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Nucleotide composition of short segments of DNA molecules
J. Mol. BioI. (1965) II, 223-237
Nucleotide Composition of Short Segments of DNA Molecules YUJI MIYAZAWA AND
C. A.
THOMAS, JR.
Biophysics Department, Johns Hopkins University BaUimore, Md, 21218, U.S.A. (Received 24 August 1964) A column has been devised which fractionates duplex DNA segments of about 200,000 molecular weight with respect to their characteristic denaturation temperature and hence composition. This fractionation has been applied to a number of different phage and bacterial DNA's and the degree of compositional heterogeneity measured. The segments show a relatively broad distribution and the composition of segments from ODe species of DNA overl ap those from another. This is in contrast with the compositions of high m olecular weight segments which show high species specificity. This fractionation procedure has been applied to A DNA, and the composition of some genetically deleted segments has been determined.
1. Introduction When the DNA molecule making up the bacterial or bacteriophage chromosome is broken by the shear often involved in the isolation procedure, the resulting duplex segments Can have different nucleotide compositions characteristic of the part of the intact chromosome that they represent. Current DNA preparations (Marmur, 1961; Berns & Thomas, 1965) produce relatively long segments, which are 10 to 400 million in molecular weight. The composition of each segment is the average of a very large number of AT and GC pairs comprising many genes. Gradient equilibrium centrifugation (Rolfe & Meselson, 1959; Sueoka , Marmur & Doty, 1959; Schildkraut, Marmur & Doty, 1962) and thermal denaturation (Marmur & Doty, 1959) experiments have shown that the composition of these long segments shows extreme species specificity. Two species of micro-organisms having DNA 's which differ in mean nucleotide composition by 10% have very few large DNA segments with the same composition (Doty, Marmur & Sueoka, 1959; Sueoka, 1961,1964). In order to draw conclusions about the coding potential and molecular systematics of DNA, one would like to know the nucleotide composition of very short segments of DNA of the length of single genes or smaller. To gain some information on this point we have fractionated. sonic fragments of DNA molecules on hydroxyapatite columns. The column was arranged in such a way as to allow denatured DNA to pass through freely while totally retaining the duplex: fragments. This column was surrounded by a water jacket, and the temperature of the column could be increased gradually to slightly over 100°C. At each temp erature a certain fraction of the duplex: segments was denatured and washed through the column. The resulting chromatogram as a function of temperature proved to be almost exactly the derivative of the increase in opti cal density as a function of 223
224
Y. MIYAZAWA AND C. A. THOMAS, JR.
temperature-the optical "melting" curve-and samples collected at higher temperatures could be shown to have a proportionately higher mole fraction of guanine cytosine. This procedure actually fractionated the short segments with respect to their nucleotide composition, and the thermal chromatogram was a reflection of the relative abundance of the compositional classes.
+
2. Methods Purification of phage and phage DNA Lysates of >.cb2+b5+ and >'cb2b6 (Kellenberger, Zichichi & Weigle, 1960,1961a,b) were prepared as previously (Macflattie & Thomas, 1964). Such lysates seldom contained more than 3 X 1010 phage/ml., but they were easily concentrated by the following procedure, suggested by Bernardi (1964, unpublished work). After treatment with about lOfLg/ml. of RNase and DNase and filtration through an HA Millipore filter covered by 5 rom of Celite, the filtrate was adjusted to 0·01111 in phosphate buffer, pH 6·8. Approximately 2 ml. (packed volume) of hydroxyapatite was added to each 100 ml. of filtrate and gently agitated for 15 min at room temperature. More than 95% of the phage became adsorbed to the hydroxyapatite crystals, which were allowed to settle and the supernatant fraction was discarded. The hydroxyapatite was resuspended in 0·020 III-phosphate buffer. A liter of 1·0 III.phosphate buffer contained the following: 0'50 mole Na2HP04, 0'50 mole NaH 2 P0 4, 10ml.l·0 III·tris, pH 7,4, 10 ml. 0·10 III-MgS0 4, 20ml. 5% NaCI. The suspension was loaded on a column and washed with the same solvent until the eluate showed no ultraviolet absorption. At this point the column was washed with 0·10 III-phosphate buffer and the optical density and titer of the fractions were measured. More than 90% of the phage was recovered by this procedure, and the ratio of the optical density at 260 mfL to plaque-forming units (optical cross-section) was measured. Values of 0·31 and 0·25 X 1O-llcm2/plaque.forming unit were observed for >.cb2+b5+ and >.cb2b5 respectively. The ratio of these numbers is nearly the ratio of the DNA contents of these two phages. A similar procedure could be applied to T2 and T5 bacteriophage, but in this work they were grown and purified by differential centrifugation as before (Thomas & Rubenstein, 1964) . . The phage DNA was extracted by rolling -with phenol at 60 tev Jtnu: (Frankel, 1963) and the phenol was removed by dialysis. The DNA which remained in the aqueous layer was further purified by chromatography on hydroxyapatite or methylated bovine serum albumin columns (Mandell & Hershey, 1960). Bacterial DNA's The DNA from bacteria was extracted with phenol after lysis by sodium lauryl sulfate and digestion with pronase (Berns & Thomas, 1965). The aqueous layer was then treated with boiled RNase and the bacterial DNA separated by chromatography on hydroxyapatite. Alternatively, DNA preparations were made by the procedure of Marmur (1961) and purified on hydroxyapatite. Fragmentation by sonic treatment The DNA solution, after purification by hydroxyapatite chromatography, contained 0·30 III-phosphate buffer. Concentrated NaCI was added to adjust it to 0,15111, and 2 to 4 mI. of this solution was chilled and bubbled with nitrogen for 10 min. Sonic treatment was performed with a Mullard Sonicator, 3/4 inch probe for 2 min at full intensity. This resulted in no increase in the optical density and no denaturation of the DNA as judged by chromatography on hydroxyapatite. The fragments have a sedimentation coefficient in the analytical centrifuge (8;°) of 6 sand 6·1 s in sucrose gradients. This corresponds to a molecular weight of 200,000 (Doty, Rice & McGill, 1958). Preparation of hydroxyapatite The following procedure is a slight modification of that proposed by Tiselius, Hjerten & Levin (1956). Approximately 400 mI. of water were poured into a 4·1. glass beaker. Into this, 2000 mI. each of 0'50 III-Na2HP04 and 0·50 M-CaCI2 was added by two separatory
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
225
funnels at a rate of 120 drops/min through Pasteur pipettes (27 drops/ml.), The contents of the beaker were mixed with a steel propeller turning at 530 rev.jrnin. At the end of this lengthy step, the precipitate was washed 4 times with distilled water and sometimes allowed to stand overnight. The supernatant fraction was again decanted and the precipitate suspended in 4000 m1. of water. 100 m1. of 40% w/w NaOH was added and the mixture boiled with simultaneous stirring for 1 hr. The supernatant fraction was again decanted and the precipitate washed with 41. of water 4 times by decantation. During this step some fine material was discarded. The phosphate buffer used in all experiments hereafter described was made by adding equal moles of Na2HP04 and NaH 2P04; the pH was near 6·8. The precipitate was resuspended in 41. of 0·01 M-phosphate buffer and brought just to boiling. The precipitate was collected by decantation and then boiled in the same solution for 5 min. The precipitate was again collected and boiled for 15 min in 0·01 M-buffer. All boiling treatments were accompanied by stirring. After each boiling step, fine material was removed by decantation. The hydroxyapatite thus obtained could be stored in 0·001 M-phosphate buffer with a small amount of chloroform. The resulting material usually formed columns having excellent flow characteristics, which possibly resulted from the loose packing of the long blade-like crystals.
Operation of hydroxyapatite columns The applicability of hydroxyapatite to the fractionation of nucleic acids has been demonstrated by Main & Cole (1957), Semenza (1957), Main, Wilkins & Cole (1959), Bernardi & Timasheff (1961) and Bernardi (1961, 1962). In our experiments the columns were used in two ways: they were developed with a linear gradient of phosphate buffer, 01' they were washed with 0·08 M-phosphate buffer stepwise at increasing temperatures. The first mode of operation was used for the purification of phage and DNA, and the second mode was used to fractionate sonic fragments with respect to composition. In these latter experiments, an arrangement depicted in Fig. 1 was employed. The temperature was maintained by flowing water from a Haake thermostatically controlled bath and measured by a sensitive thermistor connected into a Wheatstone bridge. The temperature could be measured to O·QloC and the temperature of the column could be controlled to better than 0'1 DC. In general, the column consisted of about 1 ml. of hydroxyapatite (packed volume) supported by a disc of porous polyethylene (Porex Materials Corp., Fairburn, Ga.). To load the column, sonically fragmented DNA was diluted to a phosphate concentration of 0'03 M arid slowly passed through the column at room temperature. No DNA, as assayed by radioactivity, passed through the bed. Our standard procedure was to add 1·5 ml. of 0·08 M-phosphate buffer and increase the temperature of the jacket to a predetermined value. Temperature equilibration required at least 2 min. After 5 min, the 1·5 ml. of solution was slowly drained off and another 1·5 ml. was added, held for another 5 min and drained. Experiments showed that these two washes would remove 90% of the DNA that could be removed at that temperature. The fraction of the radioactive material that could be eluted over each temperature interval is plotted against temperature as seen in Fig. 4. Generally these experiments could be done with a total of 5 X 10 4 ctsjmin.
