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Molecular weights of coliphages and coliphage DNA
J. Mol. Biol. (1970) 54, 557-565
Molecular Weights of Coliphages and Coliphage DNA III. Contour Length and Molecular Weight of DNA from Bacteriophages T4, T5 and T7, and from Bovine Papilloma Virus D. LANG Division of Biology The University of Texas at Dallas P. 0. Box 30365, Dallas, Texm 75230, U.S.A. (Received 23 March 1970) DNA has been prepared for electron microscopy at ionic strength 0.20. The lengths of DNA molecules from the following viruses were found to be: bovine papilloma virus, (2.49 _+0.05)~ (circular DNA); T7, (12.15 4 0.25)~; T5, (30.2 + 0.8)~; and T4, (52.0 + 1.0)~. The limits given are estimated systematic errors of unknown sign plus sample standard deviations. In units of T7 DNA, the relative lengths and their maximum errors are : bovine papilloma virus, 0.206 f 0.003; T5, 2.98 If: 0.06; and T4, 4.28 + 0.06; these values should equal relative molecular weights except for T4 DNA (4.71). Based on the mean value of 25.1 x IO6 daltons, obtained from three recent and independent determinations of T7 DNA molecular weight, the molar linear density of duplex DNA as prepared by standardized electron microscopy was found to be 2.07 x lOlo for DNA with common bases, 2.28 x lOlo for T4 DNA, and 2.23 x lOlo daltons/cm for T2 DNA. Consequently, molecular weights of the following viral DNA’s are: bovine papilloma virus, 5.15 x 108; T5, 74.9 x 108; T4, 119 x 106; and T2, 116 x lo6 daltons.
1. Introduction The determination of molecular weight of viral DNA by electron microscopy employs the relationship between apparent contour length, L, and molecular weight, M :
M = M’L (daltons) The molar linear density, M’ (dalton/cm), is calculated either according to the Watson-Crick B configuration of double-stranded DNA, assuming that this configuration prevails during preparation for electron microscopy, or, M’ is obtained by calibration with DNA of independently determined molecular weight. In both cases, the accuracy of a molecular-weight determination by electron microscopy is limited by errors not only of length measurements, but also of values for M’ or for the molecular weight of the calibrating DNA. This paper is part of such a calibration by known DNA’s, Recently, Bancroft & Freifelder (1970) and Dubin, Benedek, Bancroft & Freifelder (1970) made absolute determinations of molecular weights of DNA from Escherichia coli bacteriophages T4, T5 and T7. Freifelder kindly sent a sample of each original stock solution of purified phage for DNA extraction and length measurements. This was done in this laboratory with a mixture of all three phages, and also separately. The former procedure obviously permitted more precise determinations of length ratios and hence 557
558
D. LANCJ
molecular-weight ratios, since systematic magnification errors cancelled when the three DNA species were prepared simultaneously on the same grid. Addition of circular bovine papilloma virus DNA widened the range of molecular weights under study. Grids for electron microscopy were prepared by the diffusion method of Lang, Kleinschmidt & Zahn (1964) as modified by Lang, Bujard, Wolff & Russell (1967). The resulting lengths of the three DNA species will be given in absolute units and also in terms of their proportions, followed by calculation of molecular-weight ratios and an error analysis. The molar linear density of duplex DNA, prepared for electron microscopy, will be evaluated using recently published molecular weights of T’7 DNA.
2. Materials and Methods (a) Virus Bacteriophages T4, T5 and T7 were a gift from D. Freifelder. The stock solutions, each 0.3 ml., were 0.01 M-Tris buffer, 0.1 M-NaCl, 0.01 M-MgSo4 and O*OOl M-CaCle, pH 7, containing phages with optical densities (260 nm, 1 cm path) of 12 for T4 and T5, and 36 for T7. Bancroft & Freifelder (1970) and Dubin et al. (1970) used phages from the same stock solution for independent DNA molecular-weight measurements with the following results: T4, 106 x lo6 daltons; T5, 67.3 x IO6 daltons; and T7, 25.5 x lo6 daltons. (b) DNA First, the three DNA species were released simultaneously from the phages by mixing 0.10 ml. T4,0*05 ml. T5 and 0.01 ml. T7 of the above stock solutions and then adding 1.44 ml. 5 M-NaClO,, pH 7.8 (Freifelder, 1965). Using this solution, one preparation for electron microscopy was made at once and a second one 24 hr later. Second, each DNA species was released separately by adding 0.01 ml. stock solution to 0.1 ml. 5 M-NaClo+ Bovine papilloma virus DNA extracted by Bujard (1967) and T3 DNA were characterized earlier (Lang et al., 1967; Bujard, 1968). (c) Electron
microscopy
The diffusion method was applied as described by Lang et al. (1967). To 0.05 ml. of the above DNA mixture, or to each 0.11 ml. of the separately released DNA, were added 30 ml. 0.20 M-ammonium acetate, 10e3 M-EDTA, pH 6.5. The solutions were poured into Teflon-coated dishes (29 ml., see Lang & Mitani, 1970), and left there 20 min for temperature equilibration. After this, the surface was cleaned with a Teflon-coated bar and sprinkled with a few talc particles. Then cytochrome c powder (Nutritional Biochemicals Corp.) was spread from a glass needle. After a diffusion time of 15 min, portions of the surface film with adsorbed DNA were transferred to ‘I-hole Siemens-type grids, dried with ethanol, and shadowed with platinum. Carbon films served as specimen supports on the grids. Micrographs were taken on 6.5 cm x 9 cm Kodak Electron Image Plates with a Siemens Elmiskop 1A at nominal magnifications of 5000 and 10,000, after lens currents and high tension had been switched on for at least 30 min. In order to reduce that part of the magnification error which arises from grid to grid by differences in specimen position with respect to the objective lens and which amounts to + 0.8% sample S.D. (Lang et al., 1967), images of molecules were focussed by adjusting the vertical specimen position without changing the objective-lens current. For each set of 12 plates, the magnification was determined by micrographing a selected area of a carbon replica made from a cross-lined optical grating with 54,800 lines/inch. The replica was mounted on Siemens-type grids by E. F. Fullam, Inc. During the 16 days when micrographs were made, the sample S.D. of 9 independent magnification measurements wss +0.4% which reflects an excellent stability of the equipment. The plates, including the ones with the grating image, were optically enlarged 23.5 times by projection. The images were traced on paper and measured with a Minerva curvimeter (map measurer). For each molecule the length was corrected for pincushion distortion as described earlier (Lang et al., 1967); on the average, the correction was 0.5%.
