the DNA being precipitated with alcohol to sterilize it, but never dried to a water-free state. Dilutions are made in sterile 0.9% NaC1, standard and unknown being prepared in comparable dilutions. DNAsE. Commercial pancreatic deoxyribonuclease is used in a working dilution of 0.03 mg./ml, in sterile growth medium, or saline, stored in the refrigerator. SERUM ALBUMIN.Bovine serum albumin (Armour Plasma Fraction V) is dissolved at 4 g. per 100 ml. in distilled H20, adjusted to pH 7.5, and sterilized by filtration. Other purified albumin preparations from various species suffice, crude serum and other proteins do not. GROWTH MEDIUM. This should be suited to the organism used. For pneumococcus, the medium may be 1% neopeptone (Difco), 5% fresh meat infusion (lean beef ground into an equal weight of distilled water, strained, heated to 85 °, and filtered)l and 5% yeast extract (baker's yeast cake slurried into an equal weight of boiling water, filtered). When brought to pH 7.6, to 0.3% concentration of NaC1, clarified, and autoclaved, this is the basal growth medium. Additional requirements for transformation are supplied by adding K2HPO4 to a final concentration of M/40, glucose to 0.03 %, serum albumin to 0.2 %, and CaCl~ to 0.003 % (all in per cent weight per 100-ml. volume). For Hemophilus and other organisms, serum albumin has not proved necessary. Other media, such as one based on casein hydrolyzate and vitamins,:: may be used, with appropriate addition of serum albumin. SELECT:VE MEDIUM. In this example, streptomycin is the selective agent. It has also been used for Hemophilus; 9 other quantitative selection principles are described elsewhere 7,:2 for Pneumococcus. In the author's laboratory, quantitation is customarily carried out in liquid selective media containing antiserum globulin which causes single cells to grow as 9 H. E. Alexander and G. Leidy, J. Exptl. Med. 97, 17 (1953). lo H. E. Alexander and W. Redman, J. Exptl. Med. 97, 797 (1953). 11 M. H. Adams and A. S. Roe, J. Bacteriol. 49, 401 (1945). 1~R. D. Hotchkiss and J. Marmur, Proc. Natl. Acad. Sci. U.S. 40, 55 (1954); J. Marmur and R. D. Hotchklss, J. Biol. Chem. 214, 383 (1955).
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NUCLEIC ACIDS AND DERIVATIVES
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colonies. 8 Since this method has not been worked out for other bacteria as yet, the more adaptable streptomycin-blood-agar plate scoring is described here. For many organisms nutrient agar without blood would suffice. The basal neopeptone medium described is brought to 100 °, and 15 g. of agar per liter dissolved in it. After cooling to 45 °, 0.1% serum albumin, 2% sterile defibrinated whole rabbit or human blood, and streptomycin (150 -~/ml.) are added, and standard plates of about 20 ml. are poured. Such plates, made without the streptomycin, should support growth of surface colonies from single cells of the sensitive recipient strain to be used; with the drug present, only the resistant transformants or mutants should grow.
Procedure for Quantitative Transformation. Albumin-containing medium is inoculated with approximately 5000 cells/ml. (10-5 dilution of a fresh full-grown culture of transformable strain), and divided into 1.5-ml. portions in small culture tubes. After 4 hours of undisturbed incubation (in a water bath at 37 ° for accurate definition of time intervals), standard and unknown DNA's are added in a series of saline dilutions to separate cultures. Appropriate amounts are from about 1 to 2 ~,/ml. (saturation level) down to 0.01 .~,/ml. or less, in any desired dilution steps. Exactly 5 (or 10) minutes after addition of DNA, a drop of DNase solution is added to each culture to stop the formation of further transformants, and incubation is continued for 75 minutes. At the end of this time, the cultures are chilled in ice and diluted as soon as possible for streaking on selective medium. There should be a countable number of transformant colonies from 0.10 ml. evenly spread on the surface of the selective medium, using 1 : 20 dilution (at high DNA) or undiluted culture (for low DNA concentrations). The yield of colonies, in relation to those from a parallel culture treated with a standard D N A bearing the same marker, is a reliable measure of the transforming actiwity of a preparation. The slope of the linear region of the response curve indicates the active DNA concentration. The number of transformants at saturation concentration expresses the activity, or quality, level of the D N A preparation (active/total, modified by affinity constants, degree of degradation, etc.). D N A from heterologous sources can be evaluated by its ability, in known admixtures, to interfere with transformation by active specific material. The absolute yield is meaningful only when the condition and amount of inoculum, the time of growth, and the medium have been closely controlled. A larger inoculum gives a larger population, sensitive to D N A considerably earlier than the 4 hours used here, but the period of sensitivity is brief and must be rather closely outlined or it may be entirely missed.
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NUCLEIC ACIDS BY BASE COMPOSITION
715
Alternative assay methods, in which DNA is diluted to the end point at which transformation ceases to show up, ~,9.13 are possible but are quantitatively less reliable, since both statistical and physiological uncertainties produce much scatter at the dilution end point. 1~ S. Zamenhof, H. E. Alexander, and G. Leidy, J. Exptl. Med. 98, 373 (1953); A. W. Ravin, Exptl. Cell Research 7, 58 (1954). ADDENDUM The increasing evidence that many high-molecular R N A preparations tend to depolymerize spontaneously in saline suggests (May, 1956) that one should if possible determine for each R N A the denaturation increment (more or less than 33%) with reference to an arbitrarily defined native state.
[106] M e t h o d s f o r C h a r a c t e r i z a t i o n of N u c l e i c A c i d s b y Base 1 Composition
By AARON BENDICH I. Methods of Hydrolysis
Principle. The liberation of bases from nucleic acids depends on the cleavage of acid-labile glycosidic bonds. Except for the T-even bacteriophages of Escherichia coli, the DNA's of which contain the base 5-hydroxymethylcytosine 2 (which is largely destroyed during hydrolysis with HC104), hydrolysis by means of HC10~ is the method of choice for both R N A and DNA. For DNA's in general, and for the above phage DNA's in particular, hydrolysis with formic acid is also valuable. Reagents 70 to 72% HC10~ (ca. 12 N). A good analytical reagent grade is supplied by the Mallinckrodt Chemical Works (catalog No. 2766). 88% formic acid reagent grade supplied by Merck & Co.
Procedure. (a) (HC104). 3 A weighed nucleic acid preparation (dry) is intimately mixed with 70 to 72% HC104 (5 mg. per 0.1 ml. of HC104) 4 in a small glass-stoppered test tube or centrifuge tube and is heated at 100 ° for 60 minutes with occasional agitation. 5 The mixture is cooled and 1 The term " b a s e " refers to the purines and pyrimidines isolable from nucleic acids, despite the lack of demonstrable basic properties in uracil and thymine. G. R. W y a t t and S. S. Cohen, Biochem. J. 55, 774 (1953). Based mainly on A. Marshak and H. J. Vogel, J. Biol. Chem. 189, 597 (1951). 4 A 2.5% loss of thymine from insect virus D N A has been found by G. R. Wyatt [J. Gen. Physiol. 36, 201 (1952)] with this proportion of D N A to HC104. He advises the use of half this quantity of HC10~. 5 At somewhat lower temperatures, incomplete liberation of cytosine from R N A is observed together with the appearance of a cytidylic acid [P. M. Roll and I. Welicky, J. Biol. Chem. 213, 509 (1955)]. It is imperative that a temperature of 100 ° be maintained.