DETERMINATION OF THE MOLECULAR HEIGHT OF T7 DNA BY THE SEDIMENTATION EQUILIBRIUM METHOD IN A CsCl DENSITY GRADIENT. It was found (1) that an anhydrous, homogeneous DNA at equilibrium in a density gradient should form a Gaussian concentration distribution and that the variance of the Gaussian, C) should be a function of the molecular weight of the DNA.
Wavelength
The position of the camera lens determines the focal plane in the cell, as does the magnification from cell to film. The alternator on this machine is aligned so that the side should be placed at the bottom of the cell assembly.
Figure 7 • Linearity of film response to the optical density of DNA solutions placed in the li~1t path of the optical ultracentrifuge system. University of California at San Diego. This conclusion would be strengthened by an independent determination of the molecular weight of the isolated T7 DNA.
The stability of the native DNA structure, as measured by Tm', increases with increasing ratio of GC to AT in the sample (54,55). The early part of the collapse process has been shown to be reversible, as evidenced by the recovery of native optical density after rapid cooling of DNA soln. The extent to which a denatured DNA sample will renature depends on the source of the DNA (34).
Renaturation has been interpreted as a bimolecular reaction between separate complementary strands of the DNA double helix (34,36). One of the aims of this work was to study the kinetics of irreversible denaturation. Mixtures of native and denatured DNA were melted and correlated to determine whether the two methods gave similar and accurate measurements of the proportion of native material in the sample.
Samples of T7 DNA that had been partially denatured were compared to mixtures of native and fully denatured DNA by melting, banding, and sedimentation velocity procedures to learn something about the distribution of molecules in the population among possible denaturation states. The height of the minimum was somewhat variable, but the t1w peaks were never as well separated as those for a comparable mixture of native and denatured DNA. It appears that some of the material in the "native" band of partially denatured samples melts as if it is not quite natural.
Apart from possible boundary spreading, however, little difference from the sedimentation patterns of mixtures of native and denatured material ·v;as noted (see Fig. 15). The extinction coefficient and hyperchromicity of DNA after melting in formamide solution were approximately the same as those of DNA in 0.5 M NaCl aqueous solution.
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It is possible that such a subtle change in the composition of the denaturing formamide solution could significantly alter the denaturation rate during the course of the denaturation reaction. The sensitivity of the initial denaturation rate to small changes in the denaturing medium was tested by measuring the melting percentage native to DNA after five minutes in denaturing solution, varying one parameter at a time. The following experiment suggests that hydrolysis of formamide over the course of the experiment is unlikely to produce changes in the composition of the denaturing solution large enough to affect the denaturation kinetics.
This was done at times separated by fifty minutes to see if hydrolysis or any other reaction of the 90% formamide solution over this period could affect the denaturation kinetics. The denaturation kinetics of the ~m mixtures were found to be essentially identical, demonstrating the laclc of such effects. One might suspect that the heat of mixing of 'Hater and formamide may increase the temperature of the solution with the addition of the aqueous DNA solution and thus produce the observed kinetics.
It was always found that the DNA solution was added to the formamide mixture. Denaturation of T7 DNA in aqueous salt solutions at elevated temperatures i·Tas followed in much the same manner as in the formamide system. 20 show that the denaturation kinetics in aqueous solution are essentially identical to those obtained in the formamide system, again suggesting that they are a property of the DNA itself and not due to peculiarities of the denaturing system.
This indicates that the observed kinetics are not the result of an approximation of equilibrium between native and fully denatured DNA. Another possible basis for the observed denaturation kinetics of T7 DNA could be that denatured DNA or other substances that accumulate during denaturation can sometimes occur; prevent the remaining partially denatured DNA from further denaturing. That this is not the case is evident from the following experiment: a sample of T7 DNA.
