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A rapid method for the determination of the base composition of bacterial DNA
Journal of Microbiological Methods 1 (1983)305-311 Elsevier
305
JMM 00035
A rapid method for the determination of the base composition of bacterial D N A J o s e f Flossdorf Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig (F.R.G.) (Received 25 May 1983) (Revised version received 10 August 1983) (Accepted 16 August 1983)
Summary A rapid method to evaluate the (G+C)-content of bacterial DNA is described. About 2 x 108 cells are lysed by the combined action of detergents and enzymes and put into a CsCl-gradient without any purification. Up to five samples may be run at once. The high background absorption of cell-derived material other than DNA is overcome by novel collimating optics.
Key words: Micromethod - DNA base composition - Buoyant density
Introduction
Lysis of bacterial cells on a micro-scale by the action of detergents, and running the total lysates in a CsCl-gradient in an analytical ultracentrifuge, is the original procedure to evaluate the buoyant density of their DNA. It was first used by Meselson and colleagues [1, 2] to detect small density differences among molecules of D N A of Escherichia coli and, by use of the heavy nitrogen isotope 15N, to clarify by what mechanism parental D N A is replicated and distributed among progenies. The method soon evolved into a powerful tool to measure the base-composition of any D N A [3-5]. Until now, it has been unequalled with respect to speed and simplicity. Originally, the concentration distribution of UV-absorbing material was routinely recorded by taking photographs at 265 nm and by scanning them in a microdensitometer. This method is cumbersome and laborious, but background absorption by cell-derived material other than D N A as well as weak rotor illumination can simply be overcome by prolonged exposure. With the introduction of the photoelectric scanner and the replacement of the mercury lamp and the optical 0167-7012/83/$03.00 © 1983 Elsevier Science Publishers B.V.
306 filters by a high-pressure light illuminated monochromator [6-9], recording of the DNA-distribution in the centrifuge cell has become much more convenient and accurate. Similar improvements of rotor illumination, however, have been lacking, so that the new equipment was not able to overcome the background absorption of total cell lysates. It was necessary to use purified DNA, the isolation of which does not imply severe problems. However, it is lengthy and finally yields DNA in superfluous amounts, at least as compared to the few micrograms which are sufficient to measure its buoyant density. The recent development of a novel collimator for the UV-optics of the Beckman ultracentrifuge Model E [10, 11] has solved this dilemma because the rotor illumination is raised by a factor of about 45. So it is now possible to combine a simple and rapid procedure of sample preparation with the accuracy and the comfort of photoelectric recording. Examples are presented here to demonstrate that the evaluation of the (G + C)-content of bacterial DNA can be a simple laboratory routine even when only minor amounts of material are available.
Experimental procedures Organisms and cultivation Escherichia coli K 12 was grown in L-broth (Tryptone (Difco) 10%, yeast extracts 0.5%, NaCI 0.5%, glucose 0.1%, pH 7.0) at 37°C, Micrococcus luteus (DSM 20030) was grown in Caso instant-medium (Merck, Darmstadt, F.R.G.), and Cytophaga johnsonae (DSM 425) in FxAlm (Casitone (Difco) 1.0%, yeast extract 0.2%, MgSO4.7H20 0.1%, pH 7.2). Preparation of DNA The procedure given here is mainly designed for the use of disposable mini-cups (Eppendorf, Hamburg, F.R.G.) and auxiliary equipment, but other tubes may be suitable as well. It closely relies on the procedure of Marmur [12], differing only in that proteins do not need to be extracted but are hydrolyzed by enzymatic digestion [13]. The use of a suitable marker D N A with known (G + C)-content is recommended. It can either be added as purified D N A to the CsCl-solution or in the form of living cells prior to the detergent treatment. The following solutions were used: (1) Saline-citrate-buffer (SSC-buffer): 0.015 M tri-sodium citrate, 0.15 M NaCl, pH 7.0; (2) EDTA-Gardol: 7.5% Gardol (sodium lauryl sarcosinate, purchased from Serva, Heidelberg, F.R.G.) or another suitable detergent, 0.25% EDTA, pH 7.0; (3) Tris-proteinase K-solution: 1 M Tris-HC1, pH 8.0, containing 500 ~tg/ml proteinase K (from Tritirachium album, purchased from Serva, Heidelberg, F.R.G.); (4) CsCl-solution: 58.1% CsCl (w/w) in distilled water. About 108 to 2 x 108 cells of each sample and marker were harvested by centrifugation of an aliquot of the culture fluid for 5 min at 7500 x g. They were washed once by resuspension in 300 ktl of SSC-buffer followed by centrifugation. The pellet was suspended in 60 !