VOL. 15, 1591-1613 (1976)
BIOPOLY MERS
A Study of DNA Denaturation in the
U1t racentrif uge
GARY WIESEHAHN, THOMAS R. CECH,* and JOHN E. HEARST,?
Department of Chemistry, University of California, Berkeley,
California 94720
Synopsis
The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable
molecules melt early and are found a t heavier densities in the melting region.
An idealized ultracentrifuge melting transition is described. The melting transition of
singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band
a t all points in the melt. DNA's from mouse, D. melanogaster, M.lysodeikticus, T4, and
T7 also show equilibrium bands a t partially denatured densities, some of which are highly
asymmetric. Simple sequence satellite DNA shows a n all-or-none transition with no equilibrium bands a t partially denatured densities.
The temperature a t which a DNA denatures is an increasing function of the (G + C) content
of the DNA. The T, does not show a molecular-weight dependence in the range 1.2 X 106-1.5
X loi daltons (single strand) for mouse, M . lysodeikticus, or T 4 DNA. The mouse DNA
partially denatured bands do not change shape as a function of molecular weight. The T 4
DNA intermediate band develops a late-melting tail a t low molecular weight. M. lysodeikticus
DNA bands a t partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components a t each point in the melting
transition.
MATERIALS A N D METHODS
Mouse DNA was extracted from SVT2 mouse tissue culture cells or from
combined livers, brains, spleens, and testes of Balb/c mice as described
previously.' Mouse main band and satellite were separated preparatively
in Agf-Cs2S04 gradients according to Corneo et aL2 DNA from Drosophila
melanogaster was isolated from 24-hr embryos essentially as described by
Peacock et al.3 D. melanogaster 1.687 satellite DNA was purified by
banding total DNA (150 pg/ml) in 1.70 g/cm3 CsCl a t 40,000 r/min for 60
hr in a Beckman 60Ti rotor. The tubes were dripped into 40 fractions, and
the OD260 of each fraction was read on a Cary 15 spectrophotometer.
Fractions on the light side of the band were pooled and dialyzed into 0.01
M Tris-HC1, 1 mM EDTA, pH 8.0. This light-side DNA (20 yglml) was
then rebanded in Cs Formate (1.75 g/cm3) at 42,000 r/min for 60 hr in a fixed
* Present address: Department of Biology, Massachusetts Institute of Technology,
Cambridge, Mass. 02138.
To whom requests for reprint should be addressed.
1591
C 1976 by John Wiley & Sons, Inc.
WIESEHAHN, CECH, AND HEARST
1592
114I
a
11.3-
I 1 2I1 l -
i
i i h 5 b f E $ l b
Na,HPO,:
Na,PO,
= 10-X
Fig. 1. Measured pH as a function of buffer acidsalt ratio: ( 0 )measured in order of inmeasured in order of decreasing pH.
creasing pH; (0)
angle 50 rotor. The tubes were dripped and the OD260 of each fraction was
taken. The fractions containing purified 1.687 DNA were pooled and dialyzed. The DNA’s of M. lysodeikticus, singly nicked PM-2, and T4 were
gifts from P. Wollenzien, L. Liu, and A. Rosenfeld, respectively. PM-2 was
nicked according to the procedure of Wang.4 Na2HP04 and Na3P04
(Mallinckrodt), CsCl and Cs Formate (both from Harshaw, optical grade),
and Cs2SO4 (Alpha) were used without further purification. Glass-distilled
water was used in all solutions.
Measurement of pH
A radiometer pH meter equipped with a GK2321C electrode was used
for pH measurements. Stock solutions of 0.5 M Na2HP04 and 0.5 M
Na3P04 were prepared. Solutions with Na2HP04:Na3P04 ratios from 102
to 1O:lO were made from the stock solutions. One part of each of these
buffers was diluted into 11parts, 1 mM EDTA, pH 8.5, and the pH was
measured and plotted (Fig. 1). The meter was standardized with pH 10.00
buffer before each reading. Diluting into 1.70 g/cm3 CsC1,l mM EDTA,
pH 8.5 had negligible effect on the pH reading. Therefore, the pH of each
sample was taken to be the pH corresponding to the acidsalt buffer ratio
(diluted 12:l) used to make the sample. Reproducibility in pH measurement of a buffer made from the same stock solutions on the same day was
f O . O 1 pH unit. Over a period of time (90 days), however, the pH reading
of a buffer made from the same stock solutions would drift by as much as
0.05 pH unit.
