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Segmental distribution of nucleotides in the DNA of bacteriophage lambda
J. Mol. Biol. (1968) 34, 1-16
Segmental Distribution of Nucleotides in the DNA of Bacteriophage Lambda A. SKALEA, ELIZABETH BURCI
AND
A. D. HERSHEY
Carnegie Institution, Genetics Research Unit Cold Spring Harbor, N.Y., U.X.A. (Received 6 November 1967) DNA molecules isolated from phage h were fragmented by shear and fractionated in various ways, mainly with respect to buoyant density of mercury complexes in CsaSO,. Six segments of different composition were found, ranging in G + C content from 37 to 57 mole %. The lengths and left-to-right sequence of segments were determined. The segments are reasonably homogeneous internally and the boundaries between segments rather sharp.
1. Introduction The DNA of phage X contains long intramolecular segments of differing nucleotide composition that can be demonstrated qualitatively by thermal, pycnometric, and chromatographic means (Hershey, Burgi, Frankel, Goldberg & Ingraham, 1962; Hershey & Burgi, 1963; Hogness & Simmons, 1964; Hershey, 1964). Hogness & Simmons (1964) separated molecular halves from the DNA of a defective, galtransducing line of the phage, and showed by recovery of genetic markers that the fragment richer in GCt corresponds to the left portion of the genetic map (gal in hdg; genes A to J inclusive in wild type). Their conclusion has been confirmed by physical analysis of the DNA’s of the two phage lines (Skalka, Butler t Eohols, 1967; concerning the relation between hag and wild type, see also Kayajanian & Campbell, 1966, and Skalka & Burgi, 1967). One may therefore refer to a left molecular half containing, in wild-type X, 55% GC, and a right molecular half containing 45% (Hershey, 1964). We report here further details of the intramolecular nucleotide distribution. Our analysis is based on four principles. (1) Natural DNA molecules can be broken into fragments of various sizes by subjecting them to appropriate rates of hydrodynamic shear (Hershey, Burgi & Ingraham, 1962). (2) Mercuric ions combine reversibly with DNA and preferentially with parts rich in AT to form complexes the buoyant density of which depends strongly on metal content (Nandi, Wang & Davidson, 1965). (3) The resolving power of preparative density-gradient centrifugation in an angle rotor is greater than in a swinging bucket rotor (Hershey, Burgi & Davern, 1965; Flamm, Bond & Burr, 1966). (4) Owing to the complementary structure of the molecular ends, terminal fragments of h DNA can be isolated independently of their length and composition (Hershey & Burgi, 1965.) t Abbreviations used: GC, guanine + cytosine; AT, adenine + thymine; rf, molar ratio of HgCI, to nucleotides. 1 1
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
2. Materials and Methods (a) DNA preparation The DNA described in this paper came from a clear-plaque (cr) mutant of X originally received from J. Weigle. This phage has the same DNA content as Kaiser’s wild-type X (Burgi, 1963; Care, 1965). It was grown on Escherichia co&i K12 W3110, also from Weigle. DNA was extracted by the phenol method, often with the addition of sodium dodecylsulfate (Burgi, 1963), and dialyzed against 0.1 or 0.01 i%-Na,SO,, buffered if at all with 0.001 M-SOdiUm borate. The DNA was usually labeled with 3ZP at 1 pc/pg P or with 3H to give lo5 or less disintegrations/min/~g DNA. DNA from cultures grown in nutrient broth and fed r3H]thymidine contained counts in thymidylic acid only. DNA from cultures grown in glucose-mineral medium and fed tracer amounts of [3HJuraeil contained counts in deoxycytidylic acid only. Since selective labeling biases the density distribution of DNA, we usually analyzed 32P-labeled fragments, and used 3H-labeled fragments only to mark positions in the gradients. DNA concentrations were measured spectrometrically at 260 mp from the nominal extinction coefficient 0.02 cms/pg. 32P-labeled DNA was hydrolyzed at very low concentrations with pancreatic deoxyribonuclease and venom phosphodiesterase, and subjected to electrophoresis on paper strips (Wu & Kaiser, 1967; Markham & Smith, 1952). Radioactivity was found only in bands corresponding to the four nucleotides, and results conformed to the rules G = C and A = T. Measurements were usually made in duplicate. When a single figure is cited, it is an average of two or more measurements. (c) Breakage by shear DNA solutions in O-1 M-NasS04 were stirred by a razor blade rotating at speeds up to about 30,000 rev./mm (Hershey, Burgi & Ingraham, 1962; a Virtis stirrer was used for the highest speeds). Lengths of fragments were measured from sedimentation rates according to Burgi & Hershey (1963), and are expressed as fractions of the molecular length of h DNA (17.3 p, molecular weight 31 x 106; Care, 1965). The smallest length produced by stirring was 0.06; shorter fragments were produced by sonic treatment. (d) Mercury
complexes
For density-gradient centrifugation, we prepared solutions containing, per ml., 5 to 50 pg of DNA, 0.05 ml. of 0.1 M-sodium borate, and the amounts of C&SO4 and HgCl, recommended below. The manner of mixing is not critical. However, exposure of DNA to an excess of mercury causes irreversible denaturation and we consider it good practice to pour a solution containing all the other ingredients into a vessel containing the required amount of HgCl, solution. The mixtures were kept cold pending centrifugation. The resolution achieved in density-gradient centrifugation depends in a complicated way on the DNA species present in the mixture, the GC content of the components of interest, and the r, value. Optimum conditions are therefore best defined in terms of concentration of CssS04, which, moreover, is more easily controlled than the effective Hg/DNA ratio, since that depends on the purity of the DNA and other reagents. Then the proper r, is that necessary to form complexes of specified densities. For analysis of /\ DNA fragments, we used 43% by weight of Cs,SO, for optimum resolution in the low-GC part of the distribution, and 44% when we were more interested in the high-GC part. C&SO4 of nominal purity 99.9% (Kawecki Chemical Co., New York) proved satisfactory. When the mixture contained mainly unfractionated h DNA, the required r, corresponding to the stated CssSO, concentrations was about 0.24 and 0.28, respectively. When tracer amounts of labeled DNA were analyzed, we added at least 5 fig of unlabeled T2 DNA/ml., sheared to reduce its viscosity, to the mixture. Since T2 DNA combines strongly with mercury, the proper r, under these conditions is about O-35 at 43% Cs2SOI, and 0.40 at 44%. T2 DNA goes to the bottom of the tube when X DNA is visible in the gradient.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
3
(e) Centrifugution. We centrifuged 4-ml. or 6.17-g samples, overlaid with mineral oil in cellulose nitrate tubes 5/8 in. in diameter, in a Spinco type 40 or Ti50 rotor, for 48 hr at 36,000 rev./min and 4°C. If the rotor is spun for 8 hr at 45,000 rev./mm, one can often check band positions in the tubes with a radiation monitor, and then complete the run at the lower speed. A preliminary run at 44,770 rev./min in an analytical centrifuge serves the same purpose. We have no difficulty reproducing results with a given DNA preparation, but sometimes have to vary the Hg/DNA ratio to suit different preparations. (f) Joining
and disjoining
termkal fragments
To ensure that a stirred DNA sample contained only disjoined fragments, it was examined immediately after stirring or heated briefly to 65°C (in 0.01 M-NasSO,) or to 75’C (in 0.1 M-NaeSO,) to disjoin them. No joining of terminal fragments occurs during centrifug&ion under the conditions described above. For deliberate joining of terminal fragments, we annealed solutions containing 10 to 50 pg of DNA/ml. in O-6 M-Nacl as described by Hershey & Burgi (1965). On a few occasions we observed reasonably efficient joining in CseSO+ solutions, prepared as described in section (d) but lacking the buffer and HgCl,, on heating at 30°C for several hours. This procedure is convenient when the DNA is to be analyzed by density-gradient centrifugation, but we have not determined the optimal time and temperature of heating. (g) Physical
analysis
At the end of the run, we collected 25 to 40 fractions dropwise from the bottoms of the tubes into 1 nn-NaCl or O-1 M-EDTA, measured radioactivity, pooled chosen fractions, and dialyzed them first against 1 M-NaCl or 0.1 M-EDTA to remove mercury, then into 0.01 MNaeSOB. The solutions were concentrated, if necessary, under a current of air at 45’C or by dialysis against dry sucrose. A typical analysis required molecular weight measurements before and after further fragmentation by stirring, and return to a Hg-CseSO, gradient. This time the mixture usually contained, besides the 32P-labeled materials to be analyzed, 3H-labeled density markers and unlabeled T2 DNA fragments. Results of this type of analysis must be interpreted cautiously because the density of the Hg-DNA complexes depends on secondary structure as well as composition. We usually find that differently labeled samples of DNA banded in mixture are distributed on the same density scale. 3H-labeled fragments, for example, may be stored at 2 to 5 pg/ml. for weeks or months without change. On the other hand, 3aP-labeled fragments, after repeated banding, usually show a slightly higher density than their counterparts in the marker DNA added to the mixture. The shift in density can be attributed to effects of radiation on DNA concentrated in the bands, though an irreversible effect of the exposure to mercury is not excluded. Since all components of a mixture are usually displaced equally, no difficulty arises in the identification of bands. However, it is a defect of the method that the affinity of DNA fragments for mercury may depend on their history as well as their composition. Fragmentation of DNA by stirring does not itself affect the weight-average density. The smaller fragments produced in a French press or by sonic treatment do exhibit a slightly excessive density in Hg-CseSO, mixtures. This can be seen when the fragments are banded, together with a trace of unbroken DNA, in the uniform density-gradient generated in an analytical centrifuge. The unbroken DNA, visible as a pip on the densitometer tracing, does not quite coincide with the center of gravity of the very broad distribution of the fragments. Since the density shift depends on size of fragments rather than manner of production, and cannot be reversed by annealing, it may signify preferential binding of mercury at the ends of the fragments. (h) Recovery of DNA Losses of DNA during centrifugation, measured as radioactivity remaining in the tubes after collection of fractions, did not exceed 2% or so for DNA fragments produced by stirring, or 10% for DNA subjected to sonic treatment. Losses were not significantly larger when fractionated material was rebanded, always, of course, in the presence of unlabeled DNA carrier.
4
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
Losses of half the DNA were sometimes seen during concentration of solutions in a rotating evaporator, a procedure that we abandoned for that reason. However, it was reassuring in such cases to note that the recovered DNA fragments showed the expected distribution of densities in Hg-CssSO,, a result indicating that the losses were random
and the recovered material unaltered.
