Chemical and physical properties of fractionated chromatin.

Abstract

Sonicated chicken reticulocyte chromatin was fractionated into transcriptionally active and transcriptionally repressed components. The active fraction is 8% of the whole chromatin but contains 70% of the newly synthesized chromosomal RNA. This RNA has five times as many hemoglobin RNA sequences as does the RNA in the repressed fraction. The amount of the active fraction in the chromatins of several tissues correlates with their synthetic activity. The molecular weight of the DNA of the repressed fraction is approximately twice that of the active fraction. Moreover, the configuration of repressed chromatin is much more compact, consistent with a much larger sedimentation constant. The transcriptionally active fraction displays a 60 lower melting profile and is highly susceptible to DNase I relative to the repressed fraction. The active fraction contains twice as much non-histone protein and 15% less histone than the repressed fraction and is lacking the lysine-rich and much of tie arginine-rich histones. The fraction of eukaryotic chromatin which is transcriptionally active comprises only a small part of the total genome (1). It is advantageous, therefore, to be able to separate this material from the remainder of the chromatin and to identify how it differs from the much larger part that is transcriptionally inactive. For work in this direction we have chosen 12-day chick embryo reticulocyte chromatin for study, since it is actively transcribing hemoglobin messenger RNA and since it has a transcribable region which comprises a particularly small percentage of the genome and therefore produces a comparatively limited variety of mRNA molecules.Various techniques have been used to fractionate chromatin. The essential break-up of the native chromatin has been accomplished by shearing (2-5), sonication (6), DNase digestion (7), or pressure (5, 8) and the chromatin fragments have been fractionated by differential centrifugation (9), sucrose gradient centrifugation (2, 3, 5), solubility (7), and ion exchange (6) or hydroxyapatite (4) We have sought to obtain a clearer separation of transcriptionally active chromatin (TC) and inactive, repressed chromatin (RC) so that the differences might be more unambiguously assessed. This has been done by combining several fractionation and separation techniques in order to maximize efficient and reproducible separation of a small, discrete portion of the chromatin with transcriptional activity (16). This fraction shows more striking differences in both chemical and physical properties when compared to the remainder of the genome than has generally been reported. MATERIALS AND METHODSChromatin Fractionation and Separation. Blood was collected from 12-day-old chick embryos. The cells were washed in Solution 1 [0.32 M sucrose, 2 mM MgCl2, 1 mM potassium phosphate (pH 6.8)] and then lysed in 10 mM CaCl2. The nuclei were washed in a Dounce homogenizer with a solution containing 0.32 M sucrose, 1 mM MgC12, 1 mM potassium phosphate (pH 6.8), and 0.3% Triton N-101. Nuclei were st...

Morphology of Transcriptionally Active Chromatin

“…The hypothesis that condensed chromatin is usually characterized by very low or no transcriptional activity, and that transcription occurs in loosely packed forms of chromatin (including, in most cells, the nucleolar chromatin) has received support from studies of ultrathin sections in the electron microscope and from the numerous attempts to separate transcriptionally active from inactive chromatin biochemically (for references, see Anderson et al 1975;Berkowitz and Doty 1975;Krieg and Wells 1976;Rickwood and Birnie 1976;Gottesfeld 1977). Electron microscopic autoradiography has revealed that sites of RNA synthesis are enriched in dispersed chromatin regions located at the margins of condensed chromatin (Fakan andBernhard 1971, 1973;Bouteille et al 1974;Bachellerie et al 1975) and are characterized by the occurrence of distinct granular and fibrillar ribonucleoprotein (RNP) structures, such as perichromatin granules and fibrils.…”

Effect of Rate of Replication upon Transcription in Chick Embryo Limb Bud Mesenchyme Cells in Organ Culture

“…There is a large body of data showing that chromatin which is being more actively transcribed has higher ratios of non-histone protein/DNA (16,17,18,191 and lower ratios of histone/DNA [16,18,20,211 and this may account for the higher levels of transcription of the more slowly replicating chromatin DNA. However, the chromatin DNA that replicates more slowly during the first 10 min of S or the last half of S is richer in non-histone chromatin proteins and therefore may have fewer histones than the more rapidly replicating chromatin DNA.…”

“…However, the chromatin DNA that replicates more slowly during the first 10 min of S or the last half of S is richer in non-histone chromatin proteins and therefore may have fewer histones than the more rapidly replicating chromatin DNA. There is a large body of data showing that chromatin which is being more actively transcribed has higher ratios of non-histone protein/DNA (16,17,18,191 and lower ratios of histone/DNA [16,18,20,211 and this may account for the higher levels of transcription of the more slowly replicating chromatin DNA.…”

A model for cytoplasm-governed gene regulation

“…It is noteworthy that not only chromatin fractions that are disperse and active in transcription but also those that are condensed and inactive in transcription, contain RNA species (Berkowitz & Doty, 1975;Gottesfeld et al, 1975). In some cases the amount of heterochromatin-associated RNA is even higher than that of RNA associated with euchromatin (Paul & Duerksen, 1975).…”