Density Gradient Centrifugation
A typical sample was made by mixing 460 p1 of saturated CsCl, 50 p1 of
phosphate buffer, 34 p1 of H20, 11p1 of 0.05 M EDTA, and 50 pl of DNA
DNA DENATURATION
1593
in 0.015 M NaC1,0.0015 M Na citrate, 1mM EDTA (pH 7.6). The DNA
solution was added last, and the entire procedure was carried out in an ice
bucket to avoid partial denaturation of the DNA. The EDTA was found
to be necessary to inhibit degradation of single-stranded DNA. The reference was prepared exactly as the sample with 0.015 M NaC1,0.0015 M
Na citrate, 1mM EDTA substituted for the DNA solution. The refractive
index was measured at 25°C on a Zeiss refractometer, and the density
calculated from the calibration curve of Ifft et al.5 The sample and reference were immediately loaded into a centrifuge cell with 12-mm doublesector titanium centerpieces. Typically four cells were run in the AN-F
rotor with the rotor hole as reference edge. The rotor temperature was
controlled with the RTIC unit. The CsCl density was chosen so that the
native density of the DNA was in the top half of the cell. All of the runs
were at 40,000 r/min. After the initial equilibrium was reached (-40 hr),
the temperature was raised by 2°C or less. The new equilibrium position
was reached in 12 hr. If the sample was low molecular weight or had moved
a large distance in the cell, scans were taken at hourly intervals to assure
that equilibrium was reached.
Calculation of Buoyant Density
The difference in CsCl concentration between the root mean square (rms)
position (isoconcentration point)5 of the cell and band center was determined using the equation Apo = (w2/p)FAr,where i; is the arithmetic mean
distance (cm) of the DNA band and the rms position from the center of
rotation, Ar is the distance of the band center (cm) from the center of
rotation minus the distance of the rms position from the center of rotation,
o is the angular velocity, and p = 1.20 X lo9 cm5/(g-sec2)for CsCl at 25"C.5
No correction was made for the variation of p with temperature. The
pressure at band center was calculated using the equation P = (02rlp)-(9.861
X 10-7) where r is the distance from band center to the center of rotation,
1 is the distance from the meniscus to band center, is the arithmetic mean
of the density at band center and a t the meniscus, and w is the angular velocity. The factor 9.861 X
converts dyn/cm2 into atmospheres. The
effect of pressure on the buoyant density of DNA in CsCl is calculated from
P , where P is in atmospheres.6 The final
the equation A p p = 3.94 X
equation needed to determine the buoyant density is p = po Apo A p p
where p o is the starting density of the solution. The final densities are
lighter than those observed in a pure CsCl density gradient (about 0.01
g/cm3 lighter) because of the presence of 0.1 M Na+ ions in the solution.
This procedure is more laborious and tends to give less precise densities
than those found using a marker DNA in the same cell. The marker DNA
obscures intermediate states, however, so it was rarely used. When a
marker DNA was used, densities were calculated according to Schmid and
Hearst7 using IIPB = 9.35 X 10-lo (g sec2)/cm5.
+
+
1594
WIESEHAHN, CECH, AND HEARST
Molecular Weight Determination
DNA was sheared by forcing the DNA solution through a 22 or 27 gauge
needle a t maximum thumb pressure. Smaller DNA was prepared in a
decompression bomb. The DNA solution was pressurized with argon to
300 psi, equilibrated for 10 min, and then rapidly decompressed as it exited
the bomb through a needle valve.
Single-stranded DNA molecular weights were determined by boundary
sedimentation through 0.9 M NaC1,O.l M NaOH and correction to 5 ' 2 0 , ~
according to Studier.8 Alternatively, the DNA was band sedimented
through 3 M CsC1, 0.1 M KOH. Correction to S ~ Oand
, ~calculation of
molecular weights were performed according to the equation S O ~ O(Cs
,~
DNA) = 0.079 M0.397(Na DNA) (K. Martin, personal communication).
Calculation of Moments
Tracings of buoyant-density profiles were digitized manually by measuring the trace height a t l-mm intervals (0.05 mm in real space) on the
chart paper. The narrowest distribution was described by 46 points, and
the widest required 167 points. The moments of the distributions were
calculated from the digitized data using a PDP-8/E computer. No correction was made for hyperchromicity of melted DNA or for radial dilution
effect.
Curve Fitting
A nonlinear regression computer program (G2 CAL NLIN) was used to
find the values of the parameters ui, pi, and Bi which minimize the sum of
the squares
S = z[Yj - Fj(u,~,B)l~
where yj are the digitized data points and
This is just a sum of N Gaussians with the standard deviations C T ~means
,
and relative areas proportional to Bi. The program uses a modified
Gauss-Newton method to find a least squares fit.g
The value of u was set by finding the best least squares fit to the mouse
satellite peak (at 29'C so that the satellite is well separated from the main
band). All of the native components were required to have this u (0.544).
A least squares fit was found for the totally native tracings (22O and 24OC).
The averages of the relative amounts of each component from these two
fits were used as starting points for the 26°C tracing. With the u's and
relative amounts fixed, the values of the means were found by the program.
The u of any melting component was then allowed to vary. Finally, the
relative amounts of the components were allowed to vary. The position
ki,
DNA DENATURATION
1595
+
and (r of the small (G C)-rich main-band component were held fixed in
the middle of the melting transition. The program was unable to place this
component unambiguously because it represented such a small fraction
of the total sample. The (r for the totally denatured DNA was calculated
to be 0.9 from the known single-strand molecular weight and from the assumption that all of the guanines and thymines were titrated. Since there
could be some broadening due to a small density difference between separated strands, the program was allowed to vary u to find the best fit. The
value that minimized the least squares function was 0.939.