3. Results (a) Half-length
fragments
Half-length fragments of DNA may be defined operationally as breakage products produced by stirring a solution of uniform molecules at the critical speed that causes slow breakage. Then most of the molecules, if broken at all, break once, but some of the longest fragments produced by acentric breaks are broken again (Burgi & Hershey, 1961).
Fraction
no.
FIG. 1. Density distribution of half-length fragments. Upper part. A sample of s2P-labeled DNA stirred for 30 min at 12 pg/ml. and 2160 rev./min, and banded with unlabeled T2 DNA in 43.8% CszSOI at rr 0.39. Density increases from right to left. Lower part. The histogram shows the same 3aP-labeled DNA banded under the seme conditions after removal of unbroken molecules by fractional sedimentation through sucrose. Circled points show admixed DNA, 3H-labeled in cytosine, that had been stirred for 30 mm at 5 pg/ml. and 2220 rev./mm to cause complete breakage.
Egure 1 illustrates the distribution of half-length fragments of h DNA in a HgC&SO, gradient. Two samples are compared, one in which about half of the molecules are broken and one in which all are broken. The two sets of breakage products exhibit distributions that coincide when corrected for the fact that one set is labeled only in cytosine. The results show that breakage at the critical speed of stirring produces fragments of three density classes. How that happens can be understood as follows.
DISTRIBUTION
OF NUCLEOTIDES
IN
5
X
The fragments denser than unbroken DNA, which form the left band in Figure 1, comprise half the mass of the broken DNA. These are right-terminal fragments, containing 45% GC. The remainder of the broken DNA, less dense than the intact molecules, must be left-terminal fragments. Since unbroken molecules and right-terminal fragments are uniform in density, the varying density of left-terminal fragments must reflect variations in length. Lengths of fragments recovered from distributions of the sort illustrated were measured on several occasions. DNA corresponding to the center fraction of the right band in Figure 1 consists of pieces ranging in length from about 0.43 downward (weightaverage length O-35). DNA corresponding to the center fraction of the second band from the right consists of pieces ranging in length from about O-50 to 0.65 (weight average 0.56). DNA corresponding to the left band in Figure 1, like unfractionated DNA broken at the critical speed of stirring, consists of half-length fragments together with longer and shorter pieces. We obtained results consistent with the above by reversing the procedure. We first fractionated partially broken X DNA by sedimentation through sucrose, selecting authentic halves. These, spun in Hg-Cs,SO, together with marker DNA like that of Figure 1, formed only two bands, one coinciding with the left band of the marker, the other rather broad but centered between the two right bands of the marker. We conclude that the h DNA molecule consists of at least two segments differing in GC content, and that the boundary between the segments lies to the left of the molecular center. Right-terminal fragments of various lengths therefore differ little in density and form the left band in Figure 1. Left-terminal fragments are uniform in density only when short, and the short fragments form the right band in Figure 1. The remaining left-terminal fragments span the boundary separating DNA of unlike GC content and have a density that depends on length. A similar conclusion was drawn from the appearance of the bands formed in CsCl (Hershey & Burgi, 1965). (b) The AT-rich segment Figure 2 shows the density distribution of fragments of length 0.11. It reveals three components. Since the component of greatest density comprises about 10% of the DNA, or one fragment per molecule, it must come from a single stretch of low GC content spanning about 10% of the molecular length. Figure 2 also shows that the molecule contains DNA of high GC content in 44% of its length, and DNA of intermediate GC content in 46% of its length. The position of the AT-rich segment in the molecule was determined by the following experiment.
5
IO
15 Fraction
FIG. 2. Density
20
25
30
no.
distribution of fragments of length 0.11. 3ZP-labeled /min and banded wih unlabeled T2 DNA in 43% Cs,S04 at r, 0.36.
DNA
stirred
at 10,000
6
A.
SKALKA,
E.
BURG1
AND
A.
D.
HERSHEY
A solution containing 23 pg of 32P-labeled DNA/ml. was stirred for 30 minutes at the minimum speed (2600 rev./min) producing complete breakage at that concentration, and analyzed in Hg-Cs,SO, to give a result like the one shown in the lower part of Figure 1. DNA recovered from several individual fractions was freed from Hg, characterized with respect to length, restirred at 10,000 rev./min to produce fragments of length 0.11, and rebanded in Hg-C&SO, together with density markers. 30
20 1
p
IO-
IO
15
20
25
I
: I
30
t
1.5
IO
Fraction
20
2.5
d 30
I
no.
FIG. 3. Density distribution of fragments from left molecular ends. Left part. Histogram: 32P-labeled left molecular ends of length 0.43 and less, originating in the equivalent of fraction 32 in the lower histogram of Fig. 1, sheared to fragments of length 0.11. Broken line: unfractionated DNA, 3H-labeled in cytosine, sheared to the actme size. Mixture banded with unlabeled T2 DNA in 43.8% Cs,S04 at rf 0.35. Right part. Same, except 32P-labeled fragments derived from left molecular ends of weightaverage length 0.56, originating in the equivalent of fraction 25 in the lower histogram of Fig. 1.
As shown in Figure 3, left molecular ends of length 0.43 and less yielded on further breakage fragments of uniformly high GC content. The same Figure shows that left ends of length 056 yielded on further breakage mainly fragments of high GC content, about 15% of fragments rich in AT, and no fragments of characteristic composition in between. (Figure 3 also illustrates the shift in density resulting from molecular damage sustained during fractionation.) Longer left-terminal fragments (lengths 057 to 0.65, taken from the fraction corresponding to number 24 in the lower histogram of Fig. 1) yielded a result similar to that shown in the right part of Figure 3, except that the band of AT-rich DNA was somewhat larger and an additional band, containing about 10% of the DNA in fragments of intermediate CC content, was visible. Right-terminal fragments, taken from the fraction corresponding to number 13 in the lower histogram of Figure 1, yielded on further breakage mainly pieces of intermediate GC content, a considerable fraction of low GC content, and only 2% of high GC content. The last must have come from a small fraction of the original right-terminal fragments, presumably the longest ones. The main results were verified in a second experiment, in which isolated molecular ends were sheared to length 0.06 before analysis.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
7
We concluded at this stage that the A DNA molecule contains a section of length about 0.1 and high AT content interposed between a section of high GC content, length O-44, on its left, and a section of intermediate composition, length 0.46, on its right. (G) Resolution and nucleotide composition of small fragments The resolution that can be achieved by density analysis depends in two opposing ways on the size of fragments examined. As size is reduced, the efficiency of sampling of molecular segments increases, but the number of classes into which the fragments can be separated diminishes. The rate of diffusion of the fragments sets one limit, but there are other complications too (section 2(g)). Some exploratory work with fragments produced by sonic treatment and high-pressure cells was not very encouraging (Hershey & Burgi, 1966).
5
40 35
0
5
IO
15 Fraction
20
25
30
0
no.
FIG. 4. Density distribution of fragments of length O-06, and composition of DNA in the fractions. banded in 42.8% The histogram shows s2P-labeled DNA, stirred at about 30,00Orev./min, Cs2S04 at rr 0.22 (ordinate scale on the right). The curve shows nucleotide compositions of the fr&tio-ns (s&e on the left), and combines results of two experiments performed at different times that showed practically identical distributions. Many points on the curve are averages of two or three measurements, so that all measurements made (48) sre represented. Arrows indicate positions of the three bands given by fragments of length 0.11 (Fig. 2), known from other experiments in which differently labeled fragments of the two lengths were banded in mixture.
Reduction in length of fragments from 0.11 (as in Fig. 2) to 0.06 does reveal further however. This is shown in the histogram of Figure 4. The band containing Gagments of highest GC content is unchanged, as expected if these fragments originate from a single segment of uniform GC content. The tail of lowest GC content is moved slightly to the left, but its area is not greatly affected. The main effect of the reduction in size of fragments is to split the band of intermediate GC content into two equal parts, both of which must originate from the right-terminal section of the molecule. Considered by itsebf, the histogram in Figure 4 shows that when molecules of X DNA are broken into 17 pieces, the fragments assort themselves into four density classes. The four components of the distribution must come from at least four different segments in the molecules. Before pursuing the origin of the components, we report analyses of their actual nuoleotide compositions. detail,
8
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
Our preparations of h DNA contain about 50.5% GC by direct nucleotide analysis and 50% GC computed from the buoyant density in CsCl (l-709 g/ml. measured against E. co&i DNA of nominal GC content 51% and density l-71 ; Hershey, 1964). The compositions of fragments of length O-06, resolved in a Hg-Cs,SC, gradient, are shown in Figure 4. The four components of the density distribution measure 37, 43, 48.5 and 57% in GC content. Note that the 57%-GC fragments appear to be remarkably uniform in composition. A suggestion to the same effect is visible in the other major components, but they are less well resolved. The approximately linear relation between GC content and position in the density gradient is accidental in two respects. The density gradient itself, in the expanded form produced by righting the tubes, is not uniform but falls smoothly from about O-044 g/ml. /ml. near the bottom to about 0.029 near the top (computed from measurements of refractive index). Independently of the density gradient, an increase in r, from 0.2 to O-3 contracts the high-density part of the distribution and expands the low-density part. (d) The right-terminal
section
We concluded from results just described that the right-terminal section of length O-46 breaks into fragments half of which average 43% and half 48.5% in GC content. To learn something about the origin of these fragments, we looked at right molecular ends isolated in two somewhat different ways. In a first experiment, two samples of DNA, 3H-labeled in cytosine, were separately stirred to produce half-length and third-length fragments, pooled, and fractionated in Hg-Cs,SO,. About 34% of the DNA was recovered from the compact band of DNA of low GC content. After removal of Hg, these fragments were annealed with intact, unlabeled DNA at 25 pg/ml., and the mixture was sedimented into a 25ml. sucrose concentration gradient to permit separation of labeled terminal fragments joined by their cohesive sites to the unlabeled DNA. After these were recovered and detached from the unlabeled DNA by heating, they were sedimented again into sucrose to separate labeled, right-terminal fragments of different sizes (lengths O-43, O-35 and 0.26, as determined by additional sedimentation measurements.) In a second experiment directed to the same purpose, 32P-labeled DNA was sheared to fragments of length about O-3, annealed to join the molecular ends, and banded in Hg-Cs,SO,. (All applications of this technique are similar in principle; one example is shown in Fig. 6 and another is described in section 3(f).) Three bands were seen, of which the middle one, containing 46% of the DNA mainly in the form of joined ends, was recovered. The recovered DNA was heated to disjoin cohesive sites, and returned to Hg-Cs,SO, to separate right and left molecular ends. This time right ends were recovered, dialyzed free from mercury, and fractionated with respect to sedimentation rate to yield lengths 0.32, O-25 and 0.15. The two experiments provided six samples of right-terminal fragments falling into four length classes. All six samples were sheared to pieces of the same size, and analyzed by banding again in Hg-Cs,SO,. The results may be summarized as follows. (1) Right-terminal fragments of length 0.43, broken to pieces of length O-06, yielded equal amounts of the two components previously identified as containing 43 and 48.5% GC (Fig. 5(a)). (2) None of the right-terminal fragments isolated in the first experiment contained appreciable amounts of 37 or 57°/Q-GCDNA. This is illustrated for fragments of length
DISTRIBUTION
(b)
OF
NUCLEOTIDES
IN
h
9
Cd)
cc content (%) FIG. 5. Distribution of nucleotides among subsections of length 0.06 derived from right molecular ends. (a) Ends of original length 0.43; (b) ends of original length 0.32; (0) ends of original length 0.25; (d) ends of original length 0.15. The scale of CC content indicates approximate positions of the four bands given by the marker DNA included in each mixture.