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
559
3. Results The length distribution of the two mixed-DNA preparations, Figure l(a), shows three major peaks, corresponding to T7-, T5- and T4 DNA in the order of increasing length. The peak at 2.5 p is produced by added bovine papilloma virus DNA. Discrimination between intact linear molecules and fragments is not possible without arbitrariness. Suitably, one may define a peak region as the abscissa value under the peak, plus and minus three times the sample s.D., and one may then assume that all molecules within peak regions are intact. Since the sample S.D. of the length of intact viral DNA is usually &4:/, or less, a peak region would then be approximately the mean length f 12%. In this way one obtains 31% fragments by number and 19% by weight from the distribution in Figure l(a). No significant differences between corresponding mean lengths of simultaneously and separately prepared DNA species were found. Therefore, the measurements of Figure l(a) were combined with the results of the separate determinations, which were -
Length
(p)
FIG. 1. (a) Length spectrum of simultaneously prepared DNA from bovine papilloms, T7, T5, and T4 viruses, showing four major frequency peaks of full-length molecules and interspersed fragments. Length intervals are 05 p. (b) Length spectrum obtained from the values of (8) p&a the lengths from the separately prepared DNA’s. Such pooling is justified since there were no signXcant differences in lengths or accuracy. Fragments between peaks have been omitted (see text). Length intervals are 0.05 p for the 2.49 p peak and 0.5 ~1elsewhere.
T7 T6 T4
BPVt
from
DNA
151 100 61 86
Number of molecules 2.49 12.16 36.2 62-O
(P)
L &
(I4 0.08 0.40 1.3 1.3
fhmpl0
S.D.
TABLE 1
o-01 0.04 O-18 0.14
(P)
of the mean +
(s.D.
t BPV, bovine papilloma, virus.
+S.D.
Result of length meawrements
0.06 O-25 0.8 1.0
Total error of the mean+ (4
1.7%)
O-206 *0*003 1.00 2*98f0*06 4.28 &O-O6
Relative length (in units of T7 DNA)
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
561
made twice for T4- and T6 DNA and once for T7- and bovine papilloma virus DNA. The combined length spectrum, showing peak regions only, is given in Figure l(b). The mean lengths of the four DNA species are listed in Table 1, together with the experimental sample S.D. and S.D. of the mean. The sample S.D. of about 53% characterizes the precision of the method used. The S.D. of the mean of about *O-2% shows into what range the effect of random errors has been confined by measuring more than one molecule. Systematic errors, not included in these deviations, are estimated in Table 2. TABLE 2 Systematic
errors of length measurements Error (%I
1. Grating constant of oross-lined carbon replica 2. Tracing of DNA molecules on paper 3. Measurement of trace with ourvimeter Arithmetic sum Geometric sum
1.3 0.8 0.8 2.9 1.7
Tho lines of the grating replica (length standard) could not be resolved by visible light. Therefore, two identifiable particles on the replica, found by light microscopy to be 7.49 p apart, were micrographed by electron microscopy at a magnification of !%OO.By measuring the distances between the then visible grating lines, a grating constant of (5*41+0*05) IO* lines/inch ( ~s.D. of the mean) was found, 1.3% less than the manufacturer’s value of 5.4800 x lo4 lines/inch. Hence, &1+3% was listed in Table 2 as possible error. The next systematic error in Table 2 is an interpretation error which depends on the quality of the preparation and on the degree to which the DNA is one-dimensionally wrinkled. This error is usually &08% or less, as well a,s the last one, which is a personal error. Adding up: the maximum systematic error is *2*9%. But the signs of the component errors are unknown, and a more likely value is the geometric sum * 1.7%. The total error of lengths may then be the S.D. of the mean plus 1*7q/,: as given in column 6 of Table 1. With regard to the length ratios in column 7, systematic errors cancel each other. The remaining random errors, obtained by geometric addition of standard deviations of the two means involved, were multiplied by three in order t’o find the maximum error (column 7). Hence, the true value of a length ratio is most likely within the limits indicated in Table 1. Another way of estimating the accuracy is to look at the history of independent, length determinations, as shown in Figure 2. Measurements of T3 DNA from the sa,me stock solution (Fig. 2, squares), and more recently of T7 DNA (circles), were done in this laboratory by several workers over a period of almost four years using the same electron microscope but different length standards. There is no trend with time and, as expected, the lengths of T3- and T7 DNA are equal. This T7 DNA was phenolextracted from a phage strain given by B. Gomez and originally obtained from M. S. Meselson. The mean value of all determinations, 12.20 50.40 p sample S.D. 40.07 p S.D. of the mean, agrees wit’h the length of T7 DNA in Table 1 (12.15 cl). 37
562
D. LANG
-l----’
I--
--
1
14. m- =H
3
2F IO 5
m$
I966
1967
I
.
.
l;
.