RESULTS
In alkaline CsCl the buoyant density of denatured DNA is higher than
that of native DNA due to the titration of guanine and thymine. Native
DNA, destabilized by the replacement of H+ with Cs+, can be melted at
a relatively low temperature. Alkaline titrations of native and denatured
DNA have been done previously.1°-12 In each case the titration was done
by banding samples of DNA a t different pH's and the same temperature.
The double-strand to single-strand transition is such a sensitive function
of pH that partially denatured bands are rarely observed.I2 If the starting
pH is carefully chosen, however, the entire transition may be observed in
an analytical ultracentrifuge operating in the range 20"-35°C.
The experiment is analogous to an optical melting curve. During the
buoyant-density melt the shape and position of an equilibrium band are
monitored. The position of the mean of the band distribution indicates
the fraction of the base pairs which have been titrated, while the shape gives
information about the base sequence heterogeneity of the DNA being
melted. Since equilibrium must be attained at each point during the melt,
an entire melt takes from five to seven days and typically contains 12
points.
An Idealized Melting Curve
Let us assume we have a collection of DNA molecules of identical molecular weight and identical base sequence. This DNA can be banded in
alkaline CsCl that has been adjusted to a pH such that double-strand DNA
is stable at 20"C, and single-strand DNA is stable at 30°C. Let us further
assume tha't the DNA is equally stable at each point in the gradient at any
given temperature (i.e., there is no stability gradient in the cell). Finally,
let us assume that there is no temperature dependence on the buoyant
density of the DNA.
The experiment is begun at 20"C, and an equilibrium band is formed with
a Gaussian shape,13 with the variance of the DNA band being inversely
proportional to the molecular weight. The temperature is raised, and the
band is unchanged. The temperature is raised again, and the DNA begins
to denature. The thymine and guanine bases of the melted region are ti-
1596
WIESEHAHN, CECH, AND HEARST
trated by Cs+ causing the DNA to move to a higher buoyant density. If
the DNA base sequence is homogeneous, then every base pair will break
and the single strands will move to their entirely denatured positions. The
ill be larger than that of the native peak
bandwidth of the denatured peak w
due to the halving of the molecular weight. This is an all-or-none transition
(Fig. 2).
If there is heterogeneity in the DNA base sequence, only the least stable
regions of the helix will denature. The DNA will move to a new equilibrium
position, which is intermediate between its native position and its denatured position. This will be a stable equilibrium, and the DNA band will
still be a Gaussian with the same variance as before (the molecular weight
is the same as before if we neglect the contribution of the titrating Cs+ ions).
The melting will continue as the temperature is again raised. Each higher
temperature generates a higher buoyant-density DNA band with the same
shape as before. When the temperature is high enough, the most stable
regions of the molecule are melted, and the strands separate. The band
moves to its totally denatured position, and the bandwidth increases due
to the halving of the molecular weight (Fig. 3).
In reality none of the above assumptions is correct. The buoyant density
of DNA is a function of temperature.14 It will be shown that there is a
stability gradient in the cell, and DNA samples are not always made up of
identical DNA molecules.
PM-2 DNA
PM-2 DNA, which has been nicked an average of once per molecule and
separated from unnicked circles, is an example of a DNA sample with
“identical” DNA molecules. Figure 4 shows analytical tracings of PM-2
DNA at different points in the melting region. Each of the partially denatured tracings is a stable, equilibrium tracing. PM-2 DNA, therefore,
melts as expected for a DNA with sequence heterogeneity.
The PM-2 DNA can be melted with a marker DNA (M. Zysodeikticus),
which is native throughout the PM-2 melt present in the same cell. Since
the buoyant density of both the marker DNA and PM-2 DNA will increase
as the temperature is raised, the buoyant density of PM-2 DNA relative
to marker DNA will show no temperature dependence. Only the density
increase due to denaturation will be seen. Figure 5a shows the plot of
buoyant density of PM-2 in such an experiment. It looks like the ideal
curve.
Figure 5b is a plot of one-half the bandwidth at 0.606 of the peak maximum as a function of the fraction of single-stranded DNA. Clearly, this
does not look like the similar plot in the ideal case. The PM-2 DNA peak
broadens, then narrows, and finally broadens again as the DNA denatures.
The maximum bandwidth is attained when the DNA is about one-half
melted. The minimum is at about 80% melted, and the final bandwidth
is 1.48 f 0.15 times the original, native bandwidth.
The explanation for this nonideal behavior is that the assumption of no
1597
DNA DENATURATION
+-%--%
Temperature ?C)
X
0
20 40 60 80 10
Fraction Single-Stranded
Fig. 2. Ideal case of all-or-none melt. (a) Buoyant density as a function of temperature.
Function is discontinuous a t T, (25OC). (b) One-half bandwidth a t 0.606 of peak maximum
u as a function of fraction single-stranded. Native D is taken to be 1.
