Part I. Chromosomal RNA of Calf Thymus Chromatin
Calf thymus chromatin is shown to contain an associated chromosomalRNA as do the chromatins of other species. The chromosomal RNAof calf thymus chromatin is present in an amount of 1% of that of DNA.The purified material eluted from DEAE-Sephadex column at a NaClconcentration of 0.56 M as do the chromosomal RNA's of other organisms.Its average chain length by end-group assay is approximately 40 nucleotidesand it contains approximately 3-4 dihydrouridylic acid residuesper chain. Calf thumus chromosomal RNA is associated with chromosomalprotein 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 protaminewere prepared by a salt gradient dialysis. The complexes possessthe stoichiometry of one lysine or arginine residue per nucleotideresidue as determined from the biphasic melting profiles. The templateactivity of a complex in support of RNA synthesis in the presence ofexcess RNA polymerase and required substrates is proportional to itsfractional content of free DNA segments. The complexed DNA region isquantitatively blocked and does not act as template. Kinetic analysisof the template behavior reveals two different modes of inhibition bythe polypeptides. If the template is in a finely dispersed state, itis available to the enzyme as shown by the fact that the equal templateconcentrations of complex and of pure DNA are required for halfsaturation of a given amount of enzyme (K). Inhibition of RNA synthesisis, we propose, due to interference with local untwisting of DNA.If the template is in a highly aggregated state, K is drastically increasedand it is unavailable to the enzyme. The several species ofhistone molecules normally complexed with DNA in the eucaryotic organismsdiffer among themselves in content of lysine and arginine. Thepresent studies show that the arginine residues are as effective asthe 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 thepresence of 5 M urea. The complexes with subequivalent histone/DNAratio exhibit biphasic melting profiles. Tm,1 is the melting of freeDNA segments, and Tm,2 that of the histone-complexed regions. Tm,2is characteristic for each DNA-basic protein complex. Tm,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 stoichiometricratio (histone lysine plus arginine to nucleotide) of the equivalentcomplexes as detennined from the biphasic melting profiles is DNA-histoneIa 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 complexingwith various histone species. DNA-histone IV complex is inactive inpriming RNA synthesis in E.coli RNA polymerase system. Possiblestructures of the DNA-binding parts of histone molecules have beendiscussed and illustrated with CPK molecular models in the case ofhistone IV (pp. 157, 158).
