The chromosomal RNA chran.atin of the calf thymus is present in the amount of 1% of the amount of DNA. The general shape of the UV spectrum of DNA does not change due to complexation with different types of histones. Heat denaturation studies and evaluation of free DNA segments of DNA-base polypeptide complexes.
The second part describes the template properties of DNA complexes with poly-L-lysine, poly-L-arginine and protamine. The third part discusses the thermal denaturation and template properties of DNA complexes with purified histone fractions. The molecular structure of nucleohistone is discussed with the illustration of the possible structure of the DNA-binding part of the histone IV molecule by the molecular model.
Isolation of chromosomal RNA from calf thymus chromatin was performed essentially according to the method of Bonner and Widhalm (1967).
The RNA from calf thymus chromatin exhibits chromatographic properties similar to the chromosomal RNAs from other organisms. The melting temperature of the complex is high and the melting is not complete at 100°C in 5.0 x 10-4 M citrate buffer at Melting temperature of DNA-poly-L-lysine and decrease in hyperchromicity of free DNA melting.
In the course of the present studies, it has been discovered that the purity of DNA significantly affects the formation of the DNA-protamine complex. The fractional template residual activity is in fact exactly equal to the fractional free DNA residual of the complex as estimated from its melting profile (see the first section of this chapter). An estimate of the fraction of free DNA from melting data is not possible as discussed in the first section of this chapter.
This is reflected in an increase in K with increasing DNA coverage and in a relative const. Resuspending the pellets in buffer with shaking in the cold for one hour results in a homogeneous suspension. Blockade of the activity of the matrix is therefore not the result of the inaccessibility of the complexed DNA region for the RJ.~A polymerase.
PART III
In the current studies, well-defined molecular DNA complexes containing purified histone fractions are prepared by salt gradient dialysis in the pre-. Studies have also been conducted on DNA complexes formed from combinations of purified histone fractions. A crucial step in the investigation of the structure and function of a DNA-histone complex is the preparation of the complex.
Sudden mixing of DNA and histone results in imperfect complex formation, and at low ionic strength it is difficult for the components to resort to a stable equilibrium state. DNA-histone interaction is mainly through ionic reaction of DNA phosphates and lysine, arginine and histidine residues of histone. Reconstituted nucleohistone Ib and IIb show stabilization of DNA against thermal denaturation, although they do not exhibit any characteristic T, which is the opposite.
The concentration of the DNA stock solution was determined by analyzing inorganic phosphates (Ames and Dubin, 1960). For convenient determination of DNA concentration, e(Pi) at 260 mµ was also determined to be 9.9 x 103 for DNA hydrolyzate with 0.5 N perchloric acid (boiling water bath for 10 minutes). Nevertheless, the turbidity is low compared to DNA complexes with polylysine, polyarginine and protamine.
Melting profiles of DNA complexes were obtained with a Gilford multi-sample acquisition spectrophotometer Model 2000 equipped with a linear temperature programmer. Thermal denaturation of DNA double helix structure was followed by the absorbance change as monitored at 260 mµ. EDT.A also removes any trace contamination by multivalent cations, and this is important as these have large effects on the T of DNA (Dove and Davidson, 1962).
The melting profiles and their derivative curves of the DNA-histone Ia complex are shown in Figure 2. The histone/DNA ratio is expressed as the ratio of the molar concentration of histone lysine and arginine residues to the DNA nucleotide residue concentration.
DNA-hi-stone Ia complex was prepared by salt gradient dialysis in the presence of 5 M urea. Melting profiles and derivative curves for DNA-histone Ib complex are shown in Figure 3. The general features of the melting profiles and melting temperature are very similar to those of nucleohistone Ia.
The commonality of the primary structures of these two histone fractions (section B of the previous chapter) is reproduced in melting profiles of their DNA complexes. Nucleohistone IIb has higher turbidity than DNA complexes with other histones, as shown by A. Since the DNA complex with the IIb-IV combination has the highest turbidity and lowest recovery, it is likely that the higher turbidity found for nucleohistone IIb is due to its contamination with III-IV.