20
25
3c
Temperature ('C)
(bi
0
20
40
60 80
10
Fraction Single-Stranded
Fig. 3. Ideal case of heterogeneous DNA melt. (a) Buoyant density as a function of temperature. T, is taken to be 25OC. (b) u as a function of fraction single-stranded. Native
u is taken to be 1.
stability gradient in the cell is false. In the cell, there is not only a gradient
of CsC1, but there are also gradients of pressure and pH. The reason for
the pH gradient is that C S ~ P will
O ~ redistribute differently than Cs2HP04
in a centrifugal field, causing a different acid:salt ratio a t each point in the
1598
WIESEHAHN, CECH, AND HEARST
(a)
,
22"
24 5'
(C)
A
d
248'
!
25 2"
1692
1748
Fig. 4. Analytical tracings of PM-2 melt. Buffer ratio = 10:6.3. Measured pH = 11.52.
Calculated buoyant densities in this figure and in all other figures of analytical tracings are
lighter than in pure CsCl gradients because of the presence of Na+ ions.
cell. In addition, the acid dissociation constant of HP04=is pressure dependent. The result is a measurable positive pH gradient.12 Since we
cannot separate the effect of the pH gradient from the effect of the salt and
pressure gradients,15J6 we will consider the combined stability gradient
that they produce.
As the PM-2 molecule begins to melt, it moves to a higher density. Because of the stability gradient, the molecule is less stable at this new position
and more base pairs break. The molecule therefore moves to an even higher
density. The band will continue to move until the DNA (even a DNA with
heterogeneous sequence) is totally denatured unless the density of the solution increases more (as a function of radius) than the DNA increases in
density because of melting. In the P04-CsC1-PM-2DNA system the CsCl
density gradient is steep enough to stabilize intermediate states. In these
intermediate bands, however, the DNA on the high-density side of the band
will have a larger fraction of titrated base pairs than the DNA on the lowdensity side of the band. This results in the broadening of the interme-
DNA DENATURATION
0
1599
20 40 60 80 100
Fraction Single-Stranded
Fig. 5. PM-2 melt. (a) Buoyant density as a function of temperature: (0)
buffer ratio
T, was
24.7"C, each point was increased by 3.8"C so that these data could be compared with the 10:5.4
ratio data. (b) One-half bandwidth (cm f 0.0512) a t 0.606 of peak maximum as a function
of fraction single-stranded. Fraction single-stranded was calculated assuming a linear relation
between buoyant density and percent single-stranded. Error bars were determined using
repeated measurements of M. lysodeikticus marker DNA present in one of the PM-2 DNA
sample cells. u of native PM-2 DNA was found to give a molecular weight of 6 X lo6 from
PM-2 DNA using a CsCl effective gradient G = 9.0591 X
(0)
buffer ratio of
10:5.4; ( 0 )buffer ratio of 10:6.3.
= 10:5.4, T, is 28.5O, and the 10-9096 width is 2.2OC; ( 0 )buffer ratio = 106.3, actual
diate-state band. At the one-half denatured point, the DNA has the
maximum possible number of partially denatured conformations. As the
PM-2 DNA is further melted, the DNA strands are held together only by
a few (G C)-rich regions. The bandwidth decreases. When these last
stable regions are melted, the bandwidth increases again due to the halving
of the molecular weight.
The stability gradient, then, causes the increased variance of the partially
denatured DNA peak. In other words, the partially denatured DNA sees
a resultant gradient that is the effective gradient17reduced by the stability
gradient. When the DNA is totally native, the resultant gradient is equal
to the effective gradient. The DNA is not affected by the stability gradient
when all of the DNA molecules are totally native. A t the one-half denaturation point, the resultant gradient is (1/1.7)2 or about one-third of the
effective gradient. Here, when the DNA is most affected, the stability
gradient is two-thirds as large as the effective gradient. When the DNA
strands are held together only by a few (G C)-rich nuclei, they are less
affected by the stability gradient and the resultant gradient increases again.
After the strands separate, the stability gradient again has no effect on the
+
+
1600
WIESEHAHN, CECH, AND HEARST
r""
i
I
t
70
iL:T,".
.a
Temperature
CC)
Fig. 6. Optical melt of singly nicked PM-2 DNA in alkaline CsCl. Buffer ratio is 10:5.4.
The T , is 3OoCand the 1690% width is 3.1OC.
Fig. 7. Analytical tracings of mouse satellite DNA melt. Buffer ratio = 105.4.
DNA DENATURATION
1601
(f1
340
(g
30'
1.675
1.743
Fig. 8. Analytical tracings of D. melanogaster 1.687 satellite DNA melt. Buffer ratio =
10:3.6.
DNA and the resultant gradient is again equal to the effective gradient.
The final increase in the variance of the DNA peak is not due to the stability
gradient, but merely reflects the halving of the DNA molecular weight.
Figure 6 shows an optical melting curve of PM-2 in alkaline CsC1. The
optical melting transition has a width of 3.1"C from the point a t which 1Wo
of the DNA is denatured to the point a t which 90% of the DNA is denatured.
The 10-90% width of the PM-2 melt done in the centrifuge is 2.2OC. This
is consistent with the idea of a stability gradient in the centrifuge causing
a sharpening of the melting transition. Unsheared T7 DNA has a melting
behavior in alkaline CsCl gradients similar to PM-2 DNA.