~1 of SSC-buffer plus 20 ~tl of EDTA-Gardol and subjected to lysis for 10 rain at 56°C. After addition of 5 ~tl of Tris-proteinase K-solution,
307
the proteins were degraded at 56°C for 1 h. The sample was then chilled to room temperature. An aliquot of 30 ~tl was added to 0.5 ml of CsCl-solution and mixed to homogeneity by gentle shaking. As an alternative route, especially if the bacterium is resistant to the detergent treatment, the washed cells may first be treated with lysozyme. For this purpose, the pellet is resuspended in 60 ttl of SSC-buffer containing 60 ~tg of lysozyme (0.1%) and incubated at 37°C for 30 min. After adding 20 ~l of EDTA-Gardol and heating at 56°C for another 10 min, the procedure outlined above is followed. For further possible modifications in sample preparation see Marmur [12].
Ultracentrifugation Each 400 ~tl of the CsCl-solutions was run to equilibrium in 12 mm KEL-F double-sector cells with sapphire windows, three or five at a time in a four-place An-F or a six-place AN-G rotor at 25°C and at 44 000 rpm in a Beckman Model E analytical ultracentrifuge. The centrifuge was equipped with electronic speedand RTIC temperature-control, with UV-scanner, multiplexer, monochromator and high-pressure light soorce. The collimating optics had been improved by a novel illuminating system [10, 11] that enhances the intensity of rotor illumination by a factor of about 45. To avoid shearing of the DNA, it is highly recommended that the cells be only partially assembled and filled from the top by wide-lumen pipettes in the same manner that Yphantis-type cells [14, 17] are filled.
Treatment of data After 24-40 h, scans were made at a suitable wavelength between 260 and 290 nm to ensure a maximum peak height lower than 1 A unit. The radial positions of both peaks, marker (rm) and unknown DNA (rs), were measured and the difference Ar = r s - rr, and the mean radial position -f = (rs + rm)/2 calculated from these data. The difference of buoyant density between sample and marker follows from AO = (1.08 (D2/[B)TAr = 1.93 x 10-2 TAr, wherein (D E -~- 2.123 × 10 7 s -2 and [3 = 1.190 × 109 cmS.g-l.s -2 [15]. The factor 1.08 accounts for the effect of pressure on the density gradient [16, 17]. The difference in (G + C)-content A(G + C) is calculated (cmp. [18]) according to A(G + C) = 1038 AQ = 20.01 TAr. A(G + C) is the difference in base composition of sample and marker, so that finally (G + C)~ = (G + C)~ + A(G + C). Results and Discussion
The procedure described above has been used in our laboratory for more than 2 years with good success. The organisms investigated so far belonged to a wide variety of genera, among them Bacillus (in collaboration with the DSM, G6ttingen, F.R.G.), Cytophaga (in collaboration with H. Reichenbach, GBF, Braunschweig, F.R.G.), chemolithotrophic bacteria (in collaboration with the Institute of Microbiology of the Technical University of Braunschweig, F.R.G.), and Mycoplasma from different sources. As an example, Fig. la demonstrates an ultracentrifugal run which would
308 s c a r c e l y b e d o n e in this m a n n e r b u t is q u i t e i n f o r m a t i v e for the p r e s e n t p u r p o s e . Three
bacteria
were
lysed
and
mixed
together
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They
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Fig. 1. Isopycnic CsCl-gradient centrifugation of a mixture of DNAs from Cytophaga johnsonae (left peaks; (G + C) = 33%), Micrococcus luteus (right peaks; (G + C) = 74%) and Escherichia coli (intermediate peaks; (G + C) = 50%). (a) The DNAs were isolated by lyric treatment of about 2 × 108 cells of each organism and used without any purification. (b) The DNAs were isolated and purified according to Marmur [12] prior to use. Run conditions: 44 000 rpm at 25°C; scans were made after 40 h at 288 nm (a) and 270 nm (b), respectively.