Absorbancr-temperature measurements were performed with a thermostatically controlled cell in the Cary 11 spectrophotometer using the thermistor temperature-measuring device to sense the temperature of the solution being measured. Centrifugation and chromatographic procedures were the same as previously reported (Thomas & Pinkerton, 1962).
Nucleotide analysis. The digestion of DNA to nucleotides was accomplished by the sequential action of pancreatic DNase and snake venom phosphodiesterase. The DNA samples were first dialysed to 0-01 M-NaCl, 0·005 sr-tris, pH 7·3; and MgCl z was added to make them 0·002 M. A small volume of a pancreatic DNase solution (Worthington, 1500 p,gfml. dissolved in 0·10 M·NaCl, 0·1 % bovine serum albumin) was then added to give a final concentration of 10 p,g/m1. The DNA was more than 95% (perchloric acid) soluble after 2 hr. At this point 1/20 vol. of 1'0 M-tris, pH 9·0 was added and the Ca 2 + concentration adjusted to 0·005 M. No precipitation occurred after sufficient digestion with
226
Y. MIYAZA W A AND C. A. THOMAS, JR. Inlet for preheated buffer Silicone rubber
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FIG. 1. Jacketed chromatographic column. This device was used for thermal chromatography of sonic fragments of various DNA's. Later models used a larger water jacket through which the preheated buffer was conducted.
DNase. Snake venom phosphodiesterase (lot 30301, grade B, Calbiochem) was added to a. final concentration of 6 unitsjml., and incubation continued at 45°C for 2 hr. This preparetion, as well as another purified in this laboratory, had a very low level of phosphomonoesterase, since the above procedure resulted in less than 0'3% of the phosphorus being nonadsorbable on Norite. Chromatography was accomplished on a 1 X 30 cm column of Dowex-I (Bio Rad AGI-X8, 200-400 mesh, chloride form). The resin was pretreated with the following sucoession of solvents: 2 N·NaOH (twice), 2 N·HCl (twice), water, 1 M-ammonium acetate and 0·01 M-ammonium acetate. The bed was built and washed with more 0·01 M-ammonium acetate. A sample of the digest having 5 to 50 thousand cts/min was added to the top of the bed and washed with a little water. The column was developed with a linear gradient from 0·01 to 1·0 M·ammonium acetate, pH 4·7 (Hershey, 1964, personal communication). The nucleotide peaks eluting in the order C, T, A, G were quantitatively adsorbed to acidwashed Norite, deposited on a 25-mm membrane filter disc, washed with dilute glue to prevent losses, fixed to a planchet, dried and counted. Approximately 90% of the radio. active material added to the column was recovered in the form of nucleotides.
3. Results M eUing behavior of sonic fragments In order to relate our thermal denaturation studies, which have been done in 0·08 M· phosphate, with those done previously in 0·15 M-NaOI, 0·015 M-citrate (SSO), we have made studies of both whole T5 DNA molecules and sonic fragments in each sol-
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
227
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FIG. 2. Optical melting profiles oj T5 DNA. The curves show the fractional increase in the optical density at 260 mp. for unbroken T5 DNA molecules and sonic fragments in two different solvents. The measurements were made at the temperatures indicated.
vent. Appropriate comparisons for T5 DNA are shown in Fig. 2. Changing the solvent from SSC to 0·08 M-phosphate results in a decrease of 3·5°C in T m for unbroken molecules, a value which is expected on the basis of the lower ionic strength (MacHattie, 1963, unpublished work; Marmur & Doty, 1962). The sonic fragments show a. greater drop in T m (4'5°C) on passing from SSC to 0·08 M-phosphate. It is of interest to note that in either solvent the sonic fragments display transitions which occur at lower temperatures than the whole molecules from which they were derived. Apparently the stability of the duplex segments has been reduced by the sonic treatment. While no change in the thermal denaturation profile could be seen with fragments of 620,000 daltons (Marmur & Doty, 1962), it is possible that the threefold smaller size produced by our sonic treatment could be responsible for the observed decrease and broadening. This size effect is to be expected on theoretical grounds (Gibbs & DlMarzio, 1958,1959a,b; Zimm & Bragg, 1958; Magee, Gibbs & Zimm, 1963). Fractionation of duplex molecules and single polynucleotide chains on hydroxyapatite
When unbroken, undenatured DNA molecules from T2, T5 or A bacteriophage are adsorbed to columns of hydroxyapatite, and eluted by a gradient of phosphate buffer, they appear at 0·27 M-phosphate; a value which is independent of temperature up to 70°0. The chromatography does not break these DNA molecules, as evidenced by no change in sedimentation coefficient, no breakage seen in the chromatograms from the methylated bovine serum albumin columns; or, in the case of A DNA, no change in the measured lengths of the DNA molecules as seen in the electron microscope (MacHattie & Thomas, 1964). The position of the peak is not strongly affected by fragmen1:&tion ofthe duplex DNA, although some effect ean be seen with sonic fragments which are eluted earlier at 0·22 M-phosphate. Denatured DNA is eluted much earlier, at 0·10 M-phosphate, at room temperature. Upon passing to 70°0, there is a further decrease in the phosphate required for elution.
Y. MIYAZAWA AND C. A. THOMAS, JR.
228
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There appears to be no detectable dependence on molecular weight, since single polynucleotide chains derived from whole molecules and sonic fragments are eluted together. Thus this column appears to be sensitive to secondary structure but not to length. This, together with the fact that the single polynucleotide chains are eluted at lower phosphate concentrations, proves to be useful in these experiments. Melting of sonic fragments of DNA on a hydroxyapatite column After repeating at various temperatures experiments of the type shown in Fig. 3, we have selected 0·08 M-phosphate buffer as the best solvent to separate single polynucleotide chains from duplex segments near their melting points. After loading the sonic fragments in 0·03 M-phosphate and washing with 0·08 M-phosphate at room temperature, the temperature of the jacket (Fig. 1) was increased by small intervals and two 1·5-mI. fractions were collected as described in Methods. That these samples contained truly denatured DNA could be shown by rechromatography on hydroxyapatite at 60 to 70°0; the radioactive material was eluted exclusively in the range characteristic of single polynucleotide chains. Figure 4 shows the results of three independent thermal chromatograms made on different samples of ADNA of the same genotype. For purposes of comparison, a thermal chromatogram for Escherichia coli (W3110) DNA is superimposed on the same graph. Similar reproducibility has been experienced with T5 DNA and Hemophilus DNA sonic fragments. The thermal chromatograms for various DNA's are shown in Fig. 5(a). Notice that each DNA has its own melting distribution, but that there is substantial overlap between these distributions. Each of these peaks appears to be unimodal. To compare these thermal chromatograms with the thermal denaturation profile as determined by optical density, we have plotted the observed optical density against temperature for sonic fragments of each of three DNA's in the same solvent (Fig. 5(b». On the same graph, the integral form of the thermal chromatograms seen in Fig. 5(a) is drawn. There is a close similarity between the two curves in each case. The thermal
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NUCLEOTIDE COMPOSITION OF DNA MOLECULES
229
chromatograms show a slightly higher T ms which might be expected in view of the higher effective ionic strength which must prevail in the neighborhood of the highly charged hydroxyapatite crystal.