Mouse Satellite DNA
Mouse satellite DNA is an example of a relatively homogeneous base
sequence DNA.lsJg It melts with an all-or-none transition in the alkaline
CsCl density gradient. When the normal melting curve procedure of 2°C
steps in temperature is applied to mouse satellite DNA, the DNA goes from
an entirely native density to an entirely denatured density in one step. If
the melting curve is done in 0.5"C steps, part of the DNA remains native
1602
WIESEHAHN, CECH, AND HEARST
1.701
I750
Fig. 9. Analytical tracings of total D. mehogaster DNA melt. Buffer ratio = 10:4.8. At
the first temperature of this sequence (23OC) all the low-density satellites have already melted.
while the majority of the DNA shows an all-or-none transition. Figure 7
shows the stages in a melt of mouse satellite.
The small peak that remains at native satellite density (Fig. 7d) is
thought to be a stable fraction of mouse satellite DNA because its density
is the same as the native density of mouse satellite and nearly all of it melts
with an all-or-none transition when the temperature is raised by an additional 0.5OC (Fig. 7f). In a separate experiment, more than two-thirds of
the stable material was seen to melt with a rise in temperature of only
0.1oc.
The peak that appears between the separated strands of mouse satellite
(Fig. 7d) returns to a density of 1.697 when the temperature is lowered to
26°C (Fig. 7e). The area of this peak corresponds well with the area of the
main-band density DNA that is present in the sample (Fig. 7b, 7c). This
peak does not appear to be merely bulk main-band mouse DNA, however.
It shifts to a density of 1.739 with a temperature increase of only 0.5"C.
This is 72.5% of the density increase expected for complete titration, and
it occurs in an all-or-none fashion. The small peak stays between the
separated mouse satellite DNA strands for at least l0C, and then at 29.5OC
DNA DENATURATION
1603
the peak disappears under the heavy strand of mouse satellite. This DNA
acts like a covalent linkage of 70% mouse satellite with 30% of a relatively
(G C)-rich region. This peak has been seen in every melt (four times)
of isolated mouse satellite DNA. It is about 5%of the DNA in the isolated
mouse satellite DNA.
+
Drosophila 1.687 Satellite DNA
Satellite DNA of buoyant-density 1.687 isolated from D. melanogaster
shows two all-or-none transitions (Fig. 8). The first results in two separate
denatured peaks at densities of 1.701 g/cm3 and 1.762 g/cm3 (measured
native density under these conditions is 1.679 g/cm3). The second transition gives two denatured peaks at densities of 1.734 g/cm3 and 1.742 g/cm3.
The difference in T , between the two components is 4" f 2°C. This is
in agreement with the work of Peacock et ale3who separated the 1.687 g/cm3
satellite into two components with native CsCl densities of 1.686 g/cm3 and
1.688 g/cm3, and it agrees with Gall et a1.20 who showed that isolated 1.687
g/cm3 DNA melts with a biphasic transition, with the two components
melting 5.7"C apart.
Figure 8 shows the melt of isolated 1.687 g/cm3 satellite. There is no
obvious evidence of heterogeneity in either of these components. Neither
is there any evidence of (G C)-rich DNA covalently attached to satellite
DNA. This is not surprising because these satellites are known to occur
in very long ~ t r e t c h e s .Any
~ molecules consisting of (G C)-rich regions
covalently linked to satellites would also tend to be excluded because the
1.687 g/cm3 satellite was isolated by repeated neutral buoyant-density
gradients without antibiotics or heavy metals. The melt shown in Figure
8 is done in 2°C steps, and it is conceivable that a melt done in smaller steps
would reveal heterogeneity in one or both of the components.
+
+
Drosophila melanogaster DNA
Figure 9 is a melt of total DNA from D. melanogaster. This is a DNA
sample that includes a broad distribution of molecular weights and base
sequences. The bulk main-band DNA still shows the basic features of the
PM-2 DNA melt. The bandwidth first increases, then decreases, and finally increases as the DNA denatures.
Micrococc us lysodeikticus DNA
M. lysodeikticus DNA was melted a t four different molecular weights.
As shown in Table I there is no obvious molecular-weight dependence on
the melting temperature of M . lysodeikticus DNA in the single-strand
molecular-weight range of 1 X lo6 to 10 X lo6. In these alkaline CsCl
melting experiments, a change in the measured pH of a sample of 0.1 pH
unit has been found to change the melting temperature of the DNA by
WIESEHAHN, CECH, AND HEARST
1604
TABLE I
The Melting Temperatures of Four Different Molecular Weights
of M. lysodeikticus, Mouse, and T4 DNA
Single-strand
molecular
weight
(M,X 10-6)
Trn
("C)
Transition
width
("C)
M. lysodeikticus,
71% (G+C)
10.3
4.2
3.8
1.2
32.8
32.5
32.1
33.3
1.6 t
1.6 f
1.8 f
1.9 *
0.3
0.3
0.3
0.3
Mouse, 40%( G + C )
15
5.2
4.9
1.9
28.6
28.75
28.8
28.6
3.3 *
3.2 t
3.0 *
3.5 *
0.3
0.3
0.3
0.3
T4,34%( G + C )
30.3
4.7
3.3
31.9 f 1
32.5 f 1
33.25 t 0.25
<2
<2
2.2
f
0.3
about 6.5"C (obtained by melting the same DNA at slightly different pH's
and observing the change in T m ) . For this reason and because the melt
is done in 2OC steps, the observed differences in the melting temperature
data are not significant.