12-.L
l
1968
I
1969
FIG. 2. History of electron microscopic length determinations, made in this laboratory by the diffusion method at 0.2 ionic strength, for T3 DNA ( n ) snd T7 DNA (0). Both have probably equal length. The symbol (0) refers to 0.15 ionic strength and presence of formaldehyde (Lang & Mitani, 1970). The values scatter randomly with respect to the horizontal line representing the mean length of 12.20 p.
4. Discussion Figure 3 shows a survey of length measurements. It includes this and other work end also data on related viruses which are believed to have similar DNA lengths. Since the apparent DNA length depends on the ionic strength of the solutions from
Yuman paptllomo
a
*
Shape popillomo .
T3 f 9 h-
a -
I--
b
!=I c BovineQapillom~ d .m. e e lm Lti
T2 P Ir s
*
T7 1i J,U k I em m 9
r.5 st (0) I d-
&
T5 nd+ -.-
T4 t u cc
*
0
4 * 6-k-l
c-c
*
t
w
1
Length Q..d FIG. 3. Comparison of DNA lengths found by myself (values at the bottom) and by others for the viruses indicated and at the following ionic strengths: < 0.1, (A); 0.1 to (0.2, ( n ) ; and 20.2, (a). In each box, earlier determinations are entered above later ones. Errors are sample S.D.‘B and letters stand for references. LX: Crawford, Follett & Crawford, 1966; b: Kleinschmidt, Kass, Williams t Knight, 1965; c: Kellenberger, 1965; d: Lang et al., 1967; e: Bujard, 1970; f: Bendet, Schachter & Lauffer, 1962; g: Fig. 2; h: Lang 8EMitani, 1970; i: Inman, Schildkraut & Kornberg, 1965; j: Freifelder & Kleinschmidt, 1965; k: Bode & Morowitz, 1967; 1: Abelson&Thomas, 1966; m: Misre, Sinha & DasGupta, 1969; n: Frank, Zernitz & Weidel, 1963; o: Bujard, 1969; p: Cairns, 1961; q: Kleinschmidt, Lang, Jacherts & Z&n, 1962; r: Thomas & MaHattie, 1964; 8: nongluoosylated T2 DNA, Thomas, 1966; t: Chandler, 1963; u: Kleinsohmidt, 1967.
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
563
which DNA is adsorbed to the cytochrome c interface (Lang et al., 1967; Inman, 1967), different symbols are used in Figure 3 for different ionic strengths. The indicated errors are sample standard deviations. As can be seen, agreement and precision are generally best for results obtained at the higher ionic strengths, indicating that the diffusion method at ionic strength O-2 was a suitable choice for standardized length determination of DNA. As pointed out in the Introduction, utilization of length measurements for determining molecular weights requires knowledge of M’, the molar linear density of DNA in a situation corresponding to the one in which it was measured. One cannot expect that the B configuration applies necessarily to DNA prepared on grids. Conditions and steps are involved all of which may distort molecular configuration: ionic strength of the DNA solution, binding to cytochrome c and adeorption to the interface, mechanical instability of the protein film, adsorption to specimen grids, drying in ethanol, shrinkage or expansion of specimen supports in the electron beam. Indeed, DNA with two nicks on opposite strands may break, even if the nicks are 20 nucleotide pairs apart (Lang & Mitani, 1970), and lengths between 8 and 22 p of otherwise intact T3 DNA can be brought about willfully under extreme conditions. However, with the standardized method used here, results are reproducible within a few per cent, and earlier indirect evidence suggested that M’ is not more than 15% above the value calculated for the DNA B configuration (Lang & Coates, 1968). For the B configuration with 3.46 A length per pair of complementary nucleotides having the common bases adenine, thymine, guanine and cytosine (Langridge, TABLE 3 d comparison
molecular weights of NaDNA and of molar linear densities
of measured
S&mid & Hearst,? 1989
MT, (10s daltons) MT6 MT4 MT2
24.8 68.5 113.5 -
MT&G, MTP~TT MmIM,, -QhpfTI Mm/&e
2.76 4.58 -
M;, (1 O’s daltons/cm) MT5 MT4 II 62 II
2.04
Mean value of M T
1.97
1.66 -
1.89 1.98
-
Leighton Rubenstein,
(M), of their ratios
Bancroft & Freifelder, 1970 ; & 1969 Dubin et aZ., 1970
25 83 132
25.5 67.3 106 -
This paper$
-
3-32 -
2.63 4.14
2.98 4.71
5.28 -
1.59
1.58 -
(4-62)s 1.58 Cl.5519
2.06 2.29 -
2.10 1.86 1.85
-
WWO 2.23
1.94
t Molecular weights by Schmid & Hearst are increased by 7.4% (Freifelder, personal communication). $ From length ratio. 3 Values in parentheses were calculated assuming that T4- and T2 DNA have equal length. 11For comparison, reduced to non-glucosylated oytosine.