O-43 in Figure 5(a), and was more clearly evident on analysis of fragments of lengths O-35 and O-26 (not shown). Right-terminal fragments isolated in the second experiment (Fig. 5(b), (c), (d) ) did contain small amounts of 37 and 57%-GC DNA, probably owing to contamination with left and central portions of the DNA molecule. That right halves do not contain 57%.GC DNA was shown in section 3(a). (3) If right-terminal fragments contain DNA of only two classes, 43%-GC and 48-5%-G-C, the relation between length of fragment and size of either class should depend in a simple way on structure. For the length series O-43, 0.32, 0.25 and 0.15 illustrated in Figure 5, the 43%-GC class expressed as a fraction of both classes measured 50,41,29 and 33%. The same trend was evident in data not shown. Therefore most of the 43% DNA lies toward the molecular center, but some also close to the right end. Specifically, breakage of right-terminal fragments of length about O-15 into two or three pieces yields one-third 43%-GC DNA and two-thirds 48.5%. These facts, together with results presented next, lead to a precise model for the distribution of nucleotides among subsections of length O-06. (e) Xhort molecular ends We analyzed short fragments from each molecular end in the following way. 32Plabeled DNA at 50 pg/ml. in O-6 M-NaCl was stirred for 30 minutes at 10,000 rev./min and the solution was heated to 75°C and cooled slowly to permit the terminal fragments to join. After the solution had been dialyzed and banded in Hg-Cs,SO,, the
Fraction no. FIG. 6. Density distribution of fragments of length 0.14 with terminal The mixture was banded in 43.8% Cs,B04 at TV0.3.
pieces joined by annealing.
10
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
distribution in Figure 6 was seen. Here the joined ends form the center band. (A sample of the same DNA fragments similarly analyzed without annealing yielded only two bands separated by three fractions containing less than 4% of the DNA.) DNA recovered from the center band, heated to disjoin the fragments and returned to a Hg-Cs,SO, gradient, formed two widely separated bands. These yielded right and left ends of length about 0.2. Actual lengths and nucleotide compositions from two experiments are given in Table 1. Samples of each terminal fragment were sheared further by sonic treatment, yielding pieces among which about one in twelve should possess a terminal cohesive site. To analyze these, small fragments of each kind were annealed in a mixture with weakly “H-labeled molecular halves at 20 pg/ml., left with right and right with left, and sedimented through sucrose to bring the half-length fragments well down the tubes. In these tubes, 3 to 5% of the 32P-labeled DNA sedimented with the halves, and was recovered for nucleotide analysis. In a second experiment of this kind, the doubly labeled complexes were sedimented twice into sucrose before analysis. Results of the centrifugation are illustrated in Figure 7. Lengths and compositions of the recovered molecular ends are given in Table 1. Two sorts of control experiment were performed. First, we verified that the 32Plabeled fragments did not join with 3H-labeled halves when alanealed in left-left and right-right combinations. This could mean that the joining occurs by means of terminal cohesive sites as expected, or that fragments of low GC content recognize fragments of high GC content also for other reasons. The second possibility we excluded by examining 32P-labeled fragments, lacking cohesive sites, recovered from
Fraction
no.
FIG 7. Cosedimeniation of right terminal fragments of length 0.018 and left molecular halves. Upper pa&. 32P.labeled right molecular ends of length 0.19 were broken further by sonic treatment and annealed in mixture with 3H-labeled left molecular halves at 20 pg/ml. The mixture, diluted to 10 pg/ml., was sedimented into a 25ml. sucrose concentration gradient. Histogram, *aP; broken line, 3H. Centrifugation for 13 hr at 24,000 rev./min. Sedimentation from right to left. Lowe? part. Pooled fractions 4 through 16 sedimented again into sucrose. Histogram, 32P; circles, ?H.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
11
TAEZE 1 Composition of molecular ends Experiment
Left ends Length GC (%) 0.14 0.23 0.012 0.017
1 2 1 2
Right ends Length CC (%)
56.5 56.7 48.0 50.2
0.14 0.19 0.012 0,018
46.3 46-3 42.4 41.6
the two extreme bands illustrated in Figure 6. These, broken by sonic treatment and annealed with molecular halves in left-right and right-left combinations, showed no tendency to sediment with the halves. Nevertheless, after a single centrifugation in sucrose, enough 32P-labeled DNA could be recovered with the halves for nucleotide analysis. It showed the composition reported in Table 1 for the longer terminal fragments, and therefore had moved down the tube by unspecific means. From the measurements given in Table 1 we conclude that molecular ends resemble in composition their neighboring molecular sections excepting at the extreme tips, where GC content falls at both ends. More specific models can be proposed as follows. According to Table 1, left molecular ends of length 0.14 contain 56.5% GC and left molecular ends of length 0.012 contain 48% GC. If the terminal section containing 48% GC is separated by a single boundary from DNA of 57% GC, the terminal section measures only 0.01 in length. The several calculations of this sort possible from the data in Table 1 are not very exact because we did not analyze fragments of f-46 001 .
57 0.43
37 43 0.10 0.17
48.5 -42 I 0.24 0.05
FIG. 8. Distribution of nucleotides in X DNA. The CC content in mole o/Oabove the line; fractional molecular length below.
of each segment is indicated
intermediate length, but they agree in showing that the stretch of low GC content at the left end of the molecule is very short. Similar calculations from the data for right ends in Table 1 suggest that the 48*5%GC section of the molecule terminates at the right in a segment of length about 0.05 oontaining 42% GC. This model is supported by the fact (section 3(d)) that breakage of right-terminal fragments of length about 0.15 into pieces of length 0.06 yields two pieces containing 48.5% GC to one containing 43%. All our results are summarized in Figure 8. (f) The molecular center The following experiment illustrates a useful technique, permitting isolation of DNA adjacent to a section of unique composition and delineating the boundary between sections. 32P-labeled DNA was sheared to fragments of length 0.32, heated to join molecular ends, and banded in 43% Cs,SO, at r, O-24. Three bands were seen, similar to those of Figure 6 except that the right band (free left ends) contained only 15% of the DNA
12
A.
SKALKA,
E. BURG1
AND
A.
D. HERSHEY
and the left band (free right ends and those center pieces containing little of the high-GC section) 32%. Several components of the distribution were examined to verify their identity, but only one is pertinent here. Two fractions containing 8% of the DNA, taken from the high-density end of the distribution, were combined, freed from mercury, sheared to pieces of length 0.11, and rebanded as shown in Figure 9. The fragments clearly formed two bands, which
Fraction
FIG. 9. Density
distribution
no,
of fragments
from molecular
centers.
Histogram: 32P-labeled fragments of length 0.32, selected for central origin and high AT content, sheared to fragments sheared to fragments at Tf 0.35.
of length 0.11. Broken line: unfrsctionated DNA, 3H-labeled in cytosine, of the same size. Mixture banded with unlabeled T2 DNA in 43% Cs,S04
were also seen in another run of the same material. One band contained DNA of about 37% GC, the other about 43% GC. (Nucleotide compositions of these materials were unfortunately not directly determined; the estimates given are based on density and take into account the shift in density seen in all fractions recovered in this experiment.) According to the model of Figure 8, selection for high AT content among molecular thirds should yield pieces containing most of the 37%-GC section, little or none of the 57%~GC section, and usually only part of the 43%-GC section (because selection for high AT content should also select short lengths). Figure 9 conhrms these expectations, and shows that the length of the included 43%-GC section averages 1.7 times the length of the included 37%-GC section, a ratio suggesting an average length somewhat less than 0.27 for the fragments originally selected. These results show that the 37%-GC section is in fact bounded by DNA of about 43% GC on the right. All the boundaries indicated in Figure 8 are therefore reasonably sharp.