Figure 10 shows the stages in the melt of M. lysodeikticus DNA at a
molecular weight of 10.2 X lo6 (single strand). The bandwidth shows the
familiar characteristics of broadening, then narrowing, and finally broadening again as strand separation occurs. Figure 11shows the melt of M .
Zysodeikticus DNA at a molecular weight of 1.2 X 106 (single strand). The
intermediate states have larger bandwidths than at the higher molecular
weight.
The standard deviations of each of the density-gradient profiles of M .
lysodeikticus DNA at each of four molecular weights have been calculated.
These are plotted in Figure 12b as a function of the fraction of the DNA
which is titrated. The standard deviation of the native peak CN increases
as the molecular weight decreases. The standard deviation of the one-half
titrated band a l / 2 also increases, and it increases roughly in proportion to
the increase in the native standard deviation. The ratio of a l / 2 to CTNis
about 3.1, which is larger than the a112 to U N of 1.7 found for PM-2 DNA.
This higher ratio is to be expected since M . lysodeikticus D N A is not a
collection of identical molecules.
Mouse DNA
Figure 13 shows the melt of total mouse DNA. The first stage in the
denaturation is the sharpening of the main band (Fig. 13b, 13c). Next, a
shoulder is developed on the heavy-density side of the main band. Near
DNA DENATURATION
32 75"
(e)
_____ _ - -
1605
.I721
--
i \L
I105
Fig. 10. Analytical tracings of M. lysodeikticus DNA melt. The single-strand molecular
weight is 10.2 X lo6. Buffer ratio = 105.8.
the midpoint of the titration, the main band becomes bimodal. The decreased relative amount of satellite suggests that part of the satellite has
denatured. The small peak on the heavy side of the main band is a t a
density of 1.740 g/cm3. This density is consistent with its being the heavy
strand of denatured mouse satellite (Fig. 13e). As the denaturation proceeds, the main band becomes one peak with a shoulder on the light side.
The last stage is the broadening of the main band.
Table I shows that there is no detectable molecular-weight dependence
to the melt of mouse DNA. Figure 14b shows that there is also no detectable increase in the standard deviation of the one-half denatured states as
the molecular weight is decreased. In addition, the shape of the partially
denatured bands is relatively constant as a function of molecular weight.
The standard deviation of a DNA band in a neutral density gradient is
dependent on both the molecular weight of the DNA and the density heterogeneity of different DNA molecules in the band. The standard devia-
WIESEHAHN, CECH, AND HEARST
1606
1 (el
32.75'
. _.
. _.
33.75'
(9)
.
.
.
__
1.721
-
A
1.784
Fig. 11. Analytical tracings of M. lysodeikticus DNA melt. The single-strandmolecular
weight is 1.2 X lo6. Buffer ratio = 105.8.
tion of a band of partially denatured DNA is dependent on the molecular
weight of the DNA and the melting heterogeneity of different DNA molecules in the band. If the standard deviation of a band of partially denatured
DNA fails to increase as the DNA molecular weight is decreased, it means
that the melting heterogeneity is so large that it is determining the bandwidth. The melting heterogeneity is not large enough in M.Zysodeikticus
DNA to obscure the molecular-weight dependence of the partially denatured bandwidth, but in mouse DNA it is. This large melting heterogeneity
and the unique shape of the mouse DNA partially denatured bands suggests
an analysis of mouse DNA by resolving the DNA band into several components. Thi&ry21has resolved native mouse DNA into five major and
three minor components on the basis of fractionation in preparative Ag+Cs2SO4 gradients. He also found that analytical ultracentrifuge mouse
DNA CsCl bands can be resolved into five major and one minor compo-
1607
DNA DENATURATION
25
0
35
30
Temperature
("C)
20
P
c
v)
10
5
0
2
4
6
8
1
Fraction Single- Stranded
0
Fig. 12. M. lysodeikticus DNA melt. (a) Buoyant density of mean band position as a
function of temperature. The single-strand molecular weight is 10.3 X lo6. (b) Standard
10.3 X lo6;
deviation of M. lysodeikticus DNA as a function of fraction single-stranded: (0)
(X) 4.2 X lo6; (0)
3.8 X lo6; ( 0 )1.15 X lo6 (all single-strand molecular weights). Units of
standard deviations are cm + 0.0512.
nents. (We define a minor component as one with composition below 5%.)
The buoyant densities and amounts of the major components are similar
for the two techniques. We have analyzed the partially denatured bands
of mouse DNA in order to determine if their shapes could be explained on
the basis of a small number of components.
The mouse DNA band is assumed to consist of several components, each
having the shape of a Gaussian curve. For native DNA, the standard deviations of all components are assumed to be identical, and the standard
deviation is determined by the molecular weight of the DNA. Attempts
were made to fit the native mouse DNA bands starting with one Gaussian
component. Additional components were added until the average rms
deviation approached what was considered to be the experimental error
associated with the data (deviation of about 1% of the peak maximum).