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Oomposition of fractions from the thermal chromatograms
Since the melting temperature is strongly dependent on the composition of the DNA, one would expect that fractions taken at lower temperatures would be richer in A and T, whereas those taken at higher temperatures would be richer in G and O. This proves to be the case, as shown in Fig. 6. All fractions displayed a molar equivalence of A and T, G and 0, which is not surprising in view of the universal acceptance of the pairing rule. The mole fraction G + 0 is generally proportional to temperature, showing 2-0% variationj'f), This is to be compared with a value of 2'4%/"0 obtained by Marmur & Doty (1962) for a large number of DNA's from different species. In view of the significant differences in solvent and molecular weight between the two experiments, the apparent agreement between these two numbers may be fortuitous. Figure 6 shows that it is possible to fractionate and identify segments having compositions which are quite far removed from the mean value for a given DNA. Thus T5 DNA produces segments which have G + 0 contents ranging from 32 to 50%. T2 DNA has not been analysed since it is resistant to our enzymes, but it has a thermal chromatogram which is nearly superimposable with that ofT5 DNA.'\ DNA produces segments stretching from 38 to 60%, Serratia DNA from 40 to 69%, and E. coli DNA from 42 to 57%. Thus the heterogeneity is substantial, segments from one DNA overlap those from another, and the range of compositions found in a single DNA molecule covers a large fraction of the range available to the DNA's from different species (Sueoka, 1964). The fact that the composition is generally proportional to temperature means that the thermal chromatograms are direct reflexions of the heterogeneity of composition existing among the various segments. 16
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FIG. 5. (a) Thermal chromatogram8 of three different DNA's. The results wit h T2 DNA are super. imposable with t hose of T5. Those with H emophilus DNA are very similar t o T5 arid T2 DNA. (h) Optical melting curves plotted with integral form of thermal chromatoqrams. Notice that the optical m elting curves and the integral form of the thermal chromatograms shown in Fig. 5 (a) are very similar.
There are some reservations to this statement. We realize that the form of the thermal chromatograms depends slightly on the length of the duplex segments, an effect which will be discussed later. Second, when nucleotide analysis is made on the fraetions in the leading edge of the thermal chromatogram which contain less than 1% per degree, the mole fraction G + C departs from the line drawn in Fig. 6. This is not unexpe cted and probably represents a non-specific leakage of material which was damaged by the sonic treatment, or exceptionally short fragm ents which would melt at a lower temperature. These would not be expected to have compositions charact eristic of the temperature at which they were eluted. This leakage effect has been examined most extensively with E. coli DNA and T5 DNA. The points taken from the low-temperature edge of the chromatogram of T5 DNA segments have boon included as illustrative of this effect (Fig. 6). This " leakage" effect means that the
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
231
leading edge of the thermal chromatogram cannot be taken as truly indicative of the composition of the segments collected in these fractions. While this effect is much less significant in the high-temperature fractions, it is wise at this point to consider the fractions taken at the extreme edges as not simply reflecting the extremes of the compositional distribution.
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Temperature (0c) FIG. 6. ReBults of nucleotide analysis. The observed mole fraction of G + C is plotted against the mean value of the temperature over which they were collected. The solid points represent results obtained on sonic fragments which were not subjected to thermal chromatography. Nucleotide analysis made of fractions coming from the low-temperature edges of the chromatogram which contain less than l%/"C, give G + C values which fall above the line. This "leakage" effect is described in the text. (0) T5 fractions; (e) T5 unfractionated; (0) ,\ fractions; (.),\ unfractionatcd: (,6) Serratia fractions; (A) Serratia unfractionated; ('i7) E. coli fractions; (l') E. coli unfractionated.
Repeated thermal chromatography and nucleotide analysis of T5 DNA segments generate points which do not fall on the line drawn in Fig. 6, but rather on a line of lower slope. These observations are statistically significant and may indicate that there are still other effects not known which affect the temperature-composition relation as seen in these experiments.
232
Y. MIYAZAWA AND C. A. THOMAS, JR.
Comparison of the thermal chromatograms of wild type and a deleted DNA
The .\ DNA molecule containing the deletion mutations b2b5 is known to be 22% shorter than the wild-type molecule. This is shown most directly by the fact that the mutant molecules have a slower sedimentation rate (Burgi, 1963) and are seen to be shorter in the electron microscope (MacHattie & Thomas, 1964). They are lacking in genetic function (Kellenberger et al., 1960,1961a,b) and are expected to be lacking in a specific sequence of nucleotides. The following experiment demonstrates that this is the case, because the region which is deleted has a lower G C content than the average for the wild-type molecule.
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Tempercture (Oe) FIG. 7. Thermal chromatograms of wild type A together with an alternately-labeled A DNA having large deletion mutations. The distribution marked "deleted regions" was calculated by taking the difference between the two thermal chromatograms as described in the text. It represents the best estimate of the thermal chromatogram of the regions of the A DNA molecule which are missing in the b2b5 mutant.
Figure 7 shows the thermal chromatograms made with a mixture of 32P-labeled .\cb2b5 and HO-labeled .\cb2+b5+ DNA's. The ratio HO/32P is seen to increase significantly above 1·0 on the low-temperature side of the melting distribution. The control experiments ([32P] .\cb2+ b5 + versus [14 0] Acb2 + b5 +) and a repetition with the labels reversed behaved as expected. This indicates that the sections of the DNA which are missing in the b2 and b5 mutants are richer in A + T than the remainder of the molecule. By making the appropriate subtraction, we can calculate the thermal
N UCLEOTIDE C OMP O S I T I O N OF DN A M OLE C ULES
233
chromatogra m of the deleted (or deletable) regions. Denoting the fraction of the mol ecule genetically deleted by x , we have:
d1
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where d1 is the fra ction of the segments from the deletable regions which melt over t he t emperature interval i, and gr and gf ar e the fractions of HC and 32p material which are melted from the wild type and delet ed DNA's respecti vely over the t emperature interval i. Th e distribution of d l so obtained is plot t ed in Fig. 7. The result ing distribution is seen to be as broad as the others , but centered 4°C lower . Thus the average G + C content of the deleted regions should be about 8% lower than the wild-type DNA. Density of wild type and deleted A DNA mol ecules in OsOl gradients
Thi s difference should be detectable by an increased density of the deleted DNA in CsOI. To test this prediction, a number of OsClsedimentation equilibrium experiments were done with samples of ADN A to which an equal am ount ofT2 DNA was added as a density marker. In Table 1 the density differences between T2 DNA and DNA extracted from the various deleti on mutants is shown . Assumin g t hat Llp = 0·098 Ll(G + C) (Schildkraut et al., 1962), one may calculate t hat t he doubly-delet ed DNA, b2b5, is 2·3% richer in G + O. Assuming that the density or composit ion of t he wildtype molecule is t he avera ge of the va rious segments in it, one may calculate t he average composition of t he deleted regions. Since 22% of t he molecule is deleted, 78% X2·3% = 8·3% . Th e G C content of the deleted regions is 8·3% less than that
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of the wild-type molecule t ak en as a whole, in good agreement with the calculate d thermal chromatogram .
4. Discussion In t his communication we have described the operation of a chromatogra phic column which is developed by increasing the temperature. This column fractionates short segment s of the DNA duplex with resp ect to their melting point and hence nucle otide composition. Th e result is a "thermal chro mat ogram" which nucleotide analysis has shown to be a reflexion of the composition a nd abundance of the segments which can be produced by sonic treatment. This proc edure has been applied to wild-type A DNA molecules and those containing mutational deletions. The deleted segments have been shown to have a different composition from the remainder of the molecule. E xperiments which ar e in some respects similar t o t hese have been done by Bernardi (1962 and unpublished work ). Th e t hermal chrom at ography of RNA segments has been accomplished by Bolto n & McCarthy (1964). In this case E. coli messenger R NA was hybridized t o DNA immobi lized in agar bead s. By increasing t he te mperature ste pwise, fractions of R NA were eluted which differed in G + C content. The range of composit ion seen by this procedure, 13%, is slightly less than t hat observed in our experiments for E . coli DNA, which is not surp rising since it is probabl e that all messenger RNA molecules ar e not equ ally abundant.
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> Column 1: genotype of phage. Column 2: measured densities in CsCI, observed by mixing all four phage types and sedimenting to equilibrium. Column 3: percentage of the DNA missing 118 calculated from the densities using the equation 0<% = ~p X 103 (Weigle, Meselson & Paigen, 1959). Column 4: measured length of the A DNA molecules (from MacHattie & Thomas, 1964). Column 5: density difference between>' DNA and T2 DNA molecules. All runs were done at 25°C, 31,410 rev./roin for 84 to 113 hr. Column 6: the G + C content of the deleted region 118 the percentage less than the average for the Acb2+b5+ DNA. Assuming that ~p = 0·098 ~ (G + C) (Schildkraut et al. 1962), the second entry is calculated 118 follows: (0,0086-0'0070) X 1/0-098 X 84%/16% = 8'5%. Column 7: value calculated for the sixth column determined from the thermal chromatogram shown in Fig. 7.