4. Discussion Our results, summarized in the model of Figure 8, identify six sections of differing GC content in the DNA of phage h. Owing to the limited resolution possible by density analysis, lengths given for the three shorter sections are not very exact. Lengths of the three longer sections are exact except that any errors elsewhere are compensated equally in the 43%-GC and 48.5°/0-GC sections. Owing to the same limitation, any sections much shorter than the fractional length 0.06, and of course sections differing little in composition from their neighbors, will have been missed. Molecular ends are an exception because they can be directly isolated, and we examined them down to length 0.01.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
13
The model agrees with a few data not used in its construction. First, it correctly predicts a GC content of 49.7% for the molecule as a whole. Second, it permits calculation of a density distribution for half-length fragments, shown in Figure 10, that resembles the observed distributions in Figure 1. The exact shape of the theoretical distribution depends on the assumed variation in lengths, and is therefore less critical than the relation between length and density, also shown in Figure 10 and repeatedly verified in the course of our work. Third, our model is consistent with the results Length
0.56 IS 1 0.56
0.65 O-75
I.00 0.65 0.56 0.49
0.44
0.42 0.29
7 4
0 45
55
50 CC content
60
(mole%)
FIG. 10. Theoretical distribution of half-length fragments according to GC content. Assumptions: molecular structure as in Fig. 8; each molecule broken once to generate a Gaussian distribution of lengths with mean 0.5 and vaziance 0.01. The lengths corresponding to class intervals of 0.5 percentage unit in GC content were calculated from Fig. 8. Frequencies and weights of fragments in each length class were calculated from the specified distribution according to Burgi & Hershey (1961). The dashed line redistributes the fragments of highest GC content as they might appear in a density gradient.
of spectrophotometric analysis (Hirschman, Gellert, Falkow & Felsenfeld, 1967), which assign 44% GC to 0.58 of the molecular length, and 58% GC to 0.42 of the length. Another sort of check is only partly satisfactory. Inman (1966) detected three microscopically visible zones of preferential denaturation in h DNA heated in the presence of formaldehyde, and suggested that they correspond to regions of low GC content. The main one, at the center of the molecule, is evidently our 37%-GC section. Two others have midpoints at 0.73 and 0.98 molecular length from one (presumably the left) end. Inman’s right-terminal zone is probably our 42%.GC section. His third zone is not explained by our model and may signify, as he suggests, that his results depend on sequence of nucleotides as well as composition; or, of course, that our model is inaccurate. Nandi et al. (1965) and Cohen, Maitra & Hurwitz (1967) reported only two density classes among mercury complexes of half-length fragments of X DNA. Several possible reasons can be suggested for the difference between their results and ours. An excessive stirring speed, by destroying the longer left-terminal fragments, gives products with a bimodal density distribution. A central deletion in the DNA molecule has a similar effect. The hb, deletion mutant, for example, contains DNA showing only two bands after breakage into halves (Burgi, unpublished results). In this phage, the deletion has removed the 37%.GC section of the DNA and, what is more important for the density distribution, has brought the boundary between sections of high and low GC content close to the molecular center (Skalka & Burgi, 1967). Finally, resolution in a density gradient depends strongly on the ratio of mercury to DNA.
14
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
The distribution of nucleotides in ;\ DNA is remarkable equally for the great difference in composition of segments and for the sharpness of the boundaries between segments. Evidently any evolutionary factors conjectured to account for the interspecies diversity and intramolecular homogeneity of bacterial DNA’s (Sueoka, 1962; Freese, 1962) apply to ;\ DNA too, but only at the segmental level. The relative homogeneity of the DNA of E. coli, which contains 51% GC and is 100 times longer than A DNA, is brought out by the fact that it contains no segments, of the order of size of X DNA, having a GC content as high as 58% (Rownd, Nakaya & Nakamura, 1966; see also Hershey & Burgi, 1963). In h DNA, one can show to a first approximation that intrasegmental homogeneity prevails down to gene-size fragments (Hershey & Burgi, 1966). These facts seem to call for rather specific hypotheses. We begin with the assumption that nucleotide composition in DNA reflects a history of selection. Selection for a specified, uniform composition could be direct or indirect. We propose, 6rst, a scheme for indirect selection. Suppose that each species of DNA contains a small fraction of distinctive base sequences that serve critical functions. Such critical sequences might include signals directing replication of DNA and translation of its message as well as anticodons specifying functionally crucial parts of many proteins. Each species could gain by protection of its critical sequences against excessive rates of mutational change in sense, and it would simplify matters if the critical sequences shared common features. Therefore critical sequences in DNA and mutational habit might be coadapted in each species, a result brought about by selection among genes controlling mutational habit. The chosen genes might be expected to have a characteristic effect, largely unrelated to the basis of their selection, on mutation in subcritical regions of the DNA. If so, a species-specific nucleotide composition in the DNA as a whole would result. According to this scheme, nuoleotide composition in DNA reflects mainly the genetically controlled mutational equilibrium AT + GC. The last hypothesis, but not a selection mechanism, was proposed by Sueoka (1962) and Freese (1962). It has been verified in part by the demonstration that a mutator gene found in a certain strain of E. coli specifically favors the AT-tGC transversion, presumably in all parts of the DNA (Yanofsky, Cox & Horn, 1966). The same scheme of indirect selection can account for DNA structure in X if one supposes that the several segments in h DNA originated independently of one another, and came together too recently to have acquired a common species character. That possibility cannot be directly disproved and we suggest a testable alternative. According to ideas already presented, each differentiated segment in h DNA, whatever its origin, has its own set of critical nucleotide sequences, each set adapted to a different mutational habit. Suppose that mutational habit in this instance is determined directly by the structure of enzymes responsible for DNA synthesis (Speyer, 1965). Suppose too that during the lytic cycle of phage growth, each segment of the h DNA molecule is copied by a different enzyme, which in turn impresses its mark on the composition of the segment. (To make this scheme work, mutation rates during the replication of prophage would have to be relatively low, an arrangement that makes sense also for other reasons.) In this way diversification of critical sequences could be retained to serve a useful purpose, notably in the timing of phage functions (Skalka, 1966). According to the hypothesis of indirect selection, diversification of critical sequences may or may not be accompanied by a segmental distribution of nucleotides.
DISTRIBUTION
OF NUCLEOTIDES
IN h
15
Completely different hypotheses are admissible if one postulates that average nucleotide composition can itself influence DNA function. Then direct selection for the given composition is possible. For example, rate of protein synthesis could depend in a species-specific way on nucleotide composition (choice of synonymous codons) and rate of transcription could depend directly on coadaptation of polymerase and secondary structure in DNA. Here too the selection responsible for h DNA structure could be operating currently or not, and we leave to the reader formulation of specific hypotheses of direct selection applicable to present-day X. We note, however, that some relation between composition and function is seen in the fact that the section of h DNA rich in GC corresponds to the late-functioning genes A through J responsible for head and tail formation (Skalka & Burgi, 1967). If the relation is direct, all phages might be expected to show segmentally distributed nucleotides, except insofar as different means can serve similar ends. Two clues seem ambiguous. First, the average composition of X DNA is very similar to that of E. coli DNA, which may signify an adaptive economy or merely that a random assortment of DNA species is likely to contain about 50% CC. Second, known phages other than h do not seem to have DNA’s containing segmentally distributed nucleotides (Doty, Marmur & Sueoka, 1959; Hershey, Burgi, Frankel, Goldberg & Ingraham, 1962; Miyazawa & Thomas, 1965). However, few phage species have been examined in pertinent ways and it could turn out that X is just the extreme case of a general rule for phages. Segmental distribution of nucleotides has been detected in modified fertility factors from E. coli (Marmur et al., 1961; Rownd et aE., 1966). These episomes lack the species status of h but raise similar questions. The one feature we anticipated in the structure of X DNA failed to materialize. Lambda DNA is potentially, sometimes actually, circular. There was small chance, we thought, that the molecular ends should coincide with one of the boundaries between sections of unlike composition. Nevertheless, we find that left and right ends of length 0.01 differ by six percentage units in GC content. Apparently X DNA is not circular in origin or function in terms of the hypotheses just discussed. But perhaps composition and function are indeed related, and the molecular ends were chosen to beget a proper genetic map, which ought not to encourage recombination within
functional
units (Stahl, 1967).
This investigation was supported in part by U.S. Public Health Service research grant no. HD01228 from the National Institute of Child Health and Human Development. Laura Ingraham performed some early experiments. John Cairns and Joseph Speyer helped to clarify ideas. Jon Weil criticized the manuscript. REFERENCES Bnrgi, E. (1963). Proc. Nut. dcad. Sci., WC&. 49, 151. Burgi, E. & Hershey, A. D. (1961). J. Mol. Biol. 3, 458. Bnrgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309. Care, L. G. (1965). Virology, 25, 226. Cohen, S. N., Maitra, U. & Hnrwitz, J. (1967). J. Mol. Biol. 26, 19. Doty, P., Marmnr, J. & Sueoka, N. (1959). Brookhaven Symp. BioZ. 12, 1. Flamm, W. G., Bond, H. E. & Burr, H. E. (1966). Biochim. biophys. Acta, 129, 310. Freese, E. (1962). J. Theoret. BioZ. 3, 82. Hershey, A. D. (1964). Carnegie Inst. Wash. Yearboole, 63, 577. Hershey, A. D. & Burgi, E. (1963). In Hershey, A. D., Carnegie Inst. Wash. Yearbook, 62, 481.
A. SKALKA,
16
E. BURG1
AND
A. D. HERSHEY
Hershey, A. D. h Burgi, E. (1965). Proc. Nat. Acad. Sci., Wash. 53, 325. Hershey, A. D. & Burgi, E. (1966). In Hershey, A. D., Carnegie Inst. Wmh.
Yearbook,
65,
559.
Hershey, A. D., Burgi, E. & Davern, Hershey, A. D., Burgi, E., Frankel, Inst.
Wash.
Yearbook,
C. I. (1965). Biochem. Biophys. Res. Comm. 18, 675. F., Goldberg, E. & Ingraham, L. (1962). Carnegie
61, 443.
Hershey, A. D., Burgi, E. & Ingraham, L. (1962). Biophys. J. 2, 423. Hirschman, S. Z., Gellert, M., Falkow, S. & Felsenfeld, G. (1967). J. Mol. Biol. 28, 469. Hogness, D. S. & Simmons, J. R. (1964). J. Mol. Biol. 9, 411. Inman, R. B. (1966). J. Mol. BioZ. 18, 464. Kayajanian, G. & Campbell, A. (1966). ViTiroZogy,30, 482. Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 552. Marmur, J., Rownd, R., Falkow, S., Baron, L. S., Schildkraut, C. & Doty, P. (1961). Proc.
Nat.
Acad.
Sci.,
Wash. 47, 972.
Miyazawa, Y. & Thomas, C. A., Jr. (1965). J. Mol. BioZ. 11, 223. Nandi, U. S., Wang, J. C. & Davidson, N. (1965). Biochemistry, 4, 1687. Rownd, R., Nakaya, R. & Nakamura, A. (1966). J. Mol. BioZ. 17, 376. Skalka, A. (1966). Proc. Nut. Acad. Sci., Wa-sh. 55, 1190. Skalka, A. & Burgi, E. (1967). In Hershey, A. D., Carnegie In&. WC&. Yearbook, 66, 657. Skalka, A., Butler, B. & Echols, H. (1967). Proc. Nat. Acad. Sci., Wash. 58, 576. Speyer, J. F. (1965). Biochem. Biophys. Res. Comm. 21, 6. Stahl, F. W. (1967). J. Cell Physiol. 70, Supp. 1, 1. Sueoka, N. (1962). Proc. Nat. Acad. Sci., Wash. 48, 582. Wu, R. & Kaiser, A. D. (1967). Proc. Nat. Acad. Sci., Wash. 57, 170. Yanofsky, C., Cox, E. C. & Horn, V. (1966). Proc. Nat. Acad. Sci., Wash. 55, 274.