309 the ultracentrifuge cell, and DNA from Escherichia coli K 12 (strain DR 1984; (G + C) = 50%) between both. Though all organisms contribute to a non-marginal background absorption due to cell-derived material other than DNA, it is in particular C. ]ohnsonae, an intensively yellow pigmented bacterium which raises the background absorption to values as high as 0.4 A units. Nevertheless, the DNApeaks are still clearly visible. Moreover, they turn out to be rather narrow which means that the DNAs are of high molecular weight. In addition they band at just the same position as highly purified DNAs of the same organisms (compare Fig. lb) proving the DNAs to be essentially free of bound contaminations, for example proteins. Closer inspection of Fig. la shows that there is some polymeric material both at the meniscus and the bottom, but none interferes with the DNA-peaks. Occasionally we have found polymeric material clearly banding below the meniscus but outside the range covered by natural DNAs so far known. We did not prove it to be a polysaccharide but we are sure that it was neither a protein nor a nucleic acid, since the band was rather sharp and failed to exhibit the typical UV-spectra of proteins or nucleic acids. In contrast, it seemed to scatter light rather than to absorb it and, after braking the rotor, formed a fragile 'membrane' of opalescent material. From our own experience, we can tabulate the following advantages of the method described. (1) One needs only minor amounts of sample material. About I to 2 x 10s cells are sufficient to evaluate the base composition of their DNA. This is of particular value when poorly growing organisms, e.g., obligately chemolithotrophic bacteria, or clones directly taken from an agar plate have to be characterized. (2) It is rapid and simple. It does not need highly purified DNA nor, in most cases, any purification at all. The limiting factor is the capacity of the ultracentrifuge, but even here, five samples may be run at once. The accuracy is the same as would be achieved with pure DNA. (3) Since there are no separation steps involved, the method is automatically a rather sensitive test for the homogeneity of the DNA. Plasmids and, if we would deal with higher organisms, mitochondrial DNA can often be detected. Infections become instantly apparent. However, it should be emphasized that the numerical precision of any isopycnic technique is limited. Nominally, the ultracentrifuge technique yields highly reproducible results, the standard deviation of multiple estimations of AQ against the same marker is usually found to be very low. That would mean that the relative error of both Ar and -i is quite small. Furthermore, the hydrostatic pressure inside the ultracentrifuge cell may be regarded to be constant,, at least as long as we consider only its influence on the salt gradient but exclude its influence on the buoyant density of the DNA. The same holds for the value of 13. However, from this it cannot be concluded that the final meaning of A(G + C), as the actual difference of base composition between marker DNA and probe DNA, is as well defined as the small standard deviation seems to suggest. The main reason for this is the conversion of A o to A(G + (7). As can easily be seen from the literature [18], d(A(G + C))/d(o ) = 1038 is inaccurate to about 4%. This may affect the
310
calculated value of A(G + C) at least to the same extent. Therefore, even if we restrict ourselves to those cases where the base composition of the marker D N A differs from that of the sample D N A by no more than 20%, the absolute value of the (G + C)-content of the sample D N A may be as inaccurate as _+1%. This should be kept in mind when buoyant densities or (G + C)-contents are compared with data cited in the literature. We feel that a discrepancy not higher than A(A(G + C)) = 2% is acceptable. This reasoning, of course, not only holds for the method presented here but for any procedure that basically relies on the buoyant density in a CsCl-gradient. In the same sense, and to the same extent, it also holds for the evaluation of A(G + C) from the melting temperature.