The number of Gaussian components found to be necessary is six, one over
the satellite peak and five in the main band. These components are not
1608
WIESEHAHN, CECH, AND HEARST
1.688
1.755
Fig. 13. Analytical tracings of the melt of total mouse DNA. The single-strand molecular
weight is 15 X lo6. Buffer ratio = 104.8. The sensitivity of the tracings was sometimes
changed to keep the peaks on scale.
identical to those of Thi6ry in buoyant density or amount. The results are,
however, in qualitative agreement.
Can the same components used in the native band be used to explain the
partially denatured profiles? Figure 15 shows the experimental and calculated data for a six-component fit to an alkaline CsCl melt of mouse DNA.
The main-band components melt in order of increasing (G -tC) contents.
The bandwidth of each component goes through a maximum a t about its
one-half denatured density. The ratio of al/z to a~ for each main-band
component is about 2.5. The densities, standard deviations, and amounts
DNA DENATURATION
1609
(a 1
1.760
1.750
/
.2
1.730
1.720
c3
1.710
c
1.700
1.690
m
1.680
s
22
28
24
26
28
30
Temperature ("C)
32
34
),fi
od\
O
Q
16
14
12
10
0
a
2
0
2
4
6
8
1
0
Fraction Single- Stranded
Fig. 14. Mouse DNA melt. (a) Buoyant density of mean band position as a function of
temperature. The single-strand molecular weight is 15 X lo6. (b) Standard deviation of
15 X lo6; ( 0 )5.2 X lo6; (X) 4.9
mouse DNA as a function of fraction single-stranded: (0)
X lo6; (0)
1.9 X lo6 (all are single-strand molecular weights). Units of standard deviations
are cm + 0.0512.
of the components at each stage in the melt are shown in Table 11. The
calculated data seem to be in reasonable agreement with experiment. The
least satisfactory fit occurs when the DNA becomes totally denatured. The
most (G C)-rich main-band component (6) is required by the computer
to be on the light side of the 34°C tracing (Fig. 15i). It is possible that this
component is not yet totally melted.
An analysis of mouse DNA with single-strand molecular weight of 4.9
X lo6 gives similar results. It should be pointed out that better fits to some
of the partially denatured bands could be obtained by varying the relative
amounts andlor standard deviations of some of the components. The
amount of improvement was typically small and so the somewhat poorer
fit with more appropriate parameters was preferred.
+
T4 DNA
As shown in Table I, T4 DNA also shows no detectable molecular-weight
dependence on its T,. The high-molecular-weight sample went from totally native to totally denatured in the span of one 2°C jump. The inter-
1610
WIESEHAHN, CECH, AND HEARST
22.
0)
I
1)
2
3
4
5
6
34
I
2 4'
5
5 4
I
9)
62
6
29.
3
2
30.
I
349
1.688
1.755
Fig. 15. Tracings of component melt of mouse DNA (1.9 X 106 M r ) . Vertical tic marks
denote the digitized data from analytical ultracentrifuge tracings. Gaussian components
determined by least squares analysis are labeled 1-6. 1 is mouse satellite. 2-6 are main-band
Gaussian components in order of increasing native density. The line through the vertical
tics is the sum of the Gaussian components.
DNA DENATURATION
1611
TABLE I1
Densities, Standard Deviations, and Fraction of Total DNA
During the Melting of Six Different Components of Mouse DNA
1)
0
22°C
24°C
26°C
27°C
28°C
29°C
30°C
31°C
34°C
0.544a
0.544a
0.544a
0.5448
0.544a
0.544a
0.931a
0.931a
1.7302
1.759a
0.088a
0.939b
1.737
1.768
1.674
1.677
1.682
1.680
1.681
Fraction of
total DNA
0.092
0.084
0.088a
0.088a
0.083
2)
0.544a
1.681
0.184
0.544a
1.681
0.133
0.654
1.686
o.iw
0.916
1.691
0.151a
1.28
1.713
0.165
0.941
1.734
0.162
0.931a
1.741
0.159a
0.931a
0.939b
1.745
1.746
0 . 1 ~0 . 1 5 ~
3) 0
P
Fraction of
total D N A
0.544a
1.686
0.282
0.544a
1.686
0.303
0.544a
1.690
0.293a
0.598a
1.691
0.295a
0.962
1.699
0.287
1.48
1.722
0.328
0.9318
1.741
0.274a
0.931a
1.748
0.294a
0.939b
1.753
0.294a
4) 0
P
Fraction of
total D N A
0.544a
1.690
0.267
0.544a
1.690
0.272
0.5442
1.693
0.268s
0.5448
1.696
0.270a
0.584
1.699
0.262
1.13
1.707
0.228
1.22
1.728
0.274
0.931a
1.748
0.268a
0.939b
1.757
0 269a
5)
0.544a
1.695
0.141
0.5448
1.695
0.158
0.544a
1.698
0.152a
0.544a
1.701
0.153a
0.544a
1.704
0.156
0.592
1.705
0.160
1.26
1.713
0.151a
1.25
1.732
0.150a
0.939’’
1.762
0.150a
0.544s
1.701
0.034
0.5448
1.700
0.053
0.544a
1.707
0.044a
0.544a
1.707
0.044a
0.544a
1.7108
0.047a
0.544a
1.711a
0.049
0.691
1.710
0.044a
1.05
1.718
0.044a
0.939b
1.731
0.0448
0.221
0.226
0.299
0.322
0.318
0.343
0.194
0.364
0.280
3.53
3.59
6.08
8.17
P
0
P
Fraction of
total D N A
0
P
Fraction of
total D N A
6)
0
P
Fraction of
total DNA
Rms
deviation
0.088a
(mm)
Sum of
squares
(mm’)
10.14
15.33
4.27
14.58
8.07
a Parameters t h a t were not allowed to be varied by the computer program.