Segmental Distribution of Nucleotides in the DNA of Bacteriophage Lambda A. SKALEA, ELIZABETH BURCI
AND
A. D. HERSHEY
Carnegie Institution, Genetics Research Unit Cold Spring Harbor, N.Y., U.X.A. (Received 6 November 1967) DNA molecules isolated from phage h were fragmented by shear and fractionated in various ways, mainly with respect to buoyant density of mercury complexes in CsaSO,. Six segments of different composition were found, ranging in G + C content from 37 to 57 mole %. The lengths and left-to-right sequence of segments were determined. The segments are reasonably homogeneous internally and the boundaries between segments rather sharp.
1. Introduction The DNA of phage X contains long intramolecular segments of differing nucleotide composition that can be demonstrated qualitatively by thermal, pycnometric, and chromatographic means (Hershey, Burgi, Frankel, Goldberg & Ingraham, 1962; Hershey & Burgi, 1963; Hogness & Simmons, 1964; Hershey, 1964). Hogness & Simmons (1964) separated molecular halves from the DNA of a defective, galtransducing line of the phage, and showed by recovery of genetic markers that the fragment richer in GCt corresponds to the left portion of the genetic map (gal in hdg; genes A to J inclusive in wild type). Their conclusion has been confirmed by physical analysis of the DNA’s of the two phage lines (Skalka, Butler t Eohols, 1967; concerning the relation between hag and wild type, see also Kayajanian & Campbell, 1966, and Skalka & Burgi, 1967). One may therefore refer to a left molecular half containing, in wild-type X, 55% GC, and a right molecular half containing 45% (Hershey, 1964). We report here further details of the intramolecular nucleotide distribution. Our analysis is based on four principles. (1) Natural DNA molecules can be broken into fragments of various sizes by subjecting them to appropriate rates of hydrodynamic shear (Hershey, Burgi & Ingraham, 1962). (2) Mercuric ions combine reversibly with DNA and preferentially with parts rich in AT to form complexes the buoyant density of which depends strongly on metal content (Nandi, Wang & Davidson, 1965). (3) The resolving power of preparative density-gradient centrifugation in an angle rotor is greater than in a swinging bucket rotor (Hershey, Burgi & Davern, 1965; Flamm, Bond & Burr, 1966). (4) Owing to the complementary structure of the molecular ends, terminal fragments of h DNA can be isolated independently of their length and composition (Hershey & Burgi, 1965.) t Abbreviations used: GC, guanine + cytosine; AT, adenine + thymine; rf, molar ratio of HgCI, to nucleotides. 1 1
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
2. Materials and Methods (a) DNA preparation The DNA described in this paper came from a clear-plaque (cr) mutant of X originally received from J. Weigle. This phage has the same DNA content as Kaiser’s wild-type X (Burgi, 1963; Care, 1965). It was grown on Escherichia co&i K12 W3110, also from Weigle. DNA was extracted by the phenol method, often with the addition of sodium dodecylsulfate (Burgi, 1963), and dialyzed against 0.1 or 0.01 i%-Na,SO,, buffered if at all with 0.001 M-SOdiUm borate. The DNA was usually labeled with 3ZP at 1 pc/pg P or with 3H to give lo5 or less disintegrations/min/~g DNA. DNA from cultures grown in nutrient broth and fed r3H]thymidine contained counts in thymidylic acid only. DNA from cultures grown in glucose-mineral medium and fed tracer amounts of [3HJuraeil contained counts in deoxycytidylic acid only. Since selective labeling biases the density distribution of DNA, we usually analyzed 32P-labeled fragments, and used 3H-labeled fragments only to mark positions in the gradients. DNA concentrations were measured spectrometrically at 260 mp from the nominal extinction coefficient 0.02 cms/pg. 32P-labeled DNA was hydrolyzed at very low concentrations with pancreatic deoxyribonuclease and venom phosphodiesterase, and subjected to electrophoresis on paper strips (Wu & Kaiser, 1967; Markham & Smith, 1952). Radioactivity was found only in bands corresponding to the four nucleotides, and results conformed to the rules G = C and A = T. Measurements were usually made in duplicate. When a single figure is cited, it is an average of two or more measurements. (c) Breakage by shear DNA solutions in O-1 M-NasS04 were stirred by a razor blade rotating at speeds up to about 30,000 rev./mm (Hershey, Burgi & Ingraham, 1962; a Virtis stirrer was used for the highest speeds). Lengths of fragments were measured from sedimentation rates according to Burgi & Hershey (1963), and are expressed as fractions of the molecular length of h DNA (17.3 p, molecular weight 31 x 106; Care, 1965). The smallest length produced by stirring was 0.06; shorter fragments were produced by sonic treatment. (d) Mercury
complexes
For density-gradient centrifugation, we prepared solutions containing, per ml., 5 to 50 pg of DNA, 0.05 ml. of 0.1 M-sodium borate, and the amounts of C&SO4 and HgCl, recommended below. The manner of mixing is not critical. However, exposure of DNA to an excess of mercury causes irreversible denaturation and we consider it good practice to pour a solution containing all the other ingredients into a vessel containing the required amount of HgCl, solution. The mixtures were kept cold pending centrifugation. The resolution achieved in density-gradient centrifugation depends in a complicated way on the DNA species present in the mixture, the GC content of the components of interest, and the r, value. Optimum conditions are therefore best defined in terms of concentration of CssS04, which, moreover, is more easily controlled than the effective Hg/DNA ratio, since that depends on the purity of the DNA and other reagents. Then the proper r, is that necessary to form complexes of specified densities. For analysis of /\ DNA fragments, we used 43% by weight of Cs,SO, for optimum resolution in the low-GC part of the distribution, and 44% when we were more interested in the high-GC part. C&SO4 of nominal purity 99.9% (Kawecki Chemical Co., New York) proved satisfactory. When the mixture contained mainly unfractionated h DNA, the required r, corresponding to the stated CssSO, concentrations was about 0.24 and 0.28, respectively. When tracer amounts of labeled DNA were analyzed, we added at least 5 fig of unlabeled T2 DNA/ml., sheared to reduce its viscosity, to the mixture. Since T2 DNA combines strongly with mercury, the proper r, under these conditions is about O-35 at 43% Cs2SOI, and 0.40 at 44%. T2 DNA goes to the bottom of the tube when X DNA is visible in the gradient.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
3
(e) Centrifugution. We centrifuged 4-ml. or 6.17-g samples, overlaid with mineral oil in cellulose nitrate tubes 5/8 in. in diameter, in a Spinco type 40 or Ti50 rotor, for 48 hr at 36,000 rev./min and 4°C. If the rotor is spun for 8 hr at 45,000 rev./mm, one can often check band positions in the tubes with a radiation monitor, and then complete the run at the lower speed. A preliminary run at 44,770 rev./min in an analytical centrifuge serves the same purpose. We have no difficulty reproducing results with a given DNA preparation, but sometimes have to vary the Hg/DNA ratio to suit different preparations. (f) Joining
and disjoining
termkal fragments
To ensure that a stirred DNA sample contained only disjoined fragments, it was examined immediately after stirring or heated briefly to 65°C (in 0.01 M-NasSO,) or to 75’C (in 0.1 M-NaeSO,) to disjoin them. No joining of terminal fragments occurs during centrifug&ion under the conditions described above. For deliberate joining of terminal fragments, we annealed solutions containing 10 to 50 pg of DNA/ml. in O-6 M-Nacl as described by Hershey & Burgi (1965). On a few occasions we observed reasonably efficient joining in CseSO+ solutions, prepared as described in section (d) but lacking the buffer and HgCl,, on heating at 30°C for several hours. This procedure is convenient when the DNA is to be analyzed by density-gradient centrifugation, but we have not determined the optimal time and temperature of heating. (g) Physical
analysis
At the end of the run, we collected 25 to 40 fractions dropwise from the bottoms of the tubes into 1 nn-NaCl or O-1 M-EDTA, measured radioactivity, pooled chosen fractions, and dialyzed them first against 1 M-NaCl or 0.1 M-EDTA to remove mercury, then into 0.01 MNaeSOB. The solutions were concentrated, if necessary, under a current of air at 45’C or by dialysis against dry sucrose. A typical analysis required molecular weight measurements before and after further fragmentation by stirring, and return to a Hg-CseSO, gradient. This time the mixture usually contained, besides the 32P-labeled materials to be analyzed, 3H-labeled density markers and unlabeled T2 DNA fragments. Results of this type of analysis must be interpreted cautiously because the density of the Hg-DNA complexes depends on secondary structure as well as composition. We usually find that differently labeled samples of DNA banded in mixture are distributed on the same density scale. 3H-labeled fragments, for example, may be stored at 2 to 5 pg/ml. for weeks or months without change. On the other hand, 3aP-labeled fragments, after repeated banding, usually show a slightly higher density than their counterparts in the marker DNA added to the mixture. The shift in density can be attributed to effects of radiation on DNA concentrated in the bands, though an irreversible effect of the exposure to mercury is not excluded. Since all components of a mixture are usually displaced equally, no difficulty arises in the identification of bands. However, it is a defect of the method that the affinity of DNA fragments for mercury may depend on their history as well as their composition. Fragmentation of DNA by stirring does not itself affect the weight-average density. The smaller fragments produced in a French press or by sonic treatment do exhibit a slightly excessive density in Hg-CseSO, mixtures. This can be seen when the fragments are banded, together with a trace of unbroken DNA, in the uniform density-gradient generated in an analytical centrifuge. The unbroken DNA, visible as a pip on the densitometer tracing, does not quite coincide with the center of gravity of the very broad distribution of the fragments. Since the density shift depends on size of fragments rather than manner of production, and cannot be reversed by annealing, it may signify preferential binding of mercury at the ends of the fragments. (h) Recovery of DNA Losses of DNA during centrifugation, measured as radioactivity remaining in the tubes after collection of fractions, did not exceed 2% or so for DNA fragments produced by stirring, or 10% for DNA subjected to sonic treatment. Losses were not significantly larger when fractionated material was rebanded, always, of course, in the presence of unlabeled DNA carrier.
4
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
Losses of half the DNA were sometimes seen during concentration of solutions in a rotating evaporator, a procedure that we abandoned for that reason. However, it was reassuring in such cases to note that the recovered DNA fragments showed the expected distribution of densities in Hg-CssSO,, a result indicating that the losses were random
and the recovered material unaltered.