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NUCLEOTIDE COMPOSITION OF DNA MOLECULES
235
Effect of segment size on the thermal chromatogram
It is clear from Fig. 2 that the reduction of the molecular weight of the duplex molecules by sonic treatment causes a lowering of the thermal transition. Since there is little or no effect on the melting temperature with slightly larger fragments of 620,000 (Marmur & Doty, 1962), it is clear that we are in the range of strong dependence of T m on fragment size. This would mean that segments of shorter length, but richer in G + 0, would melt at the same temperature as longer segments that were richer in A + T. This fact would tend to reduce the ability of the column to discriminate with respect to composition. This would indicate that if it were possible to produce a collection of fragments of very uniform length, the discrimination with respect to composition would be even greater than shown in Fig. 6. However, it is unlikely that greater discrimination would reveal greater heterogeneity for fragments of this size. A direct measurement of the size heterogeneity of the sonic fragments has been made by a re-sedimentation experiment using 32p_ and l4O-labeled sonic fragments. 32P·Labeled fragments taken from the leading edge of a sedimentation zone were pooled with l4O-labeled fragments taken from the trailing edge of another zone (and vice versa). Upon re-sedimentation, the two labels separate from each other, showing heterogeneity in S. Interpreting this as a heterogeneity in molecular weight gives a range of 110,000 to 400,000, about a mean value of 200,000. What effect does a variation in fragment length have on the thermal chromatogram? To explore this an equal mixture of sonic fragments (6 S, 200,000) and fragments produced by passing whole molecules through a French press at 10,000 Ib.jin. 2 (7'6 S, 420,000), were sedimented through a sucrose gradient and fractions selected corresponding to 150,000, 280,000 and 620,000 molecular weight. Thermal chromatograms were made on these fractions and compared with those made on the normal unfractionated sonic fragments and samples which had received no sonic treatment and were of high molecular weight. The fragments of nominal molecular weight 620,000 produced thermal chromatograms which were almost identical with those produced by high molecular weight DNA, whereas fragments of shorter length displayed a progressive lowering and tailing of the thermal chromatogram toward the low-temperature region. The unfractionated sonic fragments produced thermal chromatograms which were about half-way between those produced by the nominal 150,000 and 280,000 samples. Thus the sample of higher molecular weight produced thermal chromatograms which are centered about higher temperatures and somewhat sharper. It should be noted that the effects mentioned here are not large. There is only a few degrees difference between the midpoints of the thermal chromatograms produced by the shortest and longest fragments tested. Whereas one would expect that heterogeneity in fragment length would tend to diminish the discrimination of the column for composition, the effect of long fragment size would be expected to produce the same effect, but for a different reason. It is likely that long fragments do not "melt" as a unit, but rather in regions (Berns & Thomas, 1961; Beer & Thomas, 1961). At present we have no way of knowing how, or how long, a partially melted fragment would be retained by the column. Although we have not studied it, we do not think that the width of the thermal chromatogram produced by very long duplexes could be the result of heterogeneity in composition, which is demonstrably a major factor in the thermal chromatograms of sonic fragments.
236
Y. MIYAZAWA AND C. A. THOMAS, JR.
Degree oj nucleotide clustering One may properly ask if the degree of heterogeneity t ha t one observes among the sonic fragments is that which would be expected by t he completel y random assembly of AT and GO pairs. The width of the t herm al chromatograms at 60% of their height is between 5 and 10°0 , with T5 and E. coli DNA showing the narrowest and ADNA showing the broadest distributions. At 2% per degree, this would imply that the standar d deviation in composit ion ranges from 5 to 10% G + O. If the segments were randomly assembled, one would expect them to have a st andard deviation in composition of between 2·7 and 2·9%. Thus the observed heterogeneity is between two and four times that expecte d from the random-assembly hypothesis. The fact that all the DNA 's examined show about the same breadth is remarkable in view of the great differences in heterogeneity of composition of longer segments of these molecules. For example, when T2 DNA molecules are broken into halves, quarters and smaller fragm ents, slight heterogeneity of density can be recognized (Thomas & Pinkerton, 1962). When T5 DNA molecules are broken (Thomas & Pinker. ton, 1963, unpublished work) , there is no detectable het erogeneity of density among the fragments, because the band widths in OsOI can be accounted for by Brownian motion alone (Baldwin, 1959; Sueoka, 1959). On the other hand, the halves and quarters of ,\ DNA molecules differ enough in density to be separ able into discrete bands in the OsOI gradient (Hershey, 1964, personal communicat ion), and are even separable to some extent by chro mat ography (Rogness & Simmons, 1964). In spite of t his, all of these molecules show about the same degree of heterogeneity of compositi on among sonic fragment s. The implications of this are not clear . These experiments show that DNA molecules from different species share many regions with the same composition; the extent of overlap dependi ng on the proximity of their mean values. Although common compositions do not imply identical sequences, a difference in composition demands a difference in sequence. Those sequences which are common to t wo species should (in principle) be found in the same thermal fraction. This may prove useful in the examinat ion of interspecies homologies. W e th ank Dr Lome A. MacHa t tie, who developed the thermist or thermometer and who performed some early expe rim ent s with T5 DNA. We als o t han k Mr Marcus Rhoades, who performed several sed iment ation experime nt s and cont ri buted information on the fracti onation of DNA over a ran ge of t emperatures. Our interest in the fra ctionation of nucleic acid s by hydroxyapatite was the result of a visit by Dr Giorgio Bernardi, who made us aware of this useful substance. This work was supported by the Atomic Energy Cornmission AT (30-1)-2119, and the National Institutes of H ealth (E 3233).
REFERENCES Baldwin, R. L. (1959). Proc. Nat. A cad. Sci ., Wash. 45, 939. Beer, M. & Thomas, C. A. (1961). J. Mo l. BioI. 3, 699. Bernardi, G. (1961) . B iochem. B iophys. R es. Oomm. 6, 54. Bernardi, G. (196 2). Biochem . J. 83, 32. Bernardi, G. & Timasheff, S. (19 61). B iochem. B iophys. R es. Oomm, 6, 58. Berna, K. 1. & Thomas, C. A . (1961). J. Mo l. B iol. 3, 289. B erns, K. 1. & Thomas. C. A. ( 1965). J . M ol. B iol, 11, in t h e press. Bolton, E. T. & McCarthy, B . J. (1964), J. lII ol. Bi ol. 8, 201. Burgi, E. (1963). P roc, N at . Acad. Sei., Wash. 49, 151. Doty, P., Marrour, J. & Sueo ka, N . (1959). Br ookhaven Symp. S oc. Biol, 12, 1. Doty, P., Rice, S. A. & McGill, B . B . (1958). Pr oc, N at. Acad . Sci ., W ash. 44, 432 .
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
237
Frankel, F. R. (1963). Proc. Nat. Acad. Sci., Wash. 49,366. Gibbs, J. H. & DiMarzio, E. A. (1958). J. Chern; Phys. 28, 1247. Gibbs, J. H. & DiMarzio, E. A. (1959a). J. Chem, Phys. 29, 679. Gibbs, J. H. & DiMarzio, E. A. (1959b). J. Chem, Phys. 30, 271. Hogness, D. S. & Simmons, J. R. (1964). J. Mol. Biol. 9, 411. Kellenberger, G. M., Zichiehi, M. L. & Weigle, J. (1960). Nature, 187, 1961. Kellenberger, G. M., Zichichi, M. L. & Weigle, J. (1961a). Proc, Nat. Acad. s«, Wash. 47, 869. Kellenberger, G. M., Zichichi, M. L. & Weigle, J. (1961b). J. Mol. Biol. 3, 399. MacHattie, L. A. & Thomas, C. A. (1964). Science, 144, 1142. Magee, W. S., Gibbs, J. H. & Zimm, B. H. (1963). Biopolymers, I, 133. Main, R. K. & Cole, L. J. (1957). Arch. Biochem, Biophys. 68, 186. Main, R. K., Wilkins, M. J. & Cole, L. J. (1959). J. Amer. Chern; Soc. 81, 6490. Mandell, J. & Hershey, A. D. (1960). Analyt. Biochem, I, 66. Marmur, J. (1961). J. Mol. Biol. 3, 208. Marmur, J. & Doty, P. (1959). Nature, 183, 1427. Marmur, J. & Doty, P. (1962). J. Mol. Biol. 5, 109. Rolfe, R. & Meselson, M. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1039. Schildkraut, C., Marmur, J. & Doty, P. (1962). J. Mol. Biol. 4, 430. Semenza, G. (1957). Ark. Kemi, II, 89. Sueoka, N. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1480. Sueoka, N. (1961). J. Mol. Biol. 3, 31. Sueoka, N. (1964). In The Bacteria, vol. 5, p. 419. New York: Academic Press. Sueoka, N., Marmur, J. & Doty, P. (1959). Nature, 183, 1429. Thomas, C. A. & Pinkerton, T. C. (1962). J. Mol. Biol. 5,356. Thomas, C. A. & Rubenstein, 1. (1964). Biophysical J. 4, 94. Tiselius, A., Hjerten, S. & Levin, O. (1956). Arch. Biochem, Biophys. 65, 132. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. Biol. I, 379. Zimm, B. H. & Bragg, J. K. (1958). J. Chem. Phys. 28, 1246.
