I. Chromosomal RNA of Calf Thymus Chromatin. II. The Template Properties of DNA-Polypeptide Complexes. III. Studies on DNA Complexes with Purified Histone Fractions
<p>Part I. Chromosomal RNA of Calf Thymus Chromatin</p> <p>Calf thymus chromatin is shown to contain an associated chromosomal RNA as do the chromatins of other species. The chromosomal RNA of calf thymus chromatin is present in an amount of 1% of that of DNA. The purified ma...
| Summary: | <p>Part I. Chromosomal RNA of Calf Thymus Chromatin</p>
<p>Calf thymus chromatin is shown to contain an associated chromosomal
RNA as do the chromatins of other species. The chromosomal RNA
of calf thymus chromatin is present in an amount of 1% of that of DNA.
The purified material eluted from DEAE-Sephadex column at a NaCl
concentration of 0.56 M as do the chromosomal RNA's of other organisms.
Its average chain length by end-group assay is approximately 40 nucleotides
and it contains approximately 3-4 dihydrouridylic acid residues
per chain. Calf thumus chromosomal RNA is associated with chromosomal
protein in a form not dissociable by high salt concentration.</p>
<p>Part II. The Template Properties of DNA-Polypeptide Complexes</p>
<p>DNA complexes with poly-L-lysine, poly-L-arginine and protamine
were prepared by a salt gradient dialysis. The complexes possess
the stoichiometry of one lysine or arginine residue per nucleotide
residue as determined from the biphasic melting profiles. The template
activity of a complex in support of RNA synthesis in the presence of
excess RNA polymerase and required substrates is proportional to its
fractional content of free DNA segments. The complexed DNA region is
quantitatively blocked and does not act as template. Kinetic analysis
of the template behavior reveals two different modes of inhibition by
the polypeptides. If the template is in a finely dispersed state, it
is available to the enzyme as shown by the fact that the equal template
concentrations of complex and of pure DNA are required for half
saturation of a given amount of enzyme (K). Inhibition of RNA synthesis
is, we propose, due to interference with local untwisting of DNA.
If the template is in a highly aggregated state, K is drastically increased
and it is unavailable to the enzyme. The several species of
histone molecules normally complexed with DNA in the eucaryotic organisms
differ among themselves in content of lysine and arginine. The
present studies show that the arginine residues are as effective as
the lysine residues in abolishing DNA template activity.</p>
<p>Part III. Studies on DNA Complexes with Purified Histone Fractions</p>
<p>Well-defined DNA complexes with calf thymus histones Ia, Ib,
IIb and IV have been prepared by a salt gradient dialysis in the
presence of 5 M urea. The complexes with subequivalent histone/DNA
ratio exhibit biphasic melting profiles. T<sub>m,1</sub> is the melting of free
DNA segments, and T<sub>m,2</sub> that of the histone-complexed regions. T<sub>m,2</sub>
is characteristic for each DNA-basic protein complex. T<sub>m,2</sub>(°C) of,
the complexes in 2.5 x 10<sup>-4</sup> M sodium EDTA, pH 8.0 is as follows: DNA,
47.2; chromatin, 74.3; DNA-histone Ia, 75.4; DNA-histone Ib, 76.3;
DNA-histone IIb, 81.5; DNA-histone IV, 83.7; DNA-protamine, 92.5; DNA-polyarginine,
98.0; and DNA-polylysine, 99.5. The stoichiometric
ratio (histone lysine plus arginine to nucleotide) of the equivalent
complexes as detennined from the biphasic melting profiles is DNA-histone
Ia and Ib, 0.8; DNA-histone IIb, 1.2 and DNA-histone IV, 1.5.
The general shape of UV spectrum of DNA is not changed by complexing
with various histone species. DNA-histone IV complex is inactive in
priming RNA synthesis in E.coli RNA polymerase system. Possible
structures of the DNA-binding parts of histone molecules have been
discussed and illustrated with CPK molecular models in the case of
histone IV (pp. 157, 158).</p> |
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