Acknowledgement I am indebted to H. Schillig and D. Hanisch for their skillful assistance during the work.
References 1
Meselson, M., Stahl, F.W. and Vinograd, J. (1957) Equilibrium sedimentation of macromolecules in density gradients. Proc. Natl. Acad. Sci. U.S.A. 43,581-588. 2 Meselson, M. and Stahl, F.W. (1958) The replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 44, 671--682. 3 Sueoka, N., Marmur, J. and Doty, P. (1959) Dependence of the density of deoxyribonucleic acids on guanine-cytosine content. Nature 183, 1429-1431. 4 Roife, R. and Meselson, M. (1959) The relative homogeneity of microbial DNA. Proc. Natl. Acad. Sci. U.S.A. 45, 1039-1043. 5 Schildkraut, C.L., Marmur, J. and Doty, P. (1962) Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCI. J. Mol. Biol. 4, 430-443. 6 Hanion, S., Lamers, K., Lauterbach, G., Johnson, R. and Schachman, H.K. (1962) Ultracentrifuge studies with absorption optics. I. An automatic photoelectric scanning absorption system. Arch. Biochem. Biophys. 99, 157-174. 7 Schachman, H.K., Gropper, L., Hanlon, S. and Putney, F. (1962) Ultracentrifuge studies with absorption optics. II. Incorporation of a monochromator and its application to the study of proteins and interacting systems. Arch. Biochem. Biophys. 99, 175-190. 8 Lamers, K., Putney, F., Steinberg, I.Z. and Schachman, H.K. (1963) Ultracentrifuge studies with absorption optics. III. A split-beam photoelectric scanning absorption system. Arch. Biochem. Biophys. 103,379--400. 9 Schachman, H.K, and Edelstein, S.J. (1966) Ultracentrifuge studies with absorption optics. IV. Molecular weight determination at the microgram level. Biochemistry 5, 2681-2705. 10 Flossdorf, J. and Schillig, H. (1979) Verbesserter Kollimator ffir die UV-Optik einer analytischen Ultrazentrifuge. Feinwerktechnik und Messtechnik 87, 93-96. 11 Flossdorf, J. (1980) Erweiterte Mel3m6glichkeiten in der analytischen Ultrazentrifugation durch die Verwendung eines neuartigen KoUimators. Makromol. Chemie 181,715-724. 12 Marmur, J. (1961) A procedure forthe isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3,208-218. 13 Gross-Bellard, M., Oudet, P. and Chambon, P. (1973) Isolation of high-molecular-weight DNA from mammalian cells. Eur. J. Biochem. 36, 32-38. 14 Yphantis, D. (1964) Equilibrium uitracentrifugation of dilute solutions. Biochemistry 3,297-317. Comp. also: Beckman Technical Bulletin E-TB-015 B. 15 Ifft, J.B., Voet, D.H. and Vinograd, J. (1961) The determination of density distributions and
311
16
17 18
density gradients in binary solutions at equilibrium in the ultracentrifuge. J. Phys. Chem. 65, 1138--1145. Hearst, J.E., Ifft, J.B. and Vinograd, J. (1961) The effects of pressure on the buoyant behavior of deoxyribonucleic acid and tobacco mosaic virus in a density gradient at equilibrium in the ultracentrifuge. Proc. Natl. Acad. Sci. U.S.A. 47, 1015-1025. Hearst, J.E. and Schmid, C.W. (1973) Density gradient sedimentation equilibrium. Methods Enzymol. 27, 111-127. De Ley, J. (1970) Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bacteriol, 101,738-754.
305
JMM 00035
A rapid method for the determination of the base composition of bacterial D N A J o s e f Flossdorf Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig (F.R.G.) (Received 25 May 1983) (Revised version received 10 August 1983) (Accepted 16 August 1983)
Summary A rapid method to evaluate the (G+C)-content of bacterial DNA is described. About 2 x 108 cells are lysed by the combined action of detergents and enzymes and put into a CsCl-gradient without any purification. Up to five samples may be run at once. The high background absorption of cell-derived material other than DNA is overcome by novel collimating optics.
