We studied the effects of ion binding and DNA composition on the helix denaturation of the DNA macromolecule. The overall goal of the research described in this thesis is to study the structure of DNA in aqueous solution in the hope of finding techniques that are sensitive to differences in structure between different molecules derived from an organism. We focused on two properties of the DNA molecule: its denaturation and its interactions with small molecules.
This heterogeneity hinders the understanding of the structure of individual genes in non-viral organisms. Optical rotation measurements show that the optical activity of the natural DNA molecule differs from that of the constituent nucleotides.
34;·CUD
Low Temperature Renaturation Temperature
Molecular heterogeneity would be expected to influence the importance of k31S compared to kZ1s. The length of the molecular strands is expected to influence the importance of k. The midpoint of the transition occurs in that state in which the free energies of the helix and the coil are equal.
Zoo and a thermometer in the cell compartment indicated that this was the accuracy of the temperature control. Agitation of the water in the cell compartment (except during absorbance measurements) provided uniform temperature.
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These solutions require no chloroform to prevent growth of organisms, presumably due to the toxicity of cacodylate and the low ionic strength. We made a spectrophotometric study of the acidification of calf thymus DNA over the range of temperatures, 0'-30', and ionic strength, 0.1-0. The general increase in absorbance in the more acidic solutions is a hyperchromic effect, indicating denaturation, and is well known from previous studies of acid and heat denaturation.
Protonation of a sigma electron pair on the N-1 ring nitrogen would have relatively little effect on the spectrum; protonation of the amino group would suppress the resonance of this group with the ring and shift the spectrum towards . The intrinsic viscosity of the reneutralized material is the same as that of the starting material according to Geiduschek and Cavalieri and Rosenberg. It is noteworthy that the isosbestic point for protonation of the undenatured material shifts from 265 ml-' at 30· to 274 ml-' at 0·,.
The general hypochromic effect in native DNA is not understood, but appears to be due to compact base packing. We will discuss this in the light of ideas later presented by Dekker (1960) and mention some recently published experimental results bearing on the question of the protonation site in DNA. This can cause the absorption spectrum to shift to longer wavelengths, which is what happens in the model compound.
This increases the aromaticity of the system and one would expect a shift in absorption to longer wavelengths. Our studies on the protonation of DNA in acid denaturation were mainly performed on calf thymus DNA. The magnitude of the observed differences is not large enough to encourage fractionation with this method.
Temperature ( °C)
We would also expect positive ion binding to be greater at low ionic strength than at high, as the phosphate groups would be less protected at low sodium ion concentrations. Rosoff and Rosenberg (1956) show that pH can be an important factor in these low ionic strength experiments. In addition, the results of Oth (1959) on changes in the sedimentation coefficient during acidification at low ionic strength (1x10-3 . ) indicate that the observed transition occurs at EZ60.
It could be argued that the increased width at low ionic strength is simply because DNA denatures at a lower rate at low temperatures. We now see that at low ionic strengths, equilibrium heating curves show significantly broader transition profiles than at high ionic strengths. The intrinsic width of the helix-coil transition increases at low ionic strength either by a change in 0"'0 or in O"'j" If electrostatic forces would decrease the stacking energy e which determines 0"'0 ' then (1'0 would approach say 1 at low ionic II strengths and the transition would be broad.
Alternatively, entropic cooperative factors would be lost if at low ionic strength the entropy of the free ends were reduced. This is the reason for the rise in melting temperature with increasing ionic strength. It is clear that we can decide on the merits of hypothesis 1 by measuring d~T at high and at low ionic strength.
51 - . iments at low ionic strength are only reproducible down to =2°, as is also the case. discussed, but even with this experimental uncertainty we can see in Table 4 that there is not enough change in d~T from 0.1 f to 3x10-4. Conductometric measurements at low ionic strength by Doty and Zubay (1958) and by Felsenfeld and Huang (1959) indicate this. The effect of Mg++ binding on DNA denaturation was first noted by R.