Nucleotide Composition of Short Segments of DNA Molecules YUJI MIYAZAWA AND
C. A.
THOMAS, JR.
Biophysics Department, Johns Hopkins University BaUimore, Md, 21218, U.S.A. (Received 24 August 1964) A column has been devised which fractionates duplex DNA segments of about 200,000 molecular weight with respect to their characteristic denaturation temperature and hence composition. This fractionation has been applied to a number of different phage and bacterial DNA's and the degree of compositional heterogeneity measured. The segments show a relatively broad distribution and the composition of segments from ODe species of DNA overl ap those from another. This is in contrast with the compositions of high m olecular weight segments which show high species specificity. This fractionation procedure has been applied to A DNA, and the composition of some genetically deleted segments has been determined.
1. Introduction When the DNA molecule making up the bacterial or bacteriophage chromosome is broken by the shear often involved in the isolation procedure, the resulting duplex segments Can have different nucleotide compositions characteristic of the part of the intact chromosome that they represent. Current DNA preparations (Marmur, 1961; Berns & Thomas, 1965) produce relatively long segments, which are 10 to 400 million in molecular weight. The composition of each segment is the average of a very large number of AT and GC pairs comprising many genes. Gradient equilibrium centrifugation (Rolfe & Meselson, 1959; Sueoka , Marmur & Doty, 1959; Schildkraut, Marmur & Doty, 1962) and thermal denaturation (Marmur & Doty, 1959) experiments have shown that the composition of these long segments shows extreme species specificity. Two species of micro-organisms having DNA 's which differ in mean nucleotide composition by 10% have very few large DNA segments with the same composition (Doty, Marmur & Sueoka, 1959; Sueoka, 1961,1964). In order to draw conclusions about the coding potential and molecular systematics of DNA, one would like to know the nucleotide composition of very short segments of DNA of the length of single genes or smaller. To gain some information on this point we have fractionated. sonic fragments of DNA molecules on hydroxyapatite columns. The column was arranged in such a way as to allow denatured DNA to pass through freely while totally retaining the duplex: fragments. This column was surrounded by a water jacket, and the temperature of the column could be increased gradually to slightly over 100°C. At each temp erature a certain fraction of the duplex: segments was denatured and washed through the column. The resulting chromatogram as a function of temperature proved to be almost exactly the derivative of the increase in opti cal density as a function of 223
224
Y. MIYAZAWA AND C. A. THOMAS, JR.
temperature-the optical "melting" curve-and samples collected at higher temperatures could be shown to have a proportionately higher mole fraction of guanine cytosine. This procedure actually fractionated the short segments with respect to their nucleotide composition, and the thermal chromatogram was a reflection of the relative abundance of the compositional classes.
+
2. Methods Purification of phage and phage DNA Lysates of >.cb2+b5+ and >'cb2b6 (Kellenberger, Zichichi & Weigle, 1960,1961a,b) were prepared as previously (Macflattie & Thomas, 1964). Such lysates seldom contained more than 3 X 1010 phage/ml., but they were easily concentrated by the following procedure, suggested by Bernardi (1964, unpublished work). After treatment with about lOfLg/ml. of RNase and DNase and filtration through an HA Millipore filter covered by 5 rom of Celite, the filtrate was adjusted to 0·01111 in phosphate buffer, pH 6·8. Approximately 2 ml. (packed volume) of hydroxyapatite was added to each 100 ml. of filtrate and gently agitated for 15 min at room temperature. More than 95% of the phage became adsorbed to the hydroxyapatite crystals, which were allowed to settle and the supernatant fraction was discarded. The hydroxyapatite was resuspended in 0·020 III-phosphate buffer. A liter of 1·0 III.phosphate buffer contained the following: 0'50 mole Na2HP04, 0'50 mole NaH 2 P0 4, 10ml.l·0 III·tris, pH 7,4, 10 ml. 0·10 III-MgS0 4, 20ml. 5% NaCI. The suspension was loaded on a column and washed with the same solvent until the eluate showed no ultraviolet absorption. At this point the column was washed with 0·10 III-phosphate buffer and the optical density and titer of the fractions were measured. More than 90% of the phage was recovered by this procedure, and the ratio of the optical density at 260 mfL to plaque-forming units (optical cross-section) was measured. Values of 0·31 and 0·25 X 1O-llcm2/plaque.forming unit were observed for >.cb2+b5+ and >.cb2b5 respectively. The ratio of these numbers is nearly the ratio of the DNA contents of these two phages. A similar procedure could be applied to T2 and T5 bacteriophage, but in this work they were grown and purified by differential centrifugation as before (Thomas & Rubenstein, 1964) . . The phage DNA was extracted by rolling -with phenol at 60 tev Jtnu: (Frankel, 1963) and the phenol was removed by dialysis. The DNA which remained in the aqueous layer was further purified by chromatography on hydroxyapatite or methylated bovine serum albumin columns (Mandell & Hershey, 1960). Bacterial DNA's The DNA from bacteria was extracted with phenol after lysis by sodium lauryl sulfate and digestion with pronase (Berns & Thomas, 1965). The aqueous layer was then treated with boiled RNase and the bacterial DNA separated by chromatography on hydroxyapatite. Alternatively, DNA preparations were made by the procedure of Marmur (1961) and purified on hydroxyapatite. Fragmentation by sonic treatment The DNA solution, after purification by hydroxyapatite chromatography, contained 0·30 III-phosphate buffer. Concentrated NaCI was added to adjust it to 0,15111, and 2 to 4 mI. of this solution was chilled and bubbled with nitrogen for 10 min. Sonic treatment was performed with a Mullard Sonicator, 3/4 inch probe for 2 min at full intensity. This resulted in no increase in the optical density and no denaturation of the DNA as judged by chromatography on hydroxyapatite. The fragments have a sedimentation coefficient in the analytical centrifuge (8;°) of 6 sand 6·1 s in sucrose gradients. This corresponds to a molecular weight of 200,000 (Doty, Rice & McGill, 1958). Preparation of hydroxyapatite The following procedure is a slight modification of that proposed by Tiselius, Hjerten & Levin (1956). Approximately 400 mI. of water were poured into a 4·1. glass beaker. Into this, 2000 mI. each of 0'50 III-Na2HP04 and 0·50 M-CaCI2 was added by two separatory
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
225
funnels at a rate of 120 drops/min through Pasteur pipettes (27 drops/ml.), The contents of the beaker were mixed with a steel propeller turning at 530 rev.jrnin. At the end of this lengthy step, the precipitate was washed 4 times with distilled water and sometimes allowed to stand overnight. The supernatant fraction was again decanted and the precipitate suspended in 4000 m1. of water. 100 m1. of 40% w/w NaOH was added and the mixture boiled with simultaneous stirring for 1 hr. The supernatant fraction was again decanted and the precipitate washed with 41. of water 4 times by decantation. During this step some fine material was discarded. The phosphate buffer used in all experiments hereafter described was made by adding equal moles of Na2HP04 and NaH 2P04; the pH was near 6·8. The precipitate was resuspended in 41. of 0·01 M-phosphate buffer and brought just to boiling. The precipitate was collected by decantation and then boiled in the same solution for 5 min. The precipitate was again collected and boiled for 15 min in 0·01 M-buffer. All boiling treatments were accompanied by stirring. After each boiling step, fine material was removed by decantation. The hydroxyapatite thus obtained could be stored in 0·001 M-phosphate buffer with a small amount of chloroform. The resulting material usually formed columns having excellent flow characteristics, which possibly resulted from the loose packing of the long blade-like crystals.
Operation of hydroxyapatite columns The applicability of hydroxyapatite to the fractionation of nucleic acids has been demonstrated by Main & Cole (1957), Semenza (1957), Main, Wilkins & Cole (1959), Bernardi & Timasheff (1961) and Bernardi (1961, 1962). In our experiments the columns were used in two ways: they were developed with a linear gradient of phosphate buffer, 01' they were washed with 0·08 M-phosphate buffer stepwise at increasing temperatures. The first mode of operation was used for the purification of phage and DNA, and the second mode was used to fractionate sonic fragments with respect to composition. In these latter experiments, an arrangement depicted in Fig. 1 was employed. The temperature was maintained by flowing water from a Haake thermostatically controlled bath and measured by a sensitive thermistor connected into a Wheatstone bridge. The temperature could be measured to O·QloC and the temperature of the column could be controlled to better than 0'1 DC. In general, the column consisted of about 1 ml. of hydroxyapatite (packed volume) supported by a disc of porous polyethylene (Porex Materials Corp., Fairburn, Ga.). To load the column, sonically fragmented DNA was diluted to a phosphate concentration of 0'03 M arid slowly passed through the column at room temperature. No DNA, as assayed by radioactivity, passed through the bed. Our standard procedure was to add 1·5 ml. of 0·08 M-phosphate buffer and increase the temperature of the jacket to a predetermined value. Temperature equilibration required at least 2 min. After 5 min, the 1·5 ml. of solution was slowly drained off and another 1·5 ml. was added, held for another 5 min and drained. Experiments showed that these two washes would remove 90% of the DNA that could be removed at that temperature. The fraction of the radioactive material that could be eluted over each temperature interval is plotted against temperature as seen in Fig. 4. Generally these experiments could be done with a total of 5 X 10 4 ctsjmin.