The turbidity of the solution increases during ring melting, especially at high histone/DNA ratio (Figure l~).
The DNA-histone Ib complex was prepared by salt gradient dialysis in the presence of 5 M urea. The increase in turbidity after 65 °C for curve 4 was too large for a reliable correction, so the true melting profile above this temperature is unclear. The biphasic nature of the melting profiles is clear, although the higher temperature transition is broader at lower histone/DNA ratios.
The melting profiles of DNA complexes with cationic proteins processed by the present method are biphasic in nature. The transition temperature of the second step is constant for all coverages and is characteristic of each particular DNA-protein complex. Comparison of the Tm of DNA complexes with different proteins thus provides information on the degree of stabilization of the DNA structure by cationic proteins.
IDr.A, the buffer capacity is low and precise control of the pH and thus the ionic strength of the melting medium becomes difficult. At this low salt concentration, the actual sodium EDT.A concentration inside the dialysis tubing will be affected by the concentration of DNA and/or DNA-histone complexes due to the Donnan phenomenon. Complexes were prepared by salt gradient dialysis in the presence of 5 M urea (in addition to DNA-protamine) at various input ratios.
All complexes were prepared by salt gradient dialysis in the presence of 5 M urea, except the DNA-protamine complex which was unsuccessful according to the procedure and was prepared by salt gradient dialysis alone. Nucleohistones Ia and Ib have the same Tm which is slightly lower than that of nucleohistones IIb or IV. DNA complexes containing combinations of histones were studied to learn more about possible interactions between different histones.
The questions are: can the multiphase melting profiles of DNA complexes with individual histone fractions be shown by a combination of histones; what will be the T of communication complexes; or the cooperativity and sharpness of the melting transition is increased. Histones Ia and IIb were mixed based on equal molar concentrations of their lysine and arginine residues. The temperature of the transition in the first step is close to the temperature of the DNA, and the transition in the second step also takes place at a constant temperature.
Like nucleohistone IIb, complexes with combination of Ia and IIb also show small turbidity changes upon heating. Histones Ia and IIb are mixed with the same concentration of their lysine and arginine residues.
The melting temperatures are in all cases between the temperatures of the two species concerned. As seen from all the melting profiles presented}, the sharpness of the melting of DNA-histone complexes is generally less than that of pure DNA. Thus, determining the ratio of free DNA to the complexed DNA from the melting profiles and the ratio of histone to total DNA constitutes one of the most direct ways to assess the stoichiometries of DNA-histone complexes.
There should be no significant amount of free histone in the complex, so the histone/DNA ratio is a measure of the proportion of histone in complex with DNA. Assuming that all lysine and arginine groups of these two histone types are bound to phosphate groups, at least 20% of DNA phosphates are free in nucleohistones Ia and Ib. A study of the interaction of toluidine blue (a cationic dye) with calf thymus nucleohistone shows that about half of the phosphates are free (Miura and Ohba, 1967).
The implications of the present finding for nucleohistone structure will be further discussed in Chap. The overall shape of the DNA spectrum is therefore not changed by complexation with histone Ia. The protamine complex of DNA, in which 70% of the amino acids are arginine, is also inactive to support RNA synthesis.
This provides a starting point for: further study of the characteristic structure of nucleohistones of DNA complexed with various histone species. The sharpness of the transition is generally less than that of DNA complexed with a single histone species. The biphasic nature of the melting profiles suggests that cooperativity of binding operates even in the presence of mixtures of histones.
If it is assumed that all lysine and arginine residues are bound to DNA, there should still be 20% of DNA phosphates which are free. It may be that a significant portion of the lysine and arginine residues that are not in the DNA-binding portion of the histone molecule are free. In view of the following properties compared to those of nucleohistones I (see previously mentioned references);.
This is shown in Figure 19 with all the cationic amino acid residues indicated by square boxes.
