Studies on the structure and function of chick-oviduct chromatin. 1. Fractionation by ECTHAM-cellulose chromatography and physico-chemical characterization.
Chick oviduct chromatin was separated into a ribonucleoprotein fraction and two chromatin fractions (early and late eluting). We utilized a gentle procedure in which moderately hydrated chromatin was subjected to chromatography on a weak ionic-exchange resin (ECTHAM-cellulose) eluted with a combined pH-salt gradient. Chemical analysis of the early (fraction I) and late (fraction II) eluting fractions revealed that their histones were identical and their nonhistone proteins were markedly different. Control experiments showed that these differences were not due to protein rearrangements during chromatin preparation and/or fractionation. The physical properties of fraction I and II differed in certain aspects. The aggregation response of fraction I to increasing concentrations of monovalent cations was five times lower than that of fraction II but the aggregation response to divalent cations was identical. Thermal denaturation assays of DNAs isolated from fractions I and II revealed identical derivative profiles of hyperchromicity vs temperature, thereby indicating similar base composition in the two fractions. Circular dichroism, spectra of the purified DNAs isolated from both fractions showed identical B-type conformations. However, DNA renaturation kinetics analyzed by computer technique indicated that fraction I DNA contained less than half the amount of highly repetitive sequences as compared to either unfractionated chromatin or fraction II. Circular dichroism spectra of fraction I and II chromatins (at room temperature) showed significant differences in a wavelength region were only DNA is optically active (i.e. 255-320 nm). These results indicated that the DNA complexed to proteins in fraction II assumed a more C-type conformation than the DNA in fraction I. The differences in the circular dichroism spectra could not be accounted for by differences in the RNAs or protein chromophores contained in fraction I and fraction II. When the circular dichroism spectra of fraction I and II were recorded at 55 degrees C, the differences between the two fractions were abolished. These results were interpreted to mean that the differences in the DNA conformations found in fractions I and II were due to the differences in their nonhistone proteins. These proteins were effective in maintaining DNA conformation differences only when they were in their native form but not when heated to 55 degree C. Comparison of the sedimentation coefficients of fractions I and II with their calculated molecular weights suggested a more extended structure in fraction I as compared to a more compact structure in fraction II. Only small differences were observed between fraction I and fraction II with respect to either buoyant density analysis in a metrizamide gradient or in the number of phosphate charges accessible to polylysine.
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D002843:Chromatin / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004276:DNA, Satellite / Q000032:analysis; D005260:Female; D006657:Histones / Q000032:analysis; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation; D010057:Oviducts / Q000032:analysis*; D012261:Ribonucleoproteins / Q000302:isolation & purification
D002843:Chromatin; D004276:DNA, Satellite; D006657:Histones; D012261:Ribonucleoproteins