Since there are no steric differences in the actions of Mg ++ and Co ++ , the binding of Co ++ is not as strong as that of Mg. This co-Inpositional selectivity in the stabilization of DNA by Mg ++ and Co ++ is Inarked and certainly experientially significant. Also, hydroxy- Inethylation of DNA with virInaldehyde reduces the binding of Mg ++, also Conductive Inetrically Undetermined.
This would require guanine to exist in its enol tautoIner, and thus can only occur in denatured DNA. This requires the adenine amino group to be non-coplanar with the adenine ring, and therefore this interaction can also only occur in denatured DNA. The observation that Mg++ is more strongly bound by denatured DNA than by native is probably incorrect (Shack and Bynum, 1959. and Dekker, 1960).
Strong complexation of Mg++ with native DNA may involve a chelate between adenine and Mg++, not as the chelate proposed by Zubay, but with a proton displaced from the N-IO amino group. Thus, on the basis of the hypothesis of selective binding, the greater stabilization of AT-rich molecules could be explained either by the Mg++ complex with adenine in natural DNA or by the cOIIlplex between Mg++. Both binding modes predict proton release upon Mg++ binding to native or denatured DNA.
These models, however, are both somewhat improbable, because there is no great difference between the action of Mg++ and that of Co++. The binding constant of Co++ for 8-hydroxyquinoline is greater than that of Mg++ by a factor of 106. If this is true in binding to DNA, then binding will increase with increasing temperature.
Temperature (oC)
Thus, none of these techniques increases the difference in stability of AT and GC base pairs, and therefore none of these techniques considers a promising attack on the illolecular heterogeneity problem. For the broadening of the transition to be due to a change in a' • we need a value of 0.1 for (1' at low ions. So even this generous estimation of the repulsive energies would be… only a explain part of the broadening obs Works at low ionic strength.
This change in free sodium ion concentration would cause a shift of the center of only I or 2 degrees. In fact, the sum of the two effects is about half as large in magnitude as the observed effect. In a homogeneous electrolyte solution, interactions span 1/2 0 1 distances of the order of the Debye length, given by K = 0.33 ]I tAro' at room temperature, in aqueous media.
Our chemical experiments therefore determined that none of the systems investigated amplified the difference in stability between Ar-rich and GC-rich DNAs. In seeking approaches to the problem of the heterogeneity of a single DNA preparation, the most interesting systems to look at so far are those that produce broad intrinsic transitions. Although specific features based on gross composition are not implicated, these intrinsic transitions can elucidate features of the internal heterogeneity of individual molecules.
It is difficult to construct a more satisfactory model due to the lack of conclusive experiments. Assays in the linear region of the response curve (about 0.07 .. rg Iml)· were used to measure biological activities. If we take the breadth of inactivation of a single marker as a measure of intrinsic breadth, then we can calculate the breadth of heterogeneity of a given sample.
Analysis of the fast component of the inactivation reaction shows that there are no noticeable differences between the characters and that the transition is quite broad. Based on the denaturation data, there is general agreement on the relative stabilities of 5r and Bm. This point of view quite reasonably explains the marked shift in the inactivation center of low ionic strength to much higher temperatures than observed optically.
The eventual loss of one of these regions prevents the renaturation of one part of the molecule. Critical experiments to test these interpretations of the inactivation of low ionic strength markers do not readily suggest them. Both reports contradict the model of the transformation system proposed by Hotchkiss, as we will show.
Next, we must say that the analysis of the transformation does not allow us to draw conclusions about the type of changes in the active molecules due to which Qoave changed the activity of the sample. This is an example of the bacteriophage ¢X174, which contains a single-stranded DNA molecule whose complement is supposed to exist in the. a bacterial cell that replicates a phage. For the DNA in Figure A, we predict a 10:10 difference in the mobility of the two subunits. If we can distinguish bands separated by 2 mm, we calculate.
In Appendix III of this thesis we have provided a discussion of the transformational analysis system which states the dilemmas we feel to be. In summary, we have proposed an analysis of the transformation assay and an experimental evaluation of this analysis. The magnitude of the change in intensity due to the weak coupling of oscillators is given by.
The significance of this experiment stems from the controversy over the stranding of the response subunit. Instead of electron microscope observation of the number of particles, we have proposed a ~uto-.