Absorbancr-temperature measurements were performed with a thermostatically controlled cell in the Cary 11 spectrophotometer using the thermistor temperature-measuring device to sense the temperature of the solution being measured. Centrifugation and chromatographic procedures were the same as previously reported (Thomas & Pinkerton, 1962).
Nucleotide analysis. The digestion of DNA to nucleotides was accomplished by the sequential action of pancreatic DNase and snake venom phosphodiesterase. The DNA samples were first dialysed to 0-01 M-NaCl, 0·005 sr-tris, pH 7·3; and MgCl z was added to make them 0·002 M. A small volume of a pancreatic DNase solution (Worthington, 1500 p,gfml. dissolved in 0·10 M·NaCl, 0·1 % bovine serum albumin) was then added to give a final concentration of 10 p,g/m1. The DNA was more than 95% (perchloric acid) soluble after 2 hr. At this point 1/20 vol. of 1'0 M-tris, pH 9·0 was added and the Ca 2 + concentration adjusted to 0·005 M. No precipitation occurred after sufficient digestion with
226
Y. MIYAZA W A AND C. A. THOMAS, JR. Inlet for preheated buffer Silicone rubber
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DNase. Snake venom phosphodiesterase (lot 30301, grade B, Calbiochem) was added to a. final concentration of 6 unitsjml., and incubation continued at 45°C for 2 hr. This preparetion, as well as another purified in this laboratory, had a very low level of phosphomonoesterase, since the above procedure resulted in less than 0'3% of the phosphorus being nonadsorbable on Norite. Chromatography was accomplished on a 1 X 30 cm column of Dowex-I (Bio Rad AGI-X8, 200-400 mesh, chloride form). The resin was pretreated with the following sucoession of solvents: 2 N·NaOH (twice), 2 N·HCl (twice), water, 1 M-ammonium acetate and 0·01 M-ammonium acetate. The bed was built and washed with more 0·01 M-ammonium acetate. A sample of the digest having 5 to 50 thousand cts/min was added to the top of the bed and washed with a little water. The column was developed with a linear gradient from 0·01 to 1·0 M·ammonium acetate, pH 4·7 (Hershey, 1964, personal communication). The nucleotide peaks eluting in the order C, T, A, G were quantitatively adsorbed to acidwashed Norite, deposited on a 25-mm membrane filter disc, washed with dilute glue to prevent losses, fixed to a planchet, dried and counted. Approximately 90% of the radio. active material added to the column was recovered in the form of nucleotides.
3. Results M eUing behavior of sonic fragments In order to relate our thermal denaturation studies, which have been done in 0·08 M· phosphate, with those done previously in 0·15 M-NaOI, 0·015 M-citrate (SSO), we have made studies of both whole T5 DNA molecules and sonic fragments in each sol-
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
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vent. Appropriate comparisons for T5 DNA are shown in Fig. 2. Changing the solvent from SSC to 0·08 M-phosphate results in a decrease of 3·5°C in T m for unbroken molecules, a value which is expected on the basis of the lower ionic strength (MacHattie, 1963, unpublished work; Marmur & Doty, 1962). The sonic fragments show a. greater drop in T m (4'5°C) on passing from SSC to 0·08 M-phosphate. It is of interest to note that in either solvent the sonic fragments display transitions which occur at lower temperatures than the whole molecules from which they were derived. Apparently the stability of the duplex segments has been reduced by the sonic treatment. While no change in the thermal denaturation profile could be seen with fragments of 620,000 daltons (Marmur & Doty, 1962), it is possible that the threefold smaller size produced by our sonic treatment could be responsible for the observed decrease and broadening. This size effect is to be expected on theoretical grounds (Gibbs & DlMarzio, 1958,1959a,b; Zimm & Bragg, 1958; Magee, Gibbs & Zimm, 1963). Fractionation of duplex molecules and single polynucleotide chains on hydroxyapatite
When unbroken, undenatured DNA molecules from T2, T5 or A bacteriophage are adsorbed to columns of hydroxyapatite, and eluted by a gradient of phosphate buffer, they appear at 0·27 M-phosphate; a value which is independent of temperature up to 70°0. The chromatography does not break these DNA molecules, as evidenced by no change in sedimentation coefficient, no breakage seen in the chromatograms from the methylated bovine serum albumin columns; or, in the case of A DNA, no change in the measured lengths of the DNA molecules as seen in the electron microscope (MacHattie & Thomas, 1964). The position of the peak is not strongly affected by fragmen1:&tion ofthe duplex DNA, although some effect ean be seen with sonic fragments which are eluted earlier at 0·22 M-phosphate. Denatured DNA is eluted much earlier, at 0·10 M-phosphate, at room temperature. Upon passing to 70°0, there is a further decrease in the phosphate required for elution.
Y. MIYAZAWA AND C. A. THOMAS, JR.
228
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There appears to be no detectable dependence on molecular weight, since single polynucleotide chains derived from whole molecules and sonic fragments are eluted together. Thus this column appears to be sensitive to secondary structure but not to length. This, together with the fact that the single polynucleotide chains are eluted at lower phosphate concentrations, proves to be useful in these experiments. Melting of sonic fragments of DNA on a hydroxyapatite column After repeating at various temperatures experiments of the type shown in Fig. 3, we have selected 0·08 M-phosphate buffer as the best solvent to separate single polynucleotide chains from duplex segments near their melting points. After loading the sonic fragments in 0·03 M-phosphate and washing with 0·08 M-phosphate at room temperature, the temperature of the jacket (Fig. 1) was increased by small intervals and two 1·5-mI. fractions were collected as described in Methods. That these samples contained truly denatured DNA could be shown by rechromatography on hydroxyapatite at 60 to 70°0; the radioactive material was eluted exclusively in the range characteristic of single polynucleotide chains. Figure 4 shows the results of three independent thermal chromatograms made on different samples of ADNA of the same genotype. For purposes of comparison, a thermal chromatogram for Escherichia coli (W3110) DNA is superimposed on the same graph. Similar reproducibility has been experienced with T5 DNA and Hemophilus DNA sonic fragments. The thermal chromatograms for various DNA's are shown in Fig. 5(a). Notice that each DNA has its own melting distribution, but that there is substantial overlap between these distributions. Each of these peaks appears to be unimodal. To compare these thermal chromatograms with the thermal denaturation profile as determined by optical density, we have plotted the observed optical density against temperature for sonic fragments of each of three DNA's in the same solvent (Fig. 5(b». On the same graph, the integral form of the thermal chromatograms seen in Fig. 5(a) is drawn. There is a close similarity between the two curves in each case. The thermal
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NUCLEOTIDE COMPOSITION OF DNA MOLECULES
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chromatograms show a slightly higher T ms which might be expected in view of the higher effective ionic strength which must prevail in the neighborhood of the highly charged hydroxyapatite crystal.
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Since the melting temperature is strongly dependent on the composition of the DNA, one would expect that fractions taken at lower temperatures would be richer in A and T, whereas those taken at higher temperatures would be richer in G and O. This proves to be the case, as shown in Fig. 6. All fractions displayed a molar equivalence of A and T, G and 0, which is not surprising in view of the universal acceptance of the pairing rule. The mole fraction G + 0 is generally proportional to temperature, showing 2-0% variationj'f), This is to be compared with a value of 2'4%/"0 obtained by Marmur & Doty (1962) for a large number of DNA's from different species. In view of the significant differences in solvent and molecular weight between the two experiments, the apparent agreement between these two numbers may be fortuitous. Figure 6 shows that it is possible to fractionate and identify segments having compositions which are quite far removed from the mean value for a given DNA. Thus T5 DNA produces segments which have G + 0 contents ranging from 32 to 50%. T2 DNA has not been analysed since it is resistant to our enzymes, but it has a thermal chromatogram which is nearly superimposable with that ofT5 DNA.'\ DNA produces segments stretching from 38 to 60%, Serratia DNA from 40 to 69%, and E. coli DNA from 42 to 57%. Thus the heterogeneity is substantial, segments from one DNA overlap those from another, and the range of compositions found in a single DNA molecule covers a large fraction of the range available to the DNA's from different species (Sueoka, 1964). The fact that the composition is generally proportional to temperature means that the thermal chromatograms are direct reflexions of the heterogeneity of composition existing among the various segments. 16
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FIG. 5. (a) Thermal chromatogram8 of three different DNA's. The results wit h T2 DNA are super. imposable with t hose of T5. Those with H emophilus DNA are very similar t o T5 arid T2 DNA. (h) Optical melting curves plotted with integral form of thermal chromatoqrams. Notice that the optical m elting curves and the integral form of the thermal chromatograms shown in Fig. 5 (a) are very similar.