Key words: Micromethod - DNA base composition - Buoyant density
Introduction
Lysis of bacterial cells on a micro-scale by the action of detergents, and running the total lysates in a CsCl-gradient in an analytical ultracentrifuge, is the original procedure to evaluate the buoyant density of their DNA. It was first used by Meselson and colleagues [1, 2] to detect small density differences among molecules of D N A of Escherichia coli and, by use of the heavy nitrogen isotope 15N, to clarify by what mechanism parental D N A is replicated and distributed among progenies. The method soon evolved into a powerful tool to measure the base-composition of any D N A [3-5]. Until now, it has been unequalled with respect to speed and simplicity. Originally, the concentration distribution of UV-absorbing material was routinely recorded by taking photographs at 265 nm and by scanning them in a microdensitometer. This method is cumbersome and laborious, but background absorption by cell-derived material other than D N A as well as weak rotor illumination can simply be overcome by prolonged exposure. With the introduction of the photoelectric scanner and the replacement of the mercury lamp and the optical 0167-7012/83/$03.00 © 1983 Elsevier Science Publishers B.V.
306 filters by a high-pressure light illuminated monochromator [6-9], recording of the DNA-distribution in the centrifuge cell has become much more convenient and accurate. Similar improvements of rotor illumination, however, have been lacking, so that the new equipment was not able to overcome the background absorption of total cell lysates. It was necessary to use purified DNA, the isolation of which does not imply severe problems. However, it is lengthy and finally yields DNA in superfluous amounts, at least as compared to the few micrograms which are sufficient to measure its buoyant density. The recent development of a novel collimator for the UV-optics of the Beckman ultracentrifuge Model E [10, 11] has solved this dilemma because the rotor illumination is raised by a factor of about 45. So it is now possible to combine a simple and rapid procedure of sample preparation with the accuracy and the comfort of photoelectric recording. Examples are presented here to demonstrate that the evaluation of the (G + C)-content of bacterial DNA can be a simple laboratory routine even when only minor amounts of material are available.
Experimental procedures Organisms and cultivation Escherichia coli K 12 was grown in L-broth (Tryptone (Difco) 10%, yeast extracts 0.5%, NaCI 0.5%, glucose 0.1%, pH 7.0) at 37°C, Micrococcus luteus (DSM 20030) was grown in Caso instant-medium (Merck, Darmstadt, F.R.G.), and Cytophaga johnsonae (DSM 425) in FxAlm (Casitone (Difco) 1.0%, yeast extract 0.2%, MgSO4.7H20 0.1%, pH 7.2). Preparation of DNA The procedure given here is mainly designed for the use of disposable mini-cups (Eppendorf, Hamburg, F.R.G.) and auxiliary equipment, but other tubes may be suitable as well. It closely relies on the procedure of Marmur [12], differing only in that proteins do not need to be extracted but are hydrolyzed by enzymatic digestion [13]. The use of a suitable marker D N A with known (G + C)-content is recommended. It can either be added as purified D N A to the CsCl-solution or in the form of living cells prior to the detergent treatment. The following solutions were used: (1) Saline-citrate-buffer (SSC-buffer): 0.015 M tri-sodium citrate, 0.15 M NaCl, pH 7.0; (2) EDTA-Gardol: 7.5% Gardol (sodium lauryl sarcosinate, purchased from Serva, Heidelberg, F.R.G.) or another suitable detergent, 0.25% EDTA, pH 7.0; (3) Tris-proteinase K-solution: 1 M Tris-HC1, pH 8.0, containing 500 ~tg/ml proteinase K (from Tritirachium album, purchased from Serva, Heidelberg, F.R.G.); (4) CsCl-solution: 58.1% CsCl (w/w) in distilled water. About 108 to 2 x 108 cells of each sample and marker were harvested by centrifugation of an aliquot of the culture fluid for 5 min at 7500 x g. They were washed once by resuspension in 300 ktl of SSC-buffer followed by centrifugation. The pellet was suspended in 60 !~1 of SSC-buffer plus 20 ~tl of EDTA-Gardol and subjected to lysis for 10 rain at 56°C. After addition of 5 ~tl of Tris-proteinase K-solution,
307
the proteins were degraded at 56°C for 1 h. The sample was then chilled to room temperature. An aliquot of 30 ~tl was added to 0.5 ml of CsCl-solution and mixed to homogeneity by gentle shaking. As an alternative route, especially if the bacterium is resistant to the detergent treatment, the washed cells may first be treated with lysozyme. For this purpose, the pellet is resuspended in 60 ttl of SSC-buffer containing 60 ~tg of lysozyme (0.1%) and incubated at 37°C for 30 min. After adding 20 ~l of EDTA-Gardol and heating at 56°C for another 10 min, the procedure outlined above is followed. For further possible modifications in sample preparation see Marmur [12].
