I. Chromosomal RNA of Calf Thymus Chromatin. II. The Template Properties of DNA-Polypeptide Complexes. III. Studies on DNA Complexes with Purified Histone Fractions

Citation

Shih, Thomas Yu-tzong (1969) I. Chromosomal RNA of Calf Thymus Chromatin. II. The Template Properties of DNA-Polypeptide Complexes. III. Studies on DNA Complexes with Purified Histone Fractions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/HQQY-2J93. https://resolver.caltech.edu/CaltechTHESIS:03312017-130624894

Abstract

Part I. Chromosomal RNA of Calf Thymus Chromatin

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.

Part II. The Template Properties of DNA-Polypeptide Complexes

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.

Part III. Studies on DNA Complexes with Purified Histone Fractions

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_m,1 is the melting of free DNA segments, and T_m,2 that of the histone-complexed regions. T_m,2 is characteristic for each DNA-basic protein complex. T_m,2(°C) of, the complexes in 2.5 x 10^-4 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).

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