564
D. LANG
Wilson, Hooper, Wilkins & Hamilton, 1960), one obtains 1.913 x lOlo dctltons/cm for N&DNA using International Atomic Weights of 1966 based on carbon isotope lzC. The molecular weights of the two possible nucleotide pairs differ by only 0.3% ; M’ should then be practically independent of base composition. In T4- and T2 DNA, supposedly without effect on configuration (Thomas, 1966), cytosine is replaced by hydroxymethylcytosine (Wyatt & Cohen, 1953), which is 100% glucosylated in T4 and 765% in T2 (Sinsheimer, 1960). M’ for these DNA’s, again in B configuration, is therefore 2,104 x lOlo (T4 DNA) and 2.064 x lOlo daltons/cm (T2 DNA). The numbers given in this paragraph are based on the assumption that the dimensions of the B configuration do not depend on base composition. The alternative is discussed by Freifelder (1970). A comparison of measured molecular weights of sodium salts of DNA (M), of their ratios, and of molar linear densities, is giveninTable 3. Listedare most recent M-values from three laboratories using different methods: S&mid & Hearst (1969), CsCl density-gradient equilibrium centrifugation; Leighton & Rubenstein (1969), autoradiography of single 32P-labeled DNA molecules: Banoroft & Freifelder (1970), Dubin et al. (1970), high-speed equilibrium centrifugation, sedimentation-diffusion measurements, and phosphorus-nitrogen determinations. The last column shows molecular weight ratios calculated from length ratios (Table 1) using M’-values of the preceding paragraph. Good agreement exists for MT,, but not for M,, and MT*, M,,. For the M ratios, good agreement exists only for ratios not containing MT,. The following reasoning might then be adopted. Since three recent and independent determinations of M,, are agreeing well, and since there is no indication that the T7 DNA’s used might be different, the mean molecular weight of (25.1 &O-4) x lo6 daltons (Table 3) is considered to be correct. This leads to rtn apparent molar linear density of M&=(25.1 &0*4)x 10s/[(1*215& 0.025) x low31 = (2.07 kO.04) x lOlo daltons/cm. Assuming independence of M’ on base composition this value should apply to other duplex DNA’s when prepared for electron microscopy by the diffusion method from 0.2 ionic strength as described, or by a similar new method reported by Lang & Mitani (1970). From the lengths in Table 1, and using Mk4 = (2.07 x 2*104/1.913) x lOlo = 2.28 x 1Oroand M& = (2.07 x 2.064/l-913) x lOlO= 2.23 x lOlo daltonslcm, one may thus calculate molecular weights as shown in Table 4. TABLE 4
Molecular weights calculated from length measurements and M,, = 25.1 x lo6 daltons DNA from:
BPVf
T6
T4
T2
(108 dZons)
5.15
74.9
119
116
t BPV, bovine papilloma virus. This paper was prepared with the technical assistance of Mr L. Lewis, Jr. and Miss B. Bruton and I would like to thank them for their help. I also thank Dr D. Freifelder who sent us bacteriophage T4, T5 and T7; also Drs H. Bujard and B. Gomez who provided
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
566
OF DNA
bovine papilloma virus DNA and another strain of T7 bacteriophage. This work was supported by National Science Foundation research grant GB0837 and by U.S. Public Health Service career development award GM-34,964 and divisional grant GM-13,234. REFERENCES Abelson, 5. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Bancroft, F. C. & Freifelder, D. (1970). J. Mol. Biol. 54, 537. Bendet, I., Schachter, E. & Lauffer, M. A. (1962). J. Mol. BioZ. 5, 76. Bode, H. R. & Morowitz, H. J. (1967). J. Mol. BioZ. 23, 191. Bujard, H. (1967). J. Virology, 1, 1135. Bujard, H. (1968). J. Mol. BioZ. 33, 503. Bujard, H. (1969). Proc. Nat. Acad. Sci., Wu&. 62, 1167. Bujard, H. (1970). J. Mol. BioZ. 49, 125. Cairns, J. (1961). J. Mol. BioZ. 3, 756. Chandler, B. (1963). M. SC. Thesis, University of Wisconsin. Crawford, L. V., Follett, E. A. C. & Crawford, E. M. (1966). J. Microscopic, Dubin, S. B., Benedek, G. B., Bancroft, F. C. & Freifelder, D. (1970). J. Mol. Frank, H., Zarnitz, M. L. & Weidel, W. (1963). 2. Naturf. 18b, 281. Freifelder, D. (1965). Biochem. Biophys. Res. Comm. 18, 141. Freifelder, D. (1970). J. Mol. BioZ. 54, 567. Freifelder, D. & Kleinschmidt, A. K. (1965). J. Mol. Biol. 14, 271. Inman, R. B. (1967). J. Mol. BioZ. 25, 239. Inman, R. B., Schildkraut, C. L. & Kornberg, A. (1965). J. Mol. BioZ. 11, Kellenberger, E. (1965). Path. Microbial. 28, 540. Kleinschmidt, A. K. (1967). Nuturwks, 54, 417. Kleinschmidt, A. K., Kass, S. J., Williams, R. C. C Knight, C. A. (1965). J.
5, 597. BioZ. 54,547.
285.
MOE. BioZ. 13,
749.
Kleinschmidt, Acta,
A. K., Lang, D., Jacherts,
D. & Zahn, R. K. (1962). Biochim.
biophy.9.
61, 857.
Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. BioZ. 23, 163. Lang, D. & Coates, P. (1968). J. Mol. BioZ. 36, 1968. Lang, D., Kleinschmidt, A. K. BEZahn, R. K. (1964). Biochim. biophye. Acta, 88, 142. Lang, D. & Mitani, M. (1970). Biopolymers, 9, 373. Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. MoZ. BioZ. 2, 19. Leighton, S. B. & Rubenstein, I. (1969). J. Mol. BioZ. 46, 313. Misra, D. N., Sinha, R. K. & DasGupta, N. N. (1969). FiroZogy, 39, 183. Schmid, C. W. & Hearst, J. E. (1969). J. MoZ. BioZ. 44, 143. Sinsheimer, R. L. (1960). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, vol. 3, p. 187, New York and London: Academic Press. Thomas, C. A., Jr. (1966). J. Gem. Phytiol. 49, No. 6, Part 2, 143. Thomas, C. A., Jr. & MaoHattie, L. A. (1964). Proc. Nat. AC&. Sci., Wash. 52, 1297. Wyatt, G. R. & Cohen, S. S. (1953). Biochem. J. 55, 774.
