Chromatin Reconstitution Research Articles

Nuclease-sensitivity of methylated DNA as a probe for chromatin reconstitution by genotoxicants

DNA in Chinese hamster ovary cells was labeled with [ 14C]thymidine and [methyl- 3H]-1-methionine in culture, and their nuclei were digested with micrococcal nuclease. Not until 10 percent of bulk DNA was digested did methylated DNA appear in the acid-soluble fraction. When these cells were exposed to UV-radiation, alkylating agents and intercalating agents in culture, the resistance of methylated DNA to digestion by the nuclease was largely or completely eliminated. The change in the sensitivity of methylated DNA to the nuclease indicates a conformational change in chromatin induced by the genotoxicants.

Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova.

On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

Phasing of nucleosomes in SV40 chromatin reconstituted in vitro.

Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones. The reconstituted chromatin sedimented with increasing S values as the histone : DNA ratios increased, and the buoyant densities in CsCl decreased concomitantly. The average repeat lengths of nucleosomes in the chromatin reconstituted at ratios of 1.0 and 1.5 were estimated to be 168 and 143 base pairs, respectively, by electrophoretic analysis of DNA fragments generated by micrococcal nuclease digestion. The chromatin generated a series of DNA bands that differed in size by about 10 nucleotides upon DNase I digestion followed by heat-denaturation. Phasing of nucleosomes was probed by the use of single-site restriction endonucleases, EcoRI, BamH1, BglI, and HpaII: the latter two cleave DNA at and near the origin of DNA replication and transcription. The form I DNA in the chromatin reconstituted at ratios of 0.5, 1.0, and 1.5 was cleaved up to 60 to 80%, 20 to 60%, and 0 to 10%, respectively. Although the frequency of cleavage by these enzymes was not very different at the ratio 0.5, the BglI site became relatively more susceptible than the other sites at the ratio 1.0. At the ratio 1.5, the DNA was almost resistant to these enzymes, though a significant amount (10%) was cleaved by BglI. These results suggest that the origin is the site unfavorable for nucleosome phasing although the region can be almost completely covered with nucleosomes at higher histone : DNA ratios. The fraction of chromatin immunoprecipitated with anti-T serum after in vitro T antigen binding also decreased with increase in the histone : DNA ratios. The results suggest that T antigen binds preferentially to the internucleosomal region. T antigen preferentially bound to the chromatin reconstituted with the DNA fragment containing the origin. Inefficient phasing of nucleosomes at the origin of DNA replication may facilitate the binding of T antigen to the origin.

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in ( i) nuclei depleted of H1 by acid extraction; ( ii) nuclei treated at pH 3.2 (without removal of H1), and ( iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.

Reconstitution of chromatin at physiological ionic strength and regulation of transcription of the reconstituted chromatin.

Rat liver and simian virus 40 (SV40) chromatin were reconstituted in vitro under physiological conditions of ionic strength and temperature. The nucleosome assembly under the conditions was mediated in the presence of chromatin extracts, rich in nicking-closing activity, from rat liver or cultured CV-1 cells. The frequency of nucleosome assembly on DNA was dependent on both the incubation time and the weight ratio of histone to DNA. The regulatory effects of host cellular histones on the transcription of SV40 DNA were investigated by using reconstituted SV40 chromatin containing or lacking histone H1. The cellular histones composing the chromatin were prepared from permissive CV-1 cells. Transcription of chromatin was analyzed in vitro using Escherichia coli DNA-dependent RNA polymerase. The rate of incorporation of ribonucleoside triphosphates into RNA synthesized on SV40 chromatin containing Hl as the template was 5 to 10% of the rate for RNA synthesized on supercoiled SV40 DNA. The rate of incorporation for SV40 chromatin lacking Hl was approximately 40 to 50% of that for SV40 DNA. RNA products transcribed from both these chromatin and SV40 DNA were fairly homogeneous 16 to 28S species with several identical peaks.

Interaction of HMG 14 and 17 with actively transcribed genes

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted of HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10–20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.