3. Results (a) Half-length
fragments
Half-length fragments of DNA may be defined operationally as breakage products produced by stirring a solution of uniform molecules at the critical speed that causes slow breakage. Then most of the molecules, if broken at all, break once, but some of the longest fragments produced by acentric breaks are broken again (Burgi & Hershey, 1961).
Fraction
no.
FIG. 1. Density distribution of half-length fragments. Upper part. A sample of s2P-labeled DNA stirred for 30 min at 12 pg/ml. and 2160 rev./min, and banded with unlabeled T2 DNA in 43.8% CszSOI at rr 0.39. Density increases from right to left. Lower part. The histogram shows the same 3aP-labeled DNA banded under the seme conditions after removal of unbroken molecules by fractional sedimentation through sucrose. Circled points show admixed DNA, 3H-labeled in cytosine, that had been stirred for 30 mm at 5 pg/ml. and 2220 rev./mm to cause complete breakage.
Egure 1 illustrates the distribution of half-length fragments of h DNA in a HgC&SO, gradient. Two samples are compared, one in which about half of the molecules are broken and one in which all are broken. The two sets of breakage products exhibit distributions that coincide when corrected for the fact that one set is labeled only in cytosine. The results show that breakage at the critical speed of stirring produces fragments of three density classes. How that happens can be understood as follows.
DISTRIBUTION
OF NUCLEOTIDES
IN
5
X
The fragments denser than unbroken DNA, which form the left band in Figure 1, comprise half the mass of the broken DNA. These are right-terminal fragments, containing 45% GC. The remainder of the broken DNA, less dense than the intact molecules, must be left-terminal fragments. Since unbroken molecules and right-terminal fragments are uniform in density, the varying density of left-terminal fragments must reflect variations in length. Lengths of fragments recovered from distributions of the sort illustrated were measured on several occasions. DNA corresponding to the center fraction of the right band in Figure 1 consists of pieces ranging in length from about 0.43 downward (weightaverage length O-35). DNA corresponding to the center fraction of the second band from the right consists of pieces ranging in length from about O-50 to 0.65 (weight average 0.56). DNA corresponding to the left band in Figure 1, like unfractionated DNA broken at the critical speed of stirring, consists of half-length fragments together with longer and shorter pieces. We obtained results consistent with the above by reversing the procedure. We first fractionated partially broken X DNA by sedimentation through sucrose, selecting authentic halves. These, spun in Hg-Cs,SO, together with marker DNA like that of Figure 1, formed only two bands, one coinciding with the left band of the marker, the other rather broad but centered between the two right bands of the marker. We conclude that the h DNA molecule consists of at least two segments differing in GC content, and that the boundary between the segments lies to the left of the molecular center. Right-terminal fragments of various lengths therefore differ little in density and form the left band in Figure 1. Left-terminal fragments are uniform in density only when short, and the short fragments form the right band in Figure 1. The remaining left-terminal fragments span the boundary separating DNA of unlike GC content and have a density that depends on length. A similar conclusion was drawn from the appearance of the bands formed in CsCl (Hershey & Burgi, 1965). (b) The AT-rich segment Figure 2 shows the density distribution of fragments of length 0.11. It reveals three components. Since the component of greatest density comprises about 10% of the DNA, or one fragment per molecule, it must come from a single stretch of low GC content spanning about 10% of the molecular length. Figure 2 also shows that the molecule contains DNA of high GC content in 44% of its length, and DNA of intermediate GC content in 46% of its length. The position of the AT-rich segment in the molecule was determined by the following experiment.
5
IO
15 Fraction
FIG. 2. Density
20
25
30
no.
distribution of fragments of length 0.11. 3ZP-labeled /min and banded wih unlabeled T2 DNA in 43% Cs,S04 at r, 0.36.
DNA
stirred
at 10,000
6
A.
SKALKA,
E.
BURG1
AND
A.
D.
HERSHEY
A solution containing 23 pg of 32P-labeled DNA/ml. was stirred for 30 minutes at the minimum speed (2600 rev./min) producing complete breakage at that concentration, and analyzed in Hg-Cs,SO, to give a result like the one shown in the lower part of Figure 1. DNA recovered from several individual fractions was freed from Hg, characterized with respect to length, restirred at 10,000 rev./min to produce fragments of length 0.11, and rebanded in Hg-C&SO, together with density markers. 30
20 1
p
IO-
IO
15
20
25
I
: I
30
t
1.5
IO
Fraction
20
2.5
d 30
I
no.
FIG. 3. Density distribution of fragments from left molecular ends. Left part. Histogram: 32P-labeled left molecular ends of length 0.43 and less, originating in the equivalent of fraction 32 in the lower histogram of Fig. 1, sheared to fragments of length 0.11. Broken line: unfractionated DNA, 3H-labeled in cytosine, sheared to the actme size. Mixture banded with unlabeled T2 DNA in 43.8% Cs,S04 at rf 0.35. Right part. Same, except 32P-labeled fragments derived from left molecular ends of weightaverage length 0.56, originating in the equivalent of fraction 25 in the lower histogram of Fig. 1.
As shown in Figure 3, left molecular ends of length 0.43 and less yielded on further breakage fragments of uniformly high GC content. The same Figure shows that left ends of length 056 yielded on further breakage mainly fragments of high GC content, about 15% of fragments rich in AT, and no fragments of characteristic composition in between. (Figure 3 also illustrates the shift in density resulting from molecular damage sustained during fractionation.) Longer left-terminal fragments (lengths 057 to 0.65, taken from the fraction corresponding to number 24 in the lower histogram of Fig. 1) yielded a result similar to that shown in the right part of Figure 3, except that the band of AT-rich DNA was somewhat larger and an additional band, containing about 10% of the DNA in fragments of intermediate CC content, was visible. Right-terminal fragments, taken from the fraction corresponding to number 13 in the lower histogram of Figure 1, yielded on further breakage mainly pieces of intermediate GC content, a considerable fraction of low GC content, and only 2% of high GC content. The last must have come from a small fraction of the original right-terminal fragments, presumably the longest ones. The main results were verified in a second experiment, in which isolated molecular ends were sheared to length 0.06 before analysis.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
7
We concluded at this stage that the A DNA molecule contains a section of length about 0.1 and high AT content interposed between a section of high GC content, length O-44, on its left, and a section of intermediate composition, length 0.46, on its right. (G) Resolution and nucleotide composition of small fragments The resolution that can be achieved by density analysis depends in two opposing ways on the size of fragments examined. As size is reduced, the efficiency of sampling of molecular segments increases, but the number of classes into which the fragments can be separated diminishes. The rate of diffusion of the fragments sets one limit, but there are other complications too (section 2(g)). Some exploratory work with fragments produced by sonic treatment and high-pressure cells was not very encouraging (Hershey & Burgi, 1966).
5
40 35
0
5
IO
15 Fraction
20
25
30
0
no.
FIG. 4. Density distribution of fragments of length O-06, and composition of DNA in the fractions. banded in 42.8% The histogram shows s2P-labeled DNA, stirred at about 30,00Orev./min, Cs2S04 at rr 0.22 (ordinate scale on the right). The curve shows nucleotide compositions of the fr&tio-ns (s&e on the left), and combines results of two experiments performed at different times that showed practically identical distributions. Many points on the curve are averages of two or three measurements, so that all measurements made (48) sre represented. Arrows indicate positions of the three bands given by fragments of length 0.11 (Fig. 2), known from other experiments in which differently labeled fragments of the two lengths were banded in mixture.
Reduction in length of fragments from 0.11 (as in Fig. 2) to 0.06 does reveal further however. This is shown in the histogram of Figure 4. The band containing Gagments of highest GC content is unchanged, as expected if these fragments originate from a single segment of uniform GC content. The tail of lowest GC content is moved slightly to the left, but its area is not greatly affected. The main effect of the reduction in size of fragments is to split the band of intermediate GC content into two equal parts, both of which must originate from the right-terminal section of the molecule. Considered by itsebf, the histogram in Figure 4 shows that when molecules of X DNA are broken into 17 pieces, the fragments assort themselves into four density classes. The four components of the distribution must come from at least four different segments in the molecules. Before pursuing the origin of the components, we report analyses of their actual nuoleotide compositions. detail,
8
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
Our preparations of h DNA contain about 50.5% GC by direct nucleotide analysis and 50% GC computed from the buoyant density in CsCl (l-709 g/ml. measured against E. co&i DNA of nominal GC content 51% and density l-71 ; Hershey, 1964). The compositions of fragments of length O-06, resolved in a Hg-Cs,SC, gradient, are shown in Figure 4. The four components of the density distribution measure 37, 43, 48.5 and 57% in GC content. Note that the 57%-GC fragments appear to be remarkably uniform in composition. A suggestion to the same effect is visible in the other major components, but they are less well resolved. The approximately linear relation between GC content and position in the density gradient is accidental in two respects. The density gradient itself, in the expanded form produced by righting the tubes, is not uniform but falls smoothly from about O-044 g/ml. /ml. near the bottom to about 0.029 near the top (computed from measurements of refractive index). Independently of the density gradient, an increase in r, from 0.2 to O-3 contracts the high-density part of the distribution and expands the low-density part. (d) The right-terminal
section
We concluded from results just described that the right-terminal section of length O-46 breaks into fragments half of which average 43% and half 48.5% in GC content. To learn something about the origin of these fragments, we looked at right molecular ends isolated in two somewhat different ways. In a first experiment, two samples of DNA, 3H-labeled in cytosine, were separately stirred to produce half-length and third-length fragments, pooled, and fractionated in Hg-Cs,SO,. About 34% of the DNA was recovered from the compact band of DNA of low GC content. After removal of Hg, these fragments were annealed with intact, unlabeled DNA at 25 pg/ml., and the mixture was sedimented into a 25ml. sucrose concentration gradient to permit separation of labeled terminal fragments joined by their cohesive sites to the unlabeled DNA. After these were recovered and detached from the unlabeled DNA by heating, they were sedimented again into sucrose to separate labeled, right-terminal fragments of different sizes (lengths O-43, O-35 and 0.26, as determined by additional sedimentation measurements.) In a second experiment directed to the same purpose, 32P-labeled DNA was sheared to fragments of length about O-3, annealed to join the molecular ends, and banded in Hg-Cs,SO,. (All applications of this technique are similar in principle; one example is shown in Fig. 6 and another is described in section 3(f).) Three bands were seen, of which the middle one, containing 46% of the DNA mainly in the form of joined ends, was recovered. The recovered DNA was heated to disjoin cohesive sites, and returned to Hg-Cs,SO, to separate right and left molecular ends. This time right ends were recovered, dialyzed free from mercury, and fractionated with respect to sedimentation rate to yield lengths 0.32, O-25 and 0.15. The two experiments provided six samples of right-terminal fragments falling into four length classes. All six samples were sheared to pieces of the same size, and analyzed by banding again in Hg-Cs,SO,. The results may be summarized as follows. (1) Right-terminal fragments of length 0.43, broken to pieces of length O-06, yielded equal amounts of the two components previously identified as containing 43 and 48.5% GC (Fig. 5(a)). (2) None of the right-terminal fragments isolated in the first experiment contained appreciable amounts of 37 or 57°/Q-GCDNA. This is illustrated for fragments of length
DISTRIBUTION
(b)
OF
NUCLEOTIDES
IN
h
9
Cd)
cc content (%) FIG. 5. Distribution of nucleotides among subsections of length 0.06 derived from right molecular ends. (a) Ends of original length 0.43; (b) ends of original length 0.32; (0) ends of original length 0.25; (d) ends of original length 0.15. The scale of CC content indicates approximate positions of the four bands given by the marker DNA included in each mixture.