Molecular Weights of Coliphages and Coliphage DNA III. Contour Length and Molecular Weight of DNA from Bacteriophages T4, T5 and T7, and from Bovine Papilloma Virus D. LANG Division of Biology The University of Texas at Dallas P. 0. Box 30365, Dallas, Texm 75230, U.S.A. (Received 23 March 1970) DNA has been prepared for electron microscopy at ionic strength 0.20. The lengths of DNA molecules from the following viruses were found to be: bovine papilloma virus, (2.49 _+0.05)~ (circular DNA); T7, (12.15 4 0.25)~; T5, (30.2 + 0.8)~; and T4, (52.0 + 1.0)~. The limits given are estimated systematic errors of unknown sign plus sample standard deviations. In units of T7 DNA, the relative lengths and their maximum errors are : bovine papilloma virus, 0.206 f 0.003; T5, 2.98 If: 0.06; and T4, 4.28 + 0.06; these values should equal relative molecular weights except for T4 DNA (4.71). Based on the mean value of 25.1 x IO6 daltons, obtained from three recent and independent determinations of T7 DNA molecular weight, the molar linear density of duplex DNA as prepared by standardized electron microscopy was found to be 2.07 x lOlo for DNA with common bases, 2.28 x lOlo for T4 DNA, and 2.23 x lOlo daltons/cm for T2 DNA. Consequently, molecular weights of the following viral DNA’s are: bovine papilloma virus, 5.15 x 108; T5, 74.9 x 108; T4, 119 x 106; and T2, 116 x lo6 daltons.
1. Introduction The determination of molecular weight of viral DNA by electron microscopy employs the relationship between apparent contour length, L, and molecular weight, M :
M = M’L (daltons) The molar linear density, M’ (dalton/cm), is calculated either according to the Watson-Crick B configuration of double-stranded DNA, assuming that this configuration prevails during preparation for electron microscopy, or, M’ is obtained by calibration with DNA of independently determined molecular weight. In both cases, the accuracy of a molecular-weight determination by electron microscopy is limited by errors not only of length measurements, but also of values for M’ or for the molecular weight of the calibrating DNA. This paper is part of such a calibration by known DNA’s, Recently, Bancroft & Freifelder (1970) and Dubin, Benedek, Bancroft & Freifelder (1970) made absolute determinations of molecular weights of DNA from Escherichia coli bacteriophages T4, T5 and T7. Freifelder kindly sent a sample of each original stock solution of purified phage for DNA extraction and length measurements. This was done in this laboratory with a mixture of all three phages, and also separately. The former procedure obviously permitted more precise determinations of length ratios and hence 557
558
D. LANCJ
molecular-weight ratios, since systematic magnification errors cancelled when the three DNA species were prepared simultaneously on the same grid. Addition of circular bovine papilloma virus DNA widened the range of molecular weights under study. Grids for electron microscopy were prepared by the diffusion method of Lang, Kleinschmidt & Zahn (1964) as modified by Lang, Bujard, Wolff & Russell (1967). The resulting lengths of the three DNA species will be given in absolute units and also in terms of their proportions, followed by calculation of molecular-weight ratios and an error analysis. The molar linear density of duplex DNA, prepared for electron microscopy, will be evaluated using recently published molecular weights of T’7 DNA.
2. Materials and Methods (a) Virus Bacteriophages T4, T5 and T7 were a gift from D. Freifelder. The stock solutions, each 0.3 ml., were 0.01 M-Tris buffer, 0.1 M-NaCl, 0.01 M-MgSo4 and O*OOl M-CaCle, pH 7, containing phages with optical densities (260 nm, 1 cm path) of 12 for T4 and T5, and 36 for T7. Bancroft & Freifelder (1970) and Dubin et al. (1970) used phages from the same stock solution for independent DNA molecular-weight measurements with the following results: T4, 106 x lo6 daltons; T5, 67.3 x IO6 daltons; and T7, 25.5 x lo6 daltons. (b) DNA First, the three DNA species were released simultaneously from the phages by mixing 0.10 ml. T4,0*05 ml. T5 and 0.01 ml. T7 of the above stock solutions and then adding 1.44 ml. 5 M-NaClO,, pH 7.8 (Freifelder, 1965). Using this solution, one preparation for electron microscopy was made at once and a second one 24 hr later. Second, each DNA species was released separately by adding 0.01 ml. stock solution to 0.1 ml. 5 M-NaClo+ Bovine papilloma virus DNA extracted by Bujard (1967) and T3 DNA were characterized earlier (Lang et al., 1967; Bujard, 1968). (c) Electron
microscopy
The diffusion method was applied as described by Lang et al. (1967). To 0.05 ml. of the above DNA mixture, or to each 0.11 ml. of the separately released DNA, were added 30 ml. 0.20 M-ammonium acetate, 10e3 M-EDTA, pH 6.5. The solutions were poured into Teflon-coated dishes (29 ml., see Lang & Mitani, 1970), and left there 20 min for temperature equilibration. After this, the surface was cleaned with a Teflon-coated bar and sprinkled with a few talc particles. Then cytochrome c powder (Nutritional Biochemicals Corp.) was spread from a glass needle. After a diffusion time of 15 min, portions of the surface film with adsorbed DNA were transferred to ‘I-hole Siemens-type grids, dried with ethanol, and shadowed with platinum. Carbon films served as specimen supports on the grids. Micrographs were taken on 6.5 cm x 9 cm Kodak Electron Image Plates with a Siemens Elmiskop 1A at nominal magnifications of 5000 and 10,000, after lens currents and high tension had been switched on for at least 30 min. In order to reduce that part of the magnification error which arises from grid to grid by differences in specimen position with respect to the objective lens and which amounts to + 0.8% sample S.D. (Lang et al., 1967), images of molecules were focussed by adjusting the vertical specimen position without changing the objective-lens current. For each set of 12 plates, the magnification was determined by micrographing a selected area of a carbon replica made from a cross-lined optical grating with 54,800 lines/inch. The replica was mounted on Siemens-type grids by E. F. Fullam, Inc. During the 16 days when micrographs were made, the sample S.D. of 9 independent magnification measurements wss +0.4% which reflects an excellent stability of the equipment. The plates, including the ones with the grating image, were optically enlarged 23.5 times by projection. The images were traced on paper and measured with a Minerva curvimeter (map measurer). For each molecule the length was corrected for pincushion distortion as described earlier (Lang et al., 1967); on the average, the correction was 0.5%.