Ultracentrifugation Each 400 ~tl of the CsCl-solutions was run to equilibrium in 12 mm KEL-F double-sector cells with sapphire windows, three or five at a time in a four-place An-F or a six-place AN-G rotor at 25°C and at 44 000 rpm in a Beckman Model E analytical ultracentrifuge. The centrifuge was equipped with electronic speedand RTIC temperature-control, with UV-scanner, multiplexer, monochromator and high-pressure light soorce. The collimating optics had been improved by a novel illuminating system [10, 11] that enhances the intensity of rotor illumination by a factor of about 45. To avoid shearing of the DNA, it is highly recommended that the cells be only partially assembled and filled from the top by wide-lumen pipettes in the same manner that Yphantis-type cells [14, 17] are filled.
Treatment of data After 24-40 h, scans were made at a suitable wavelength between 260 and 290 nm to ensure a maximum peak height lower than 1 A unit. The radial positions of both peaks, marker (rm) and unknown DNA (rs), were measured and the difference Ar = r s - rr, and the mean radial position -f = (rs + rm)/2 calculated from these data. The difference of buoyant density between sample and marker follows from AO = (1.08 (D2/[B)TAr = 1.93 x 10-2 TAr, wherein (D E -~- 2.123 × 10 7 s -2 and [3 = 1.190 × 109 cmS.g-l.s -2 [15]. The factor 1.08 accounts for the effect of pressure on the density gradient [16, 17]. The difference in (G + C)-content A(G + C) is calculated (cmp. [18]) according to A(G + C) = 1038 AQ = 20.01 TAr. A(G + C) is the difference in base composition of sample and marker, so that finally (G + C)~ = (G + C)~ + A(G + C). Results and Discussion
The procedure described above has been used in our laboratory for more than 2 years with good success. The organisms investigated so far belonged to a wide variety of genera, among them Bacillus (in collaboration with the DSM, G6ttingen, F.R.G.), Cytophaga (in collaboration with H. Reichenbach, GBF, Braunschweig, F.R.G.), chemolithotrophic bacteria (in collaboration with the Institute of Microbiology of the Technical University of Braunschweig, F.R.G.), and Mycoplasma from different sources. As an example, Fig. la demonstrates an ultracentrifugal run which would
308 s c a r c e l y b e d o n e in this m a n n e r b u t is q u i t e i n f o r m a t i v e for the p r e s e n t p u r p o s e . Three
bacteria
were
lysed
and
mixed
together
in a CsCl-gradient.
They
were
cho-
to c o v e r n e a r l y the full r a n g e o f p o s s i b l e b a s e c o m p o s i t i o n o f DNA: DNA f r o m Cytophaga johnsonae (DSM 425; (G + C) = 33%) b a n d s n e a r the m e n i s c u s , DNA f r o m Micrococcus luteus (DSM 20030; (G + C) = 74%) n e a r the b o t t o m of sen
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.-TZ__2
Fig. 1. Isopycnic CsCl-gradient centrifugation of a mixture of DNAs from Cytophaga johnsonae (left peaks; (G + C) = 33%), Micrococcus luteus (right peaks; (G + C) = 74%) and Escherichia coli (intermediate peaks; (G + C) = 50%). (a) The DNAs were isolated by lyric treatment of about 2 × 108 cells of each organism and used without any purification. (b) The DNAs were isolated and purified according to Marmur [12] prior to use. Run conditions: 44 000 rpm at 25°C; scans were made after 40 h at 288 nm (a) and 270 nm (b), respectively.