There are some reservations to this statement. We realize that the form of the thermal chromatograms depends slightly on the length of the duplex segments, an effect which will be discussed later. Second, when nucleotide analysis is made on the fraetions in the leading edge of the thermal chromatogram which contain less than 1% per degree, the mole fraction G + C departs from the line drawn in Fig. 6. This is not unexpe cted and probably represents a non-specific leakage of material which was damaged by the sonic treatment, or exceptionally short fragm ents which would melt at a lower temperature. These would not be expected to have compositions charact eristic of the temperature at which they were eluted. This leakage effect has been examined most extensively with E. coli DNA and T5 DNA. The points taken from the low-temperature edge of the chromatogram of T5 DNA segments have boon included as illustrative of this effect (Fig. 6). This " leakage" effect means that the
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
231
leading edge of the thermal chromatogram cannot be taken as truly indicative of the composition of the segments collected in these fractions. While this effect is much less significant in the high-temperature fractions, it is wise at this point to consider the fractions taken at the extreme edges as not simply reflecting the extremes of the compositional distribution.
0'70
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Temperature (0c) FIG. 6. ReBults of nucleotide analysis. The observed mole fraction of G + C is plotted against the mean value of the temperature over which they were collected. The solid points represent results obtained on sonic fragments which were not subjected to thermal chromatography. Nucleotide analysis made of fractions coming from the low-temperature edges of the chromatogram which contain less than l%/"C, give G + C values which fall above the line. This "leakage" effect is described in the text. (0) T5 fractions; (e) T5 unfractionated; (0) ,\ fractions; (.),\ unfractionatcd: (,6) Serratia fractions; (A) Serratia unfractionated; ('i7) E. coli fractions; (l') E. coli unfractionated.
Repeated thermal chromatography and nucleotide analysis of T5 DNA segments generate points which do not fall on the line drawn in Fig. 6, but rather on a line of lower slope. These observations are statistically significant and may indicate that there are still other effects not known which affect the temperature-composition relation as seen in these experiments.
232
Y. MIYAZAWA AND C. A. THOMAS, JR.
Comparison of the thermal chromatograms of wild type and a deleted DNA
The .\ DNA molecule containing the deletion mutations b2b5 is known to be 22% shorter than the wild-type molecule. This is shown most directly by the fact that the mutant molecules have a slower sedimentation rate (Burgi, 1963) and are seen to be shorter in the electron microscope (MacHattie & Thomas, 1964). They are lacking in genetic function (Kellenberger et al., 1960,1961a,b) and are expected to be lacking in a specific sequence of nucleotides. The following experiment demonstrates that this is the case, because the region which is deleted has a lower G C content than the average for the wild-type molecule.
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Tempercture (Oe) FIG. 7. Thermal chromatograms of wild type A together with an alternately-labeled A DNA having large deletion mutations. The distribution marked "deleted regions" was calculated by taking the difference between the two thermal chromatograms as described in the text. It represents the best estimate of the thermal chromatogram of the regions of the A DNA molecule which are missing in the b2b5 mutant.
Figure 7 shows the thermal chromatograms made with a mixture of 32P-labeled .\cb2b5 and HO-labeled .\cb2+b5+ DNA's. The ratio HO/32P is seen to increase significantly above 1·0 on the low-temperature side of the melting distribution. The control experiments ([32P] .\cb2+ b5 + versus [14 0] Acb2 + b5 +) and a repetition with the labels reversed behaved as expected. This indicates that the sections of the DNA which are missing in the b2 and b5 mutants are richer in A + T than the remainder of the molecule. By making the appropriate subtraction, we can calculate the thermal
N UCLEOTIDE C OMP O S I T I O N OF DN A M OLE C ULES
233
chromatogra m of the deleted (or deletable) regions. Denoting the fraction of the mol ecule genetically deleted by x , we have:
d1
= ;1 [ g cl -
91p (1 - x) ]
where d1 is the fra ction of the segments from the deletable regions which melt over t he t emperature interval i, and gr and gf ar e the fractions of HC and 32p material which are melted from the wild type and delet ed DNA's respecti vely over the t emperature interval i. Th e distribution of d l so obtained is plot t ed in Fig. 7. The result ing distribution is seen to be as broad as the others , but centered 4°C lower . Thus the average G + C content of the deleted regions should be about 8% lower than the wild-type DNA. Density of wild type and deleted A DNA mol ecules in OsOl gradients
Thi s difference should be detectable by an increased density of the deleted DNA in CsOI. To test this prediction, a number of OsClsedimentation equilibrium experiments were done with samples of ADN A to which an equal am ount ofT2 DNA was added as a density marker. In Table 1 the density differences between T2 DNA and DNA extracted from the various deleti on mutants is shown . Assumin g t hat Llp = 0·098 Ll(G + C) (Schildkraut et al., 1962), one may calculate t hat t he doubly-delet ed DNA, b2b5, is 2·3% richer in G + O. Assuming that the density or composit ion of t he wildtype molecule is t he avera ge of the va rious segments in it, one may calculate t he average composition of t he deleted regions. Since 22% of t he molecule is deleted, 78% X2·3% = 8·3% . Th e G C content of the deleted regions is 8·3% less than that
22%
+
of the wild-type molecule t ak en as a whole, in good agreement with the calculate d thermal chromatogram .
4. Discussion In t his communication we have described the operation of a chromatogra phic column which is developed by increasing the temperature. This column fractionates short segment s of the DNA duplex with resp ect to their melting point and hence nucle otide composition. Th e result is a "thermal chro mat ogram" which nucleotide analysis has shown to be a reflexion of the composition a nd abundance of the segments which can be produced by sonic treatment. This proc edure has been applied to wild-type A DNA molecules and those containing mutational deletions. The deleted segments have been shown to have a different composition from the remainder of the molecule. E xperiments which ar e in some respects similar t o t hese have been done by Bernardi (1962 and unpublished work ). Th e t hermal chrom at ography of RNA segments has been accomplished by Bolto n & McCarthy (1964). In this case E. coli messenger R NA was hybridized t o DNA immobi lized in agar bead s. By increasing t he te mperature ste pwise, fractions of R NA were eluted which differed in G + C content. The range of composit ion seen by this procedure, 13%, is slightly less than t hat observed in our experiments for E . coli DNA, which is not surp rising since it is probabl e that all messenger RNA molecules ar e not equ ally abundant.
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TABLE
1
2
Density of phage
Acb2+bfj+ lcb2bfj+ Acb2+bfj Acb2b5
1·498 1·482 1·492 1·476
4
5
Length of DNA molecule
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3
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0% 16% 6% 22%
1
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-
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> Column 1: genotype of phage. Column 2: measured densities in CsCI, observed by mixing all four phage types and sedimenting to equilibrium. Column 3: percentage of the DNA missing 118 calculated from the densities using the equation 0<% = ~p X 103 (Weigle, Meselson & Paigen, 1959). Column 4: measured length of the A DNA molecules (from MacHattie & Thomas, 1964). Column 5: density difference between>' DNA and T2 DNA molecules. All runs were done at 25°C, 31,410 rev./roin for 84 to 113 hr. Column 6: the G + C content of the deleted region 118 the percentage less than the average for the Acb2+b5+ DNA. Assuming that ~p = 0·098 ~ (G + C) (Schildkraut et al. 1962), the second entry is calculated 118 follows: (0,0086-0'0070) X 1/0-098 X 84%/16% = 8'5%. Column 7: value calculated for the sixth column determined from the thermal chromatogram shown in Fig. 7.