40 J.Lg/roll as measured from melting data 'i·ras compared to the denaturation rate under identical conditions but at 4 J.Lg/ml and as measured from banding data. 23, there is no difference between the denaturation kinetics of the tv1o samples, although there is. Although this small difference in denaturation rate may be due to experimental error, it may also be due to the viscosity effect noted by Schilillraut et al.
The fact that a tenfold difference in concentration only slightly affects the denaturation rate strongly argues that the denaturation reaction is basically unimolecular. The kinetics of denaturation of T7 DNA in the presence of .004 M EDTA is similar to that in the absence of EDTA. This suggests that the presence of small amounts of divalent metal ions is probably not an important factor in producing the observed kinetics.
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All results above · Obtained here with solutions that were not stirred or shaken during denaturation except briefly and gently during the initial dilution of DNA in the denaturing mixture. Comparisons of the kinetics of denaturation of stirred and unstirred samples under different, identical conditions are shown in Fig. The stirring speed was not measured, but was not turbulent and almost constant throughout the experiment.
Strong indications were obtained that the degree of denaturation increases with mixing speed, but this is sufficient for a study with several quantitative methods for generating and evaluating shear. It does not matter when the mixing starts during the denaturation process; alvmys increases the rate. Thus, the sample denatured without stirring up to the point "<. Denaturation appears to stop rapidly and completely denature after the start of stirring (Figure 26).
The rate of denaturation of DNA samples at concentrations of 40 ~ g/ml and 4 ~ g/ml are both accelerated by mixing. Mixing apparently does not itself produce denaturation in a sample that is not already denaturing. Vigorous mixing does not induce denaturation in DNA samples under formamide and salt conditions, which by themselves are not sufficient to initiate denaturation.
Time; . min
After removing the DNA from the denaturation conditions, these DNA molecules appear to at least partially restore their original structure in the intermediate states of denaturation. The rate of denaturation is very sensitive to slight changes in the environment caused by reactions of the formarrid-1.-1ater solvent mixture. The possibility that the observed denaturation kinetics is affected by renaturation of irreversibly denatured molecules during DNA isolation from denaturing mixtures can be ruled out with data from several sources: .. l) The most obvious evidence comes from mixing experiments, p. 78.
One can hope that the fact that this DNA is isolated from material that is thought to be biologically homogeneous can ensure homogeneity of the product. The fact that stirring during denaturation produces kinetics in the approximation first order may be an indication that stirring minimizes the effects of position and overall molecular configuration on the rate of denaturation of the individual native regions. Renatured T7 DNA melts sharply at the characteristic native melting temperature but the percentage increase in OD at melting depends on the degree of renaturation of the sample.
The band is slightly sls:ev;ed tm;ard heavier densities in the lower third of the curve. Although there may be significant errors in these approximations, the rate of disappearance of the denatured peak appears to follow second-order kinetics quite closely, as shown in Fig. near 86~£ natives in both trials.
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Tim e ; min
The figures in Table l also give the error in the densitometer calculation of a film, i·7here x - x is the effective gap Hidth of the densitometer, U. Let us not consider the real case for the density gradient >I here comes light in the solution normal to the \·7i'.'ldOV7s and thus crosses the cell on the path x - x. The absorption distribution for the case of light crossing a Gaussian concentration distribution along the path x - x.
It should be noted that the above calculations were made with the assumption that Q = o, so that the light enters the solution normally with the windows. It seems probable from the above results that, for small deviations, the apparent decrease in molecular weight due to bending of light is almost exclusively a function of the maximum value of . Thus, the optical error due to bending light can be greatly reduced by inserting the appropriate wedge window at the bottom of the cell.
These figures represent the x-shift from the salt gradient alone, but there is also a refractive index gradient due to compression. 44,770 rpm from shifting the focused slit image behind the camera lens in the UV system. At speeds around 44~770 rpm, the total x-shift of the light as it passes through the solution will be somewhere between that calculated for the salt gradient alone and some 30% greater than that.