b All u’s were assumed to be equal for the 34°C data. The computer program
chose u to give the best least squares fit t o the data.
mediate-molecular-weight sample also went from totally native to largely
denatured in one 2°C jump. The denatured peak had a partially melted
light density tail, however. When the temperature was lowered, the bulk
of the DNA stayed at a totally denatured density, while part of the DNA
(-30%) returned to a native density. The lowest molecular-weight sample
showed an early melting fraction as well as a late melting fraction. When
the temperature was lowered during the early part of its denaturation, the
bulk of the DNA returned to native density while a small part (-5%) was
totally denatured (data not shown). Thus phage DNA can be fractionated
by shearing to low molecular weight and separating the early and late
melting fractions in alkaline CsC1.
T, as a Function of (G + C) Content
Figure 16 shows a plot of the pH required to melt the DNA samples
studied here a t 25°C in alkaline CsCl versus the melting temperature in
Cit/NaCl for these same DNA samples. The pH’s are those corresponding
WIESEHAHN, CECH, AND HEARST
1612
pH
I
T
1
00
d5
,T
,,
in
SSC
1
I
90
95
I
I00
(Oc)
Fig. 16. pH needed to melt various DNA's a t 25OC in alkaline CsCl as a function of T, in
Cit/NaCl (0.15 M NaCl, 0.015 M sodium citrate): 1-D. melanogaster 1.686 DNA; 2--0.
melanogaster 1.688DNA; 3-T4 DNA; 4--0. melanogaster total DNA; 5-mouse satellite
DNA, 6-PM-2 DNA; 7-mouse total DNA &A DNA; 9-T7 DNA; 10-M. lysodeikticus
DNA.
to the buffer ratios in Figure 1. Melting experiments with T, at temperatures other than 25OC were corrected to 25OC using the conversion factor
of 0.1 pH unit per 6.5"C change in melting temperature. The slope of the
line in Figure 16 is 0.01 pH unitPC.
DISCUSSION
DNA melting heterogeneity can be observed in the centrifuge. This leads
to the hope of preparative fractionation of DNA on the basis of melting
behavior.22 The stable fraction of mouse satellite DNA indicates a longer
order heterogeneity than has previously been seen in this DNA. This
long-range heterogeneity (2 X lo7 daltons double strand) suggests the
possibility that satellites may vary from chromosome to chromosome in
the mouse karyotype.
The heterogeneity in mouse main-band DNA is similarly long range.
There is no obvious change in the melting heterogeneity of mouse DNA in
the molecular-weight range studied here. Even though the mouse DNA
melt can be treated formally as the melt of six components, these components obviously do not consist of identical molecules (PM-2 DNA type).
Their increase in standard deviation at one-half denaturation is clearly
greater than that of PM-2 DNA.
The authors thank D. Wandres for help with data analysis, Dr. I. Tinoco for the use of the
PDP-8B computer, and Dr. D. Appleby for helpful discussions. They also thank Mrs. M.
Malone, Mrs. E. Rotondo, Mrs. D. Miner, and Mr. M. Tuttle for technical assistance.
DNA DENATURATION
1613
This work was supported by United States Public Health Service Grants GM-11180 and
GM-15661. T.C. was supported by a National Science Foundation Graduate Fellowship.
References
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U.S. 49,12-17.
12. Wang, J. C. (1974) J. Mol. Biol. 89,783-801.
13. Meselson, M., Stahl, F. W. & Vinograd, J. (1957) Proc. Nat. Acad. Sci. U S . 43,581-588.
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16. Chapman, Jr., R. E. & Sturtevant, J. M. (1970) Biopolymers 9,445-457.
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18. Southern, E. M. (1975) J. Mol. Biol. 94,51-69.
19. Biro, P. A., Carr-Brown, A., Southern, E. M., & Walker, P. M. B. (1975) J . Mol. Biol.
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20. Gall, J. G., Cohen, E. H. & Polan, M. L. (1971) Chromosoma 33,319-344.
21. ThiBry, J.-P. (1974) These de Doctorat d’Etat, UniversitB Paris VII.
22. Cech, T. R., Wiesehahn, G. & Hearst, J. E. (1976) Biochemistry 15,1865-1873.
Received October 30,1975
Accepted March 3,1976