O-43 in Figure 5(a), and was more clearly evident on analysis of fragments of lengths O-35 and O-26 (not shown). Right-terminal fragments isolated in the second experiment (Fig. 5(b), (c), (d) ) did contain small amounts of 37 and 57%-GC DNA, probably owing to contamination with left and central portions of the DNA molecule. That right halves do not contain 57%.GC DNA was shown in section 3(a). (3) If right-terminal fragments contain DNA of only two classes, 43%-GC and 48-5%-G-C, the relation between length of fragment and size of either class should depend in a simple way on structure. For the length series O-43, 0.32, 0.25 and 0.15 illustrated in Figure 5, the 43%-GC class expressed as a fraction of both classes measured 50,41,29 and 33%. The same trend was evident in data not shown. Therefore most of the 43% DNA lies toward the molecular center, but some also close to the right end. Specifically, breakage of right-terminal fragments of length about O-15 into two or three pieces yields one-third 43%-GC DNA and two-thirds 48.5%. These facts, together with results presented next, lead to a precise model for the distribution of nucleotides among subsections of length O-06. (e) Xhort molecular ends We analyzed short fragments from each molecular end in the following way. 32Plabeled DNA at 50 pg/ml. in O-6 M-NaCl was stirred for 30 minutes at 10,000 rev./min and the solution was heated to 75°C and cooled slowly to permit the terminal fragments to join. After the solution had been dialyzed and banded in Hg-Cs,SO,, the
Fraction no. FIG. 6. Density distribution of fragments of length 0.14 with terminal The mixture was banded in 43.8% Cs,B04 at TV0.3.
pieces joined by annealing.
10
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
distribution in Figure 6 was seen. Here the joined ends form the center band. (A sample of the same DNA fragments similarly analyzed without annealing yielded only two bands separated by three fractions containing less than 4% of the DNA.) DNA recovered from the center band, heated to disjoin the fragments and returned to a Hg-Cs,SO, gradient, formed two widely separated bands. These yielded right and left ends of length about 0.2. Actual lengths and nucleotide compositions from two experiments are given in Table 1. Samples of each terminal fragment were sheared further by sonic treatment, yielding pieces among which about one in twelve should possess a terminal cohesive site. To analyze these, small fragments of each kind were annealed in a mixture with weakly “H-labeled molecular halves at 20 pg/ml., left with right and right with left, and sedimented through sucrose to bring the half-length fragments well down the tubes. In these tubes, 3 to 5% of the 32P-labeled DNA sedimented with the halves, and was recovered for nucleotide analysis. In a second experiment of this kind, the doubly labeled complexes were sedimented twice into sucrose before analysis. Results of the centrifugation are illustrated in Figure 7. Lengths and compositions of the recovered molecular ends are given in Table 1. Two sorts of control experiment were performed. First, we verified that the 32Plabeled fragments did not join with 3H-labeled halves when alanealed in left-left and right-right combinations. This could mean that the joining occurs by means of terminal cohesive sites as expected, or that fragments of low GC content recognize fragments of high GC content also for other reasons. The second possibility we excluded by examining 32P-labeled fragments, lacking cohesive sites, recovered from
Fraction
no.
FIG 7. Cosedimeniation of right terminal fragments of length 0.018 and left molecular halves. Upper pa&. 32P.labeled right molecular ends of length 0.19 were broken further by sonic treatment and annealed in mixture with 3H-labeled left molecular halves at 20 pg/ml. The mixture, diluted to 10 pg/ml., was sedimented into a 25ml. sucrose concentration gradient. Histogram, *aP; broken line, 3H. Centrifugation for 13 hr at 24,000 rev./min. Sedimentation from right to left. Lowe? part. Pooled fractions 4 through 16 sedimented again into sucrose. Histogram, 32P; circles, ?H.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
11
TAEZE 1 Composition of molecular ends Experiment
Left ends Length GC (%) 0.14 0.23 0.012 0.017
1 2 1 2
Right ends Length CC (%)
56.5 56.7 48.0 50.2
0.14 0.19 0.012 0,018
46.3 46-3 42.4 41.6
the two extreme bands illustrated in Figure 6. These, broken by sonic treatment and annealed with molecular halves in left-right and right-left combinations, showed no tendency to sediment with the halves. Nevertheless, after a single centrifugation in sucrose, enough 32P-labeled DNA could be recovered with the halves for nucleotide analysis. It showed the composition reported in Table 1 for the longer terminal fragments, and therefore had moved down the tube by unspecific means. From the measurements given in Table 1 we conclude that molecular ends resemble in composition their neighboring molecular sections excepting at the extreme tips, where GC content falls at both ends. More specific models can be proposed as follows. According to Table 1, left molecular ends of length 0.14 contain 56.5% GC and left molecular ends of length 0.012 contain 48% GC. If the terminal section containing 48% GC is separated by a single boundary from DNA of 57% GC, the terminal section measures only 0.01 in length. The several calculations of this sort possible from the data in Table 1 are not very exact because we did not analyze fragments of f-46 001 .
57 0.43
37 43 0.10 0.17
48.5 -42 I 0.24 0.05
FIG. 8. Distribution of nucleotides in X DNA. The CC content in mole o/Oabove the line; fractional molecular length below.
of each segment is indicated
intermediate length, but they agree in showing that the stretch of low GC content at the left end of the molecule is very short. Similar calculations from the data for right ends in Table 1 suggest that the 48*5%GC section of the molecule terminates at the right in a segment of length about 0.05 oontaining 42% GC. This model is supported by the fact (section 3(d)) that breakage of right-terminal fragments of length about 0.15 into pieces of length 0.06 yields two pieces containing 48.5% GC to one containing 43%. All our results are summarized in Figure 8. (f) The molecular center The following experiment illustrates a useful technique, permitting isolation of DNA adjacent to a section of unique composition and delineating the boundary between sections. 32P-labeled DNA was sheared to fragments of length 0.32, heated to join molecular ends, and banded in 43% Cs,SO, at r, O-24. Three bands were seen, similar to those of Figure 6 except that the right band (free left ends) contained only 15% of the DNA
12
A.
SKALKA,
E. BURG1
AND
A.
D. HERSHEY
and the left band (free right ends and those center pieces containing little of the high-GC section) 32%. Several components of the distribution were examined to verify their identity, but only one is pertinent here. Two fractions containing 8% of the DNA, taken from the high-density end of the distribution, were combined, freed from mercury, sheared to pieces of length 0.11, and rebanded as shown in Figure 9. The fragments clearly formed two bands, which
Fraction
FIG. 9. Density
distribution
no,
of fragments
from molecular
centers.
Histogram: 32P-labeled fragments of length 0.32, selected for central origin and high AT content, sheared to fragments sheared to fragments at Tf 0.35.
of length 0.11. Broken line: unfrsctionated DNA, 3H-labeled in cytosine, of the same size. Mixture banded with unlabeled T2 DNA in 43% Cs,S04
were also seen in another run of the same material. One band contained DNA of about 37% GC, the other about 43% GC. (Nucleotide compositions of these materials were unfortunately not directly determined; the estimates given are based on density and take into account the shift in density seen in all fractions recovered in this experiment.) According to the model of Figure 8, selection for high AT content among molecular thirds should yield pieces containing most of the 37%-GC section, little or none of the 57%~GC section, and usually only part of the 43%-GC section (because selection for high AT content should also select short lengths). Figure 9 conhrms these expectations, and shows that the length of the included 43%-GC section averages 1.7 times the length of the included 37%-GC section, a ratio suggesting an average length somewhat less than 0.27 for the fragments originally selected. These results show that the 37%-GC section is in fact bounded by DNA of about 43% GC on the right. All the boundaries indicated in Figure 8 are therefore reasonably sharp.
4. Discussion Our results, summarized in the model of Figure 8, identify six sections of differing GC content in the DNA of phage h. Owing to the limited resolution possible by density analysis, lengths given for the three shorter sections are not very exact. Lengths of the three longer sections are exact except that any errors elsewhere are compensated equally in the 43%-GC and 48.5°/0-GC sections. Owing to the same limitation, any sections much shorter than the fractional length 0.06, and of course sections differing little in composition from their neighbors, will have been missed. Molecular ends are an exception because they can be directly isolated, and we examined them down to length 0.01.