309 the ultracentrifuge cell, and DNA from Escherichia coli K 12 (strain DR 1984; (G + C) = 50%) between both. Though all organisms contribute to a non-marginal background absorption due to cell-derived material other than DNA, it is in particular C. ]ohnsonae, an intensively yellow pigmented bacterium which raises the background absorption to values as high as 0.4 A units. Nevertheless, the DNApeaks are still clearly visible. Moreover, they turn out to be rather narrow which means that the DNAs are of high molecular weight. In addition they band at just the same position as highly purified DNAs of the same organisms (compare Fig. lb) proving the DNAs to be essentially free of bound contaminations, for example proteins. Closer inspection of Fig. la shows that there is some polymeric material both at the meniscus and the bottom, but none interferes with the DNA-peaks. Occasionally we have found polymeric material clearly banding below the meniscus but outside the range covered by natural DNAs so far known. We did not prove it to be a polysaccharide but we are sure that it was neither a protein nor a nucleic acid, since the band was rather sharp and failed to exhibit the typical UV-spectra of proteins or nucleic acids. In contrast, it seemed to scatter light rather than to absorb it and, after braking the rotor, formed a fragile 'membrane' of opalescent material. From our own experience, we can tabulate the following advantages of the method described. (1) One needs only minor amounts of sample material. About I to 2 x 10s cells are sufficient to evaluate the base composition of their DNA. This is of particular value when poorly growing organisms, e.g., obligately chemolithotrophic bacteria, or clones directly taken from an agar plate have to be characterized. (2) It is rapid and simple. It does not need highly purified DNA nor, in most cases, any purification at all. The limiting factor is the capacity of the ultracentrifuge, but even here, five samples may be run at once. The accuracy is the same as would be achieved with pure DNA. (3) Since there are no separation steps involved, the method is automatically a rather sensitive test for the homogeneity of the DNA. Plasmids and, if we would deal with higher organisms, mitochondrial DNA can often be detected. Infections become instantly apparent. However, it should be emphasized that the numerical precision of any isopycnic technique is limited. Nominally, the ultracentrifuge technique yields highly reproducible results, the standard deviation of multiple estimations of AQ against the same marker is usually found to be very low. That would mean that the relative error of both Ar and -i is quite small. Furthermore, the hydrostatic pressure inside the ultracentrifuge cell may be regarded to be constant,, at least as long as we consider only its influence on the salt gradient but exclude its influence on the buoyant density of the DNA. The same holds for the value of 13. However, from this it cannot be concluded that the final meaning of A(G + C), as the actual difference of base composition between marker DNA and probe DNA, is as well defined as the small standard deviation seems to suggest. The main reason for this is the conversion of A o to A(G + (7). As can easily be seen from the literature [18], d(A(G + C))/d(o ) = 1038 is inaccurate to about 4%. This may affect the
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calculated value of A(G + C) at least to the same extent. Therefore, even if we restrict ourselves to those cases where the base composition of the marker D N A differs from that of the sample D N A by no more than 20%, the absolute value of the (G + C)-content of the sample D N A may be as inaccurate as _+1%. This should be kept in mind when buoyant densities or (G + C)-contents are compared with data cited in the literature. We feel that a discrepancy not higher than A(A(G + C)) = 2% is acceptable. This reasoning, of course, not only holds for the method presented here but for any procedure that basically relies on the buoyant density in a CsCl-gradient. In the same sense, and to the same extent, it also holds for the evaluation of A(G + C) from the melting temperature.
Acknowledgement I am indebted to H. Schillig and D. Hanisch for their skillful assistance during the work.
References 1
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