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
559
3. Results The length distribution of the two mixed-DNA preparations, Figure l(a), shows three major peaks, corresponding to T7-, T5- and T4 DNA in the order of increasing length. The peak at 2.5 p is produced by added bovine papilloma virus DNA. Discrimination between intact linear molecules and fragments is not possible without arbitrariness. Suitably, one may define a peak region as the abscissa value under the peak, plus and minus three times the sample s.D., and one may then assume that all molecules within peak regions are intact. Since the sample S.D. of the length of intact viral DNA is usually &4:/, or less, a peak region would then be approximately the mean length f 12%. In this way one obtains 31% fragments by number and 19% by weight from the distribution in Figure l(a). No significant differences between corresponding mean lengths of simultaneously and separately prepared DNA species were found. Therefore, the measurements of Figure l(a) were combined with the results of the separate determinations, which were -
Length
(p)
FIG. 1. (a) Length spectrum of simultaneously prepared DNA from bovine papilloms, T7, T5, and T4 viruses, showing four major frequency peaks of full-length molecules and interspersed fragments. Length intervals are 05 p. (b) Length spectrum obtained from the values of (8) p&a the lengths from the separately prepared DNA’s. Such pooling is justified since there were no signXcant differences in lengths or accuracy. Fragments between peaks have been omitted (see text). Length intervals are 0.05 p for the 2.49 p peak and 0.5 ~1elsewhere.
T7 T6 T4
BPVt
from
DNA
151 100 61 86
Number of molecules 2.49 12.16 36.2 62-O
(P)
L &
(I4 0.08 0.40 1.3 1.3
fhmpl0
S.D.
TABLE 1
o-01 0.04 O-18 0.14
(P)
of the mean +
(s.D.
t BPV, bovine papilloma, virus.
+S.D.
Result of length meawrements
0.06 O-25 0.8 1.0
Total error of the mean+ (4
1.7%)
O-206 *0*003 1.00 2*98f0*06 4.28 &O-O6
Relative length (in units of T7 DNA)
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
561
made twice for T4- and T6 DNA and once for T7- and bovine papilloma virus DNA. The combined length spectrum, showing peak regions only, is given in Figure l(b). The mean lengths of the four DNA species are listed in Table 1, together with the experimental sample S.D. and S.D. of the mean. The sample S.D. of about 53% characterizes the precision of the method used. The S.D. of the mean of about *O-2% shows into what range the effect of random errors has been confined by measuring more than one molecule. Systematic errors, not included in these deviations, are estimated in Table 2. TABLE 2 Systematic
errors of length measurements Error (%I
1. Grating constant of oross-lined carbon replica 2. Tracing of DNA molecules on paper 3. Measurement of trace with ourvimeter Arithmetic sum Geometric sum
1.3 0.8 0.8 2.9 1.7
Tho lines of the grating replica (length standard) could not be resolved by visible light. Therefore, two identifiable particles on the replica, found by light microscopy to be 7.49 p apart, were micrographed by electron microscopy at a magnification of !%OO.By measuring the distances between the then visible grating lines, a grating constant of (5*41+0*05) IO* lines/inch ( ~s.D. of the mean) was found, 1.3% less than the manufacturer’s value of 5.4800 x lo4 lines/inch. Hence, &1+3% was listed in Table 2 as possible error. The next systematic error in Table 2 is an interpretation error which depends on the quality of the preparation and on the degree to which the DNA is one-dimensionally wrinkled. This error is usually &08% or less, as well a,s the last one, which is a personal error. Adding up: the maximum systematic error is *2*9%. But the signs of the component errors are unknown, and a more likely value is the geometric sum * 1.7%. The total error of lengths may then be the S.D. of the mean plus 1*7q/,: as given in column 6 of Table 1. With regard to the length ratios in column 7, systematic errors cancel each other. The remaining random errors, obtained by geometric addition of standard deviations of the two means involved, were multiplied by three in order t’o find the maximum error (column 7). Hence, the true value of a length ratio is most likely within the limits indicated in Table 1. Another way of estimating the accuracy is to look at the history of independent, length determinations, as shown in Figure 2. Measurements of T3 DNA from the sa,me stock solution (Fig. 2, squares), and more recently of T7 DNA (circles), were done in this laboratory by several workers over a period of almost four years using the same electron microscope but different length standards. There is no trend with time and, as expected, the lengths of T3- and T7 DNA are equal. This T7 DNA was phenolextracted from a phage strain given by B. Gomez and originally obtained from M. S. Meselson. The mean value of all determinations, 12.20 50.40 p sample S.D. 40.07 p S.D. of the mean, agrees wit’h the length of T7 DNA in Table 1 (12.15 cl). 37
562
D. LANG
-l----’
I--
--
1
14. m- =H
3
2F IO 5
m$
I966
1967
I
.
.
l;
.