DISTRIBUTION
OF
NUCLEOTIDES
IN
h
13
The model agrees with a few data not used in its construction. First, it correctly predicts a GC content of 49.7% for the molecule as a whole. Second, it permits calculation of a density distribution for half-length fragments, shown in Figure 10, that resembles the observed distributions in Figure 1. The exact shape of the theoretical distribution depends on the assumed variation in lengths, and is therefore less critical than the relation between length and density, also shown in Figure 10 and repeatedly verified in the course of our work. Third, our model is consistent with the results Length
0.56 IS 1 0.56
0.65 O-75
I.00 0.65 0.56 0.49
0.44
0.42 0.29
7 4
0 45
55
50 CC content
60
(mole%)
FIG. 10. Theoretical distribution of half-length fragments according to GC content. Assumptions: molecular structure as in Fig. 8; each molecule broken once to generate a Gaussian distribution of lengths with mean 0.5 and vaziance 0.01. The lengths corresponding to class intervals of 0.5 percentage unit in GC content were calculated from Fig. 8. Frequencies and weights of fragments in each length class were calculated from the specified distribution according to Burgi & Hershey (1961). The dashed line redistributes the fragments of highest GC content as they might appear in a density gradient.
of spectrophotometric analysis (Hirschman, Gellert, Falkow & Felsenfeld, 1967), which assign 44% GC to 0.58 of the molecular length, and 58% GC to 0.42 of the length. Another sort of check is only partly satisfactory. Inman (1966) detected three microscopically visible zones of preferential denaturation in h DNA heated in the presence of formaldehyde, and suggested that they correspond to regions of low GC content. The main one, at the center of the molecule, is evidently our 37%-GC section. Two others have midpoints at 0.73 and 0.98 molecular length from one (presumably the left) end. Inman’s right-terminal zone is probably our 42%.GC section. His third zone is not explained by our model and may signify, as he suggests, that his results depend on sequence of nucleotides as well as composition; or, of course, that our model is inaccurate. Nandi et al. (1965) and Cohen, Maitra & Hurwitz (1967) reported only two density classes among mercury complexes of half-length fragments of X DNA. Several possible reasons can be suggested for the difference between their results and ours. An excessive stirring speed, by destroying the longer left-terminal fragments, gives products with a bimodal density distribution. A central deletion in the DNA molecule has a similar effect. The hb, deletion mutant, for example, contains DNA showing only two bands after breakage into halves (Burgi, unpublished results). In this phage, the deletion has removed the 37%.GC section of the DNA and, what is more important for the density distribution, has brought the boundary between sections of high and low GC content close to the molecular center (Skalka & Burgi, 1967). Finally, resolution in a density gradient depends strongly on the ratio of mercury to DNA.
14
A. SKALKA,
E. BURG1
AND
A. D. HERSHEY
The distribution of nucleotides in ;\ DNA is remarkable equally for the great difference in composition of segments and for the sharpness of the boundaries between segments. Evidently any evolutionary factors conjectured to account for the interspecies diversity and intramolecular homogeneity of bacterial DNA’s (Sueoka, 1962; Freese, 1962) apply to ;\ DNA too, but only at the segmental level. The relative homogeneity of the DNA of E. coli, which contains 51% GC and is 100 times longer than A DNA, is brought out by the fact that it contains no segments, of the order of size of X DNA, having a GC content as high as 58% (Rownd, Nakaya & Nakamura, 1966; see also Hershey & Burgi, 1963). In h DNA, one can show to a first approximation that intrasegmental homogeneity prevails down to gene-size fragments (Hershey & Burgi, 1966). These facts seem to call for rather specific hypotheses. We begin with the assumption that nucleotide composition in DNA reflects a history of selection. Selection for a specified, uniform composition could be direct or indirect. We propose, 6rst, a scheme for indirect selection. Suppose that each species of DNA contains a small fraction of distinctive base sequences that serve critical functions. Such critical sequences might include signals directing replication of DNA and translation of its message as well as anticodons specifying functionally crucial parts of many proteins. Each species could gain by protection of its critical sequences against excessive rates of mutational change in sense, and it would simplify matters if the critical sequences shared common features. Therefore critical sequences in DNA and mutational habit might be coadapted in each species, a result brought about by selection among genes controlling mutational habit. The chosen genes might be expected to have a characteristic effect, largely unrelated to the basis of their selection, on mutation in subcritical regions of the DNA. If so, a species-specific nucleotide composition in the DNA as a whole would result. According to this scheme, nuoleotide composition in DNA reflects mainly the genetically controlled mutational equilibrium AT + GC. The last hypothesis, but not a selection mechanism, was proposed by Sueoka (1962) and Freese (1962). It has been verified in part by the demonstration that a mutator gene found in a certain strain of E. coli specifically favors the AT-tGC transversion, presumably in all parts of the DNA (Yanofsky, Cox & Horn, 1966). The same scheme of indirect selection can account for DNA structure in X if one supposes that the several segments in h DNA originated independently of one another, and came together too recently to have acquired a common species character. That possibility cannot be directly disproved and we suggest a testable alternative. According to ideas already presented, each differentiated segment in h DNA, whatever its origin, has its own set of critical nucleotide sequences, each set adapted to a different mutational habit. Suppose that mutational habit in this instance is determined directly by the structure of enzymes responsible for DNA synthesis (Speyer, 1965). Suppose too that during the lytic cycle of phage growth, each segment of the h DNA molecule is copied by a different enzyme, which in turn impresses its mark on the composition of the segment. (To make this scheme work, mutation rates during the replication of prophage would have to be relatively low, an arrangement that makes sense also for other reasons.) In this way diversification of critical sequences could be retained to serve a useful purpose, notably in the timing of phage functions (Skalka, 1966). According to the hypothesis of indirect selection, diversification of critical sequences may or may not be accompanied by a segmental distribution of nucleotides.
DISTRIBUTION
OF NUCLEOTIDES
IN h
15
Completely different hypotheses are admissible if one postulates that average nucleotide composition can itself influence DNA function. Then direct selection for the given composition is possible. For example, rate of protein synthesis could depend in a species-specific way on nucleotide composition (choice of synonymous codons) and rate of transcription could depend directly on coadaptation of polymerase and secondary structure in DNA. Here too the selection responsible for h DNA structure could be operating currently or not, and we leave to the reader formulation of specific hypotheses of direct selection applicable to present-day X. We note, however, that some relation between composition and function is seen in the fact that the section of h DNA rich in GC corresponds to the late-functioning genes A through J responsible for head and tail formation (Skalka & Burgi, 1967). If the relation is direct, all phages might be expected to show segmentally distributed nucleotides, except insofar as different means can serve similar ends. Two clues seem ambiguous. First, the average composition of X DNA is very similar to that of E. coli DNA, which may signify an adaptive economy or merely that a random assortment of DNA species is likely to contain about 50% CC. Second, known phages other than h do not seem to have DNA’s containing segmentally distributed nucleotides (Doty, Marmur & Sueoka, 1959; Hershey, Burgi, Frankel, Goldberg & Ingraham, 1962; Miyazawa & Thomas, 1965). However, few phage species have been examined in pertinent ways and it could turn out that X is just the extreme case of a general rule for phages. Segmental distribution of nucleotides has been detected in modified fertility factors from E. coli (Marmur et al., 1961; Rownd et aE., 1966). These episomes lack the species status of h but raise similar questions. The one feature we anticipated in the structure of X DNA failed to materialize. Lambda DNA is potentially, sometimes actually, circular. There was small chance, we thought, that the molecular ends should coincide with one of the boundaries between sections of unlike composition. Nevertheless, we find that left and right ends of length 0.01 differ by six percentage units in GC content. Apparently X DNA is not circular in origin or function in terms of the hypotheses just discussed. But perhaps composition and function are indeed related, and the molecular ends were chosen to beget a proper genetic map, which ought not to encourage recombination within
functional
units (Stahl, 1967).
This investigation was supported in part by U.S. Public Health Service research grant no. HD01228 from the National Institute of Child Health and Human Development. Laura Ingraham performed some early experiments. John Cairns and Joseph Speyer helped to clarify ideas. Jon Weil criticized the manuscript. REFERENCES Bnrgi, E. (1963). Proc. Nut. dcad. Sci., WC&. 49, 151. Burgi, E. & Hershey, A. D. (1961). J. Mol. Biol. 3, 458. Bnrgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309. Care, L. G. (1965). Virology, 25, 226. Cohen, S. N., Maitra, U. & Hnrwitz, J. (1967). J. Mol. Biol. 26, 19. Doty, P., Marmnr, J. & Sueoka, N. (1959). Brookhaven Symp. BioZ. 12, 1. Flamm, W. G., Bond, H. E. & Burr, H. E. (1966). Biochim. biophys. Acta, 129, 310. Freese, E. (1962). J. Theoret. BioZ. 3, 82. Hershey, A. D. (1964). Carnegie Inst. Wash. Yearboole, 63, 577. Hershey, A. D. & Burgi, E. (1963). In Hershey, A. D., Carnegie Inst. Wash. Yearbook, 62, 481.
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Hershey, A. D. h Burgi, E. (1965). Proc. Nat. Acad. Sci., Wash. 53, 325. Hershey, A. D. & Burgi, E. (1966). In Hershey, A. D., Carnegie Inst. Wmh.
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C. I. (1965). Biochem. Biophys. Res. Comm. 18, 675. F., Goldberg, E. & Ingraham, L. (1962). Carnegie
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Hershey, A. D., Burgi, E. & Ingraham, L. (1962). Biophys. J. 2, 423. Hirschman, S. Z., Gellert, M., Falkow, S. & Felsenfeld, G. (1967). J. Mol. Biol. 28, 469. Hogness, D. S. & Simmons, J. R. (1964). J. Mol. Biol. 9, 411. Inman, R. B. (1966). J. Mol. BioZ. 18, 464. Kayajanian, G. & Campbell, A. (1966). ViTiroZogy,30, 482. Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 552. Marmur, J., Rownd, R., Falkow, S., Baron, L. S., Schildkraut, C. & Doty, P. (1961). Proc.
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Miyazawa, Y. & Thomas, C. A., Jr. (1965). J. Mol. BioZ. 11, 223. Nandi, U. S., Wang, J. C. & Davidson, N. (1965). Biochemistry, 4, 1687. Rownd, R., Nakaya, R. & Nakamura, A. (1966). J. Mol. BioZ. 17, 376. Skalka, A. (1966). Proc. Nut. Acad. Sci., Wa-sh. 55, 1190. Skalka, A. & Burgi, E. (1967). In Hershey, A. D., Carnegie In&. WC&. Yearbook, 66, 657. Skalka, A., Butler, B. & Echols, H. (1967). Proc. Nat. Acad. Sci., Wash. 58, 576. Speyer, J. F. (1965). Biochem. Biophys. Res. Comm. 21, 6. Stahl, F. W. (1967). J. Cell Physiol. 70, Supp. 1, 1. Sueoka, N. (1962). Proc. Nat. Acad. Sci., Wash. 48, 582. Wu, R. & Kaiser, A. D. (1967). Proc. Nat. Acad. Sci., Wash. 57, 170. Yanofsky, C., Cox, E. C. & Horn, V. (1966). Proc. Nat. Acad. Sci., Wash. 55, 274.