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NUCLEOTIDE COMPOSITION OF DNA MOLECULES
235
Effect of segment size on the thermal chromatogram
It is clear from Fig. 2 that the reduction of the molecular weight of the duplex molecules by sonic treatment causes a lowering of the thermal transition. Since there is little or no effect on the melting temperature with slightly larger fragments of 620,000 (Marmur & Doty, 1962), it is clear that we are in the range of strong dependence of T m on fragment size. This would mean that segments of shorter length, but richer in G + 0, would melt at the same temperature as longer segments that were richer in A + T. This fact would tend to reduce the ability of the column to discriminate with respect to composition. This would indicate that if it were possible to produce a collection of fragments of very uniform length, the discrimination with respect to composition would be even greater than shown in Fig. 6. However, it is unlikely that greater discrimination would reveal greater heterogeneity for fragments of this size. A direct measurement of the size heterogeneity of the sonic fragments has been made by a re-sedimentation experiment using 32p_ and l4O-labeled sonic fragments. 32P·Labeled fragments taken from the leading edge of a sedimentation zone were pooled with l4O-labeled fragments taken from the trailing edge of another zone (and vice versa). Upon re-sedimentation, the two labels separate from each other, showing heterogeneity in S. Interpreting this as a heterogeneity in molecular weight gives a range of 110,000 to 400,000, about a mean value of 200,000. What effect does a variation in fragment length have on the thermal chromatogram? To explore this an equal mixture of sonic fragments (6 S, 200,000) and fragments produced by passing whole molecules through a French press at 10,000 Ib.jin. 2 (7'6 S, 420,000), were sedimented through a sucrose gradient and fractions selected corresponding to 150,000, 280,000 and 620,000 molecular weight. Thermal chromatograms were made on these fractions and compared with those made on the normal unfractionated sonic fragments and samples which had received no sonic treatment and were of high molecular weight. The fragments of nominal molecular weight 620,000 produced thermal chromatograms which were almost identical with those produced by high molecular weight DNA, whereas fragments of shorter length displayed a progressive lowering and tailing of the thermal chromatogram toward the low-temperature region. The unfractionated sonic fragments produced thermal chromatograms which were about half-way between those produced by the nominal 150,000 and 280,000 samples. Thus the sample of higher molecular weight produced thermal chromatograms which are centered about higher temperatures and somewhat sharper. It should be noted that the effects mentioned here are not large. There is only a few degrees difference between the midpoints of the thermal chromatograms produced by the shortest and longest fragments tested. Whereas one would expect that heterogeneity in fragment length would tend to diminish the discrimination of the column for composition, the effect of long fragment size would be expected to produce the same effect, but for a different reason. It is likely that long fragments do not "melt" as a unit, but rather in regions (Berns & Thomas, 1961; Beer & Thomas, 1961). At present we have no way of knowing how, or how long, a partially melted fragment would be retained by the column. Although we have not studied it, we do not think that the width of the thermal chromatogram produced by very long duplexes could be the result of heterogeneity in composition, which is demonstrably a major factor in the thermal chromatograms of sonic fragments.
236
Y. MIYAZAWA AND C. A. THOMAS, JR.
Degree oj nucleotide clustering One may properly ask if the degree of heterogeneity t ha t one observes among the sonic fragments is that which would be expected by t he completel y random assembly of AT and GO pairs. The width of the t herm al chromatograms at 60% of their height is between 5 and 10°0 , with T5 and E. coli DNA showing the narrowest and ADNA showing the broadest distributions. At 2% per degree, this would imply that the standar d deviation in composit ion ranges from 5 to 10% G + O. If the segments were randomly assembled, one would expect them to have a st andard deviation in composition of between 2·7 and 2·9%. Thus the observed heterogeneity is between two and four times that expecte d from the random-assembly hypothesis. The fact that all the DNA 's examined show about the same breadth is remarkable in view of the great differences in heterogeneity of composition of longer segments of these molecules. For example, when T2 DNA molecules are broken into halves, quarters and smaller fragm ents, slight heterogeneity of density can be recognized (Thomas & Pinkerton, 1962). When T5 DNA molecules are broken (Thomas & Pinker. ton, 1963, unpublished work) , there is no detectable het erogeneity of density among the fragments, because the band widths in OsOI can be accounted for by Brownian motion alone (Baldwin, 1959; Sueoka, 1959). On the other hand, the halves and quarters of ,\ DNA molecules differ enough in density to be separ able into discrete bands in the OsOI gradient (Hershey, 1964, personal communicat ion), and are even separable to some extent by chro mat ography (Rogness & Simmons, 1964). In spite of t his, all of these molecules show about the same degree of heterogeneity of compositi on among sonic fragment s. The implications of this are not clear . These experiments show that DNA molecules from different species share many regions with the same composition; the extent of overlap dependi ng on the proximity of their mean values. Although common compositions do not imply identical sequences, a difference in composition demands a difference in sequence. Those sequences which are common to t wo species should (in principle) be found in the same thermal fraction. This may prove useful in the examinat ion of interspecies homologies. W e th ank Dr Lome A. MacHa t tie, who developed the thermist or thermometer and who performed some early expe rim ent s with T5 DNA. We als o t han k Mr Marcus Rhoades, who performed several sed iment ation experime nt s and cont ri buted information on the fracti onation of DNA over a ran ge of t emperatures. Our interest in the fra ctionation of nucleic acid s by hydroxyapatite was the result of a visit by Dr Giorgio Bernardi, who made us aware of this useful substance. This work was supported by the Atomic Energy Cornmission AT (30-1)-2119, and the National Institutes of H ealth (E 3233).
REFERENCES Baldwin, R. L. (1959). Proc. Nat. A cad. Sci ., Wash. 45, 939. Beer, M. & Thomas, C. A. (1961). J. Mo l. BioI. 3, 699. Bernardi, G. (1961) . B iochem. B iophys. R es. Oomm. 6, 54. Bernardi, G. (196 2). Biochem . J. 83, 32. Bernardi, G. & Timasheff, S. (19 61). B iochem. B iophys. R es. Oomm, 6, 58. Berna, K. 1. & Thomas, C. A . (1961). J. Mo l. B iol. 3, 289. B erns, K. 1. & Thomas. C. A. ( 1965). J . M ol. B iol, 11, in t h e press. Bolton, E. T. & McCarthy, B . J. (1964), J. lII ol. Bi ol. 8, 201. Burgi, E. (1963). P roc, N at . Acad. Sei., Wash. 49, 151. Doty, P., Marrour, J. & Sueo ka, N . (1959). Br ookhaven Symp. S oc. Biol, 12, 1. Doty, P., Rice, S. A. & McGill, B . B . (1958). Pr oc, N at. Acad . Sci ., W ash. 44, 432 .
NUCLEOTIDE COMPOSITION OF DNA MOLECULES
237
Frankel, F. R. (1963). Proc. Nat. Acad. Sci., Wash. 49,366. Gibbs, J. H. & DiMarzio, E. A. (1958). J. Chern; Phys. 28, 1247. Gibbs, J. H. & DiMarzio, E. A. (1959a). J. Chem, Phys. 29, 679. Gibbs, J. H. & DiMarzio, E. A. (1959b). J. Chem, Phys. 30, 271. Hogness, D. S. & Simmons, J. R. (1964). J. Mol. Biol. 9, 411. Kellenberger, G. M., Zichiehi, M. L. & Weigle, J. (1960). Nature, 187, 1961. Kellenberger, G. M., Zichichi, M. L. & Weigle, J. (1961a). Proc, Nat. Acad. s«, Wash. 47, 869. Kellenberger, G. M., Zichichi, M. L. & Weigle, J. (1961b). J. Mol. Biol. 3, 399. MacHattie, L. A. & Thomas, C. A. (1964). Science, 144, 1142. Magee, W. S., Gibbs, J. H. & Zimm, B. H. (1963). Biopolymers, I, 133. Main, R. K. & Cole, L. J. (1957). Arch. Biochem, Biophys. 68, 186. Main, R. K., Wilkins, M. J. & Cole, L. J. (1959). J. Amer. Chern; Soc. 81, 6490. Mandell, J. & Hershey, A. D. (1960). Analyt. Biochem, I, 66. Marmur, J. (1961). J. Mol. Biol. 3, 208. Marmur, J. & Doty, P. (1959). Nature, 183, 1427. Marmur, J. & Doty, P. (1962). J. Mol. Biol. 5, 109. Rolfe, R. & Meselson, M. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1039. Schildkraut, C., Marmur, J. & Doty, P. (1962). J. Mol. Biol. 4, 430. Semenza, G. (1957). Ark. Kemi, II, 89. Sueoka, N. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1480. Sueoka, N. (1961). J. Mol. Biol. 3, 31. Sueoka, N. (1964). In The Bacteria, vol. 5, p. 419. New York: Academic Press. Sueoka, N., Marmur, J. & Doty, P. (1959). Nature, 183, 1429. Thomas, C. A. & Pinkerton, T. C. (1962). J. Mol. Biol. 5,356. Thomas, C. A. & Rubenstein, 1. (1964). Biophysical J. 4, 94. Tiselius, A., Hjerten, S. & Levin, O. (1956). Arch. Biochem, Biophys. 65, 132. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. Biol. I, 379. Zimm, B. H. & Bragg, J. K. (1958). J. Chem. Phys. 28, 1246.