12-.L
l
1968
I
1969
FIG. 2. History of electron microscopic length determinations, made in this laboratory by the diffusion method at 0.2 ionic strength, for T3 DNA ( n ) snd T7 DNA (0). Both have probably equal length. The symbol (0) refers to 0.15 ionic strength and presence of formaldehyde (Lang & Mitani, 1970). The values scatter randomly with respect to the horizontal line representing the mean length of 12.20 p.
4. Discussion Figure 3 shows a survey of length measurements. It includes this and other work end also data on related viruses which are believed to have similar DNA lengths. Since the apparent DNA length depends on the ionic strength of the solutions from
Yuman paptllomo
a
*
Shape popillomo .
T3 f 9 h-
a -
I--
b
!=I c BovineQapillom~ d .m. e e lm Lti
T2 P Ir s
*
T7 1i J,U k I em m 9
r.5 st (0) I d-
&
T5 nd+ -.-
T4 t u cc
*
0
4 * 6-k-l
c-c
*
t
w
1
Length Q..d FIG. 3. Comparison of DNA lengths found by myself (values at the bottom) and by others for the viruses indicated and at the following ionic strengths: < 0.1, (A); 0.1 to (0.2, ( n ) ; and 20.2, (a). In each box, earlier determinations are entered above later ones. Errors are sample S.D.‘B and letters stand for references. LX: Crawford, Follett & Crawford, 1966; b: Kleinschmidt, Kass, Williams t Knight, 1965; c: Kellenberger, 1965; d: Lang et al., 1967; e: Bujard, 1970; f: Bendet, Schachter & Lauffer, 1962; g: Fig. 2; h: Lang 8EMitani, 1970; i: Inman, Schildkraut & Kornberg, 1965; j: Freifelder & Kleinschmidt, 1965; k: Bode & Morowitz, 1967; 1: Abelson&Thomas, 1966; m: Misre, Sinha & DasGupta, 1969; n: Frank, Zernitz & Weidel, 1963; o: Bujard, 1969; p: Cairns, 1961; q: Kleinschmidt, Lang, Jacherts & Z&n, 1962; r: Thomas & MaHattie, 1964; 8: nongluoosylated T2 DNA, Thomas, 1966; t: Chandler, 1963; u: Kleinsohmidt, 1967.
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
OF DNA
563
which DNA is adsorbed to the cytochrome c interface (Lang et al., 1967; Inman, 1967), different symbols are used in Figure 3 for different ionic strengths. The indicated errors are sample standard deviations. As can be seen, agreement and precision are generally best for results obtained at the higher ionic strengths, indicating that the diffusion method at ionic strength O-2 was a suitable choice for standardized length determination of DNA. As pointed out in the Introduction, utilization of length measurements for determining molecular weights requires knowledge of M’, the molar linear density of DNA in a situation corresponding to the one in which it was measured. One cannot expect that the B configuration applies necessarily to DNA prepared on grids. Conditions and steps are involved all of which may distort molecular configuration: ionic strength of the DNA solution, binding to cytochrome c and adeorption to the interface, mechanical instability of the protein film, adsorption to specimen grids, drying in ethanol, shrinkage or expansion of specimen supports in the electron beam. Indeed, DNA with two nicks on opposite strands may break, even if the nicks are 20 nucleotide pairs apart (Lang & Mitani, 1970), and lengths between 8 and 22 p of otherwise intact T3 DNA can be brought about willfully under extreme conditions. However, with the standardized method used here, results are reproducible within a few per cent, and earlier indirect evidence suggested that M’ is not more than 15% above the value calculated for the DNA B configuration (Lang & Coates, 1968). For the B configuration with 3.46 A length per pair of complementary nucleotides having the common bases adenine, thymine, guanine and cytosine (Langridge, TABLE 3 d comparison
molecular weights of NaDNA and of molar linear densities
of measured
S&mid & Hearst,? 1989
MT, (10s daltons) MT6 MT4 MT2
24.8 68.5 113.5 -
MT&G, MTP~TT MmIM,, -QhpfTI Mm/&e
2.76 4.58 -
M;, (1 O’s daltons/cm) MT5 MT4 II 62 II
2.04
Mean value of M T
1.97
1.66 -
1.89 1.98
-
Leighton Rubenstein,
(M), of their ratios
Bancroft & Freifelder, 1970 ; & 1969 Dubin et aZ., 1970
25 83 132
25.5 67.3 106 -
This paper$
-
3-32 -
2.63 4.14
2.98 4.71
5.28 -
1.59
1.58 -
(4-62)s 1.58 Cl.5519
2.06 2.29 -
2.10 1.86 1.85
-
WWO 2.23
1.94
t Molecular weights by Schmid & Hearst are increased by 7.4% (Freifelder, personal communication). $ From length ratio. 3 Values in parentheses were calculated assuming that T4- and T2 DNA have equal length. 11For comparison, reduced to non-glucosylated oytosine.