Transcription of globin genes in reticulocyte chromatin

FEBS LettersVolume 105, Issue 1 p. 131-136 Full-length articleFree Access Transcription of globin genes in reticulocyte chromatin Daphne Maryanka, Daphne Maryanka Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorGraham J. Cowling, Graham J. Cowling Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorJames Allan, James Allan Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorStephen J. Fey, Stephen J. Fey Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorPiroska Huvos, Piroska Huvos Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorHannah J. Gould, Hannah J. Gould Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this author Daphne Maryanka, Daphne Maryanka Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorGraham J. Cowling, Graham J. Cowling Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorJames Allan, James Allan Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorStephen J. Fey, Stephen J. Fey Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorPiroska Huvos, Piroska Huvos Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this authorHannah J. Gould, Hannah J. Gould Department of Biophysics, King's College, 26-29 Drury Lane, London WC2B 5RL, EnglandSearch for more papers by this author First published: September 01, 1979 https://doi.org/10.1016/0014-5793(79)80902-3Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume105, Issue1September 01, 1979Pages 131-136 ReferencesRelatedInformation

Modification of rat liver chromatin by [formula omitted] or [formula omitted] and template activity for RNA synthesis by Escherichia coli RNA polymerase after reconstitution

Rat liver chromatin was fractionated into DNA, histones and non-histone chromosomal proteins and each component was modified with N- methyl-N′- nitro-N- nitrosoguanidine or N- ethyl-N′- nitro-N- nitrosoguanidine . The radioactivity of 14C-labeled alkyl or guanidino moieties of both compounds bound significantly to both histones and non-histone chromosomal proteins and the binding of N- methyl-N′- nitro-N- nitrosoguanidine was higher than N- ethyl-N′- nitro-N- nitrosoguanidine . However the binding of both compounds to DNA was very low and its significance was hard to evaluate. All of the three components, one of which was modified, were reconstituted into chromatin, then, [ 3H]UMP incorporation into acid insoluble material using Escherichia coli RNA polymerase (EC 2.7.7.6) was measured. Only with the reconstituted chromatin containing histones modified either by N- methyl-N′- nitro-N- nitrosoguanidine or N- ethyl-N′- nitro-N- nitrosoguanidine , the template activity increased drastically; i.e., about 10 or 5 times higher than that with the unmodified reconstituted chromatin, respectively. However, any remarkable alteration in the electrophoretic pattern of protein fraction of the reconstituted chromatin could not be found. The results obtained in this study are discussed in the context that the modified histones could give rise to change in the mutual interaction of chromosomal components during the reconstitution of chromatin accompanied with the increase of chromatin template activity.

Human cytomegalovirus-induced chromatin factors responsible for changes in template activity and structure of infected cell chromatin

Human cytomegalovirus (HCMV) infection in human embryo lung cells caused rapid changes in conformation of chromatin as judged from circular dichroism (the ellipticity at 272 run) of the infected cell nuclei or chromatin determined at intervals after infection. The infection also caused a rapid increase in chromatin template activity determined by (i) the incorporation of [ 3H]UTP into RNA in the presence of saturating levels of E. coli RNA polymerase and (ii) the number of binding sites for ethidium bromide in chromatin. These conformational and functional changes did not occur when cycloheximide was present in the cultures or when cells were infected with UV-irradiated virus, suggesting the dependency of the chromatin changes on HCMV-coded functions expressed after infection. Chromatin reconstitution experiments in which NaCl extracts from HCMV-infected cell chromatin were added to uninfected cell chromatin and the reconstituted chromatin was subjected to the determination of ellipticity at 272 nm and dye-binding capacity showed that HCMV induced at least two chromatin factors; one responsible for an increase in ellipticity at 272 nm of chromatin was selectively extractable with 0.35 – 0.6 M NaCl from HCMV-infected cell chromatin and the other responsible for an increase in dye-binding capacity (corresponding to chromatin template activity) was extractable with 0.35 M NaCl. These chromatin factors had properties of DNA-binding proteins; the former was dissociated from DNA at 0.6 M NaCl and the latter at 0.35 M NaCl.

Mode of chromatin reconstitution. Elements controlling globin gene transcription.