564
D. LANG
Wilson, Hooper, Wilkins & Hamilton, 1960), one obtains 1.913 x lOlo dctltons/cm for N&DNA using International Atomic Weights of 1966 based on carbon isotope lzC. The molecular weights of the two possible nucleotide pairs differ by only 0.3% ; M’ should then be practically independent of base composition. In T4- and T2 DNA, supposedly without effect on configuration (Thomas, 1966), cytosine is replaced by hydroxymethylcytosine (Wyatt & Cohen, 1953), which is 100% glucosylated in T4 and 765% in T2 (Sinsheimer, 1960). M’ for these DNA’s, again in B configuration, is therefore 2,104 x lOlo (T4 DNA) and 2.064 x lOlo daltons/cm (T2 DNA). The numbers given in this paragraph are based on the assumption that the dimensions of the B configuration do not depend on base composition. The alternative is discussed by Freifelder (1970). A comparison of measured molecular weights of sodium salts of DNA (M), of their ratios, and of molar linear densities, is giveninTable 3. Listedare most recent M-values from three laboratories using different methods: S&mid & Hearst (1969), CsCl density-gradient equilibrium centrifugation; Leighton & Rubenstein (1969), autoradiography of single 32P-labeled DNA molecules: Banoroft & Freifelder (1970), Dubin et al. (1970), high-speed equilibrium centrifugation, sedimentation-diffusion measurements, and phosphorus-nitrogen determinations. The last column shows molecular weight ratios calculated from length ratios (Table 1) using M’-values of the preceding paragraph. Good agreement exists for MT,, but not for M,, and MT*, M,,. For the M ratios, good agreement exists only for ratios not containing MT,. The following reasoning might then be adopted. Since three recent and independent determinations of M,, are agreeing well, and since there is no indication that the T7 DNA’s used might be different, the mean molecular weight of (25.1 &O-4) x lo6 daltons (Table 3) is considered to be correct. This leads to rtn apparent molar linear density of M&=(25.1 &0*4)x 10s/[(1*215& 0.025) x low31 = (2.07 kO.04) x lOlo daltons/cm. Assuming independence of M’ on base composition this value should apply to other duplex DNA’s when prepared for electron microscopy by the diffusion method from 0.2 ionic strength as described, or by a similar new method reported by Lang & Mitani (1970). From the lengths in Table 1, and using Mk4 = (2.07 x 2*104/1.913) x lOlo = 2.28 x 1Oroand M& = (2.07 x 2.064/l-913) x lOlO= 2.23 x lOlo daltonslcm, one may thus calculate molecular weights as shown in Table 4. TABLE 4
Molecular weights calculated from length measurements and M,, = 25.1 x lo6 daltons DNA from:
BPVf
T6
T4
T2
(108 dZons)
5.15
74.9
119
116
t BPV, bovine papilloma virus. This paper was prepared with the technical assistance of Mr L. Lewis, Jr. and Miss B. Bruton and I would like to thank them for their help. I also thank Dr D. Freifelder who sent us bacteriophage T4, T5 and T7; also Drs H. Bujard and B. Gomez who provided
MOLECULAR
WEIGHT
PER
UNIT
LENGTH
566
OF DNA
bovine papilloma virus DNA and another strain of T7 bacteriophage. This work was supported by National Science Foundation research grant GB0837 and by U.S. Public Health Service career development award GM-34,964 and divisional grant GM-13,234. REFERENCES Abelson, 5. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Bancroft, F. C. & Freifelder, D. (1970). J. Mol. Biol. 54, 537. Bendet, I., Schachter, E. & Lauffer, M. A. (1962). J. Mol. BioZ. 5, 76. Bode, H. R. & Morowitz, H. J. (1967). J. Mol. BioZ. 23, 191. Bujard, H. (1967). J. Virology, 1, 1135. Bujard, H. (1968). J. Mol. BioZ. 33, 503. Bujard, H. (1969). Proc. Nat. Acad. Sci., Wu&. 62, 1167. Bujard, H. (1970). J. Mol. BioZ. 49, 125. Cairns, J. (1961). J. Mol. BioZ. 3, 756. Chandler, B. (1963). M. SC. Thesis, University of Wisconsin. Crawford, L. V., Follett, E. A. C. & Crawford, E. M. (1966). J. Microscopic, Dubin, S. B., Benedek, G. B., Bancroft, F. C. & Freifelder, D. (1970). J. Mol. Frank, H., Zarnitz, M. L. & Weidel, W. (1963). 2. Naturf. 18b, 281. Freifelder, D. (1965). Biochem. Biophys. Res. Comm. 18, 141. Freifelder, D. (1970). J. Mol. BioZ. 54, 567. Freifelder, D. & Kleinschmidt, A. K. (1965). J. Mol. Biol. 14, 271. Inman, R. B. (1967). J. Mol. BioZ. 25, 239. Inman, R. B., Schildkraut, C. L. & Kornberg, A. (1965). J. Mol. BioZ. 11, Kellenberger, E. (1965). Path. Microbial. 28, 540. Kleinschmidt, A. K. (1967). Nuturwks, 54, 417. Kleinschmidt, A. K., Kass, S. J., Williams, R. C. C Knight, C. A. (1965). J.
5, 597. BioZ. 54,547.
285.
MOE. BioZ. 13,
749.
Kleinschmidt, Acta,
A. K., Lang, D., Jacherts,
D. & Zahn, R. K. (1962). Biochim.
biophy.9.
61, 857.
Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. BioZ. 23, 163. Lang, D. & Coates, P. (1968). J. Mol. BioZ. 36, 1968. Lang, D., Kleinschmidt, A. K. BEZahn, R. K. (1964). Biochim. biophye. Acta, 88, 142. Lang, D. & Mitani, M. (1970). Biopolymers, 9, 373. Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. MoZ. BioZ. 2, 19. Leighton, S. B. & Rubenstein, I. (1969). J. Mol. BioZ. 46, 313. Misra, D. N., Sinha, R. K. & DasGupta, N. N. (1969). FiroZogy, 39, 183. Schmid, C. W. & Hearst, J. E. (1969). J. MoZ. BioZ. 44, 143. Sinsheimer, R. L. (1960). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, vol. 3, p. 187, New York and London: Academic Press. Thomas, C. A., Jr. (1966). J. Gem. Phytiol. 49, No. 6, Part 2, 143. Thomas, C. A., Jr. & MaoHattie, L. A. (1964). Proc. Nat. AC&. Sci., Wash. 52, 1297. Wyatt, G. R. & Cohen, S. S. (1953). Biochem. J. 55, 774.