The mode of reconstitution has been determined for the factors that control globin gene transcription in chicken reticulocyte chromatin. During chromatin reconstitution, elements in the nonhistone protein fraction that control the globin gene reassociate with the DNA before the histones. From our results, it appears that it is necessary for these elements to reassociate with the DNA before the histones for the globin gene to be transcribed.

Myocardial chromatin activation in experimental hyperthyroidism in rats. Role of nuclear non-histone proteins.

HomeCirculation ResearchVol. 42, No. 3Myocardial chromatin activation in experimental hyperthyroidism in rats. Role of nuclear non-histone proteins. Free AccessAbstractPDF/EPUBAboutView PDFSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessAbstractPDF/EPUBMyocardial chromatin activation in experimental hyperthyroidism in rats. Role of nuclear non-histone proteins. C J Limas and C Chan-Stier C J LimasC J Limas Search for more papers by this author and C Chan-StierC Chan-Stier Search for more papers by this author Originally published1 Mar 1978https://doi.org/10.1161/01.RES.42.3.311Circulation Research. 1978;42:311–316 Previous Back to top Next FiguresReferencesRelatedDetailsCited By Moussavi R (1990) Neonatal hyperthyroidism enhances growth of the heart but not of skeletal muscle, Mechanisms of Ageing and Development, 10.1016/0047-6374(90)90140-B, 52:1, (1-10), Online publication date: 1-Mar-1990. Zimmer H, Seesko R, Zierhut W and Pechan I (1987) Cardiac Effects of ß-Adrenergic Blockers in Combination with Metabolic Interventions Pharmacological Aspects of Heart Disease, 10.1007/978-1-4613-2057-9_19, (233-247), . Zimmer H and Peffer H (1986) Metabolic aspects of the development of experimental cardiac hypertrophy Controversial issues in cardiac pathophysiology, 10.1007/978-3-662-11374-5_13, (127-137), . Limas C (1984) Subcellular Changes in Compensated and Failing Hypertrophied Hearts Functional Aspects of the Normal, Hypertrophied, and Failing Heart, 10.1007/978-1-4613-3825-3_10, (151-170), . Morkin E, Flink I and Goldman S (1983) Biochemical and physiologic effects of thyroid hormone on cardiac performance, Progress in Cardiovascular Diseases, 10.1016/0033-0620(83)90004-X, 25:5, (435-464), Online publication date: 1-Mar-1983. Limas C (1982) Enhanced myocardial RNA synthesis in spontaneously hypertensive rats Possible role of high-mobility group non-histone proteins, Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 10.1016/0167-4781(82)90007-0, 696:1, (37-43), Online publication date: 1-Jan-1982. Lester L, Sodt P, Rich B, Lucky A, Hutcheon N and Arcilla R (1982) Cardiac abnormalities in children with hyperthyroidism, Pediatric Cardiology, 10.1007/BF02332112, 2:3, (215-223), Online publication date: 1-Sep-1982. Limas C (1981) Phosphorylation of myocardial nuclei enhances their susceptibility to deoxyribonuclease I, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(81)91972-0, 100:3, (1347-1352), Online publication date: 1-Jun-1981. Zak R (1981) Contractile Function as a Determinant of Muscle Growth Cell and Muscle Motility, 10.1007/978-1-4684-8196-9_1, (1-33), . Smallridge R (1980) Thyroid Hormone Effects on the Heart Hearts and Heart-Like Organs, 10.1016/B978-0-12-119402-4.50009-9, (93-160), . Zak R, Kizu A and Bugaisky L (1979) Cardiac hypertrophy: Its characteristics as a growth process, The American Journal of Cardiology, 10.1016/0002-9149(79)90226-1, 44:5, (941-946), Online publication date: 1-Oct-1979. March 1, 1978Vol 42, Issue 3 Advertisement Article InformationMetrics Copyright © 1978 by American Heart Associationhttps://doi.org/10.1161/01.RES.42.3.311 Originally publishedMarch 1, 1978 PDF download Advertisement

Reconstitution of chromatin: assembly of the nucleosome.

The order of reassociation of the four histones H2a, H2b, H3 and H4 to the DNA during the reconstitution of chromatin was determined. At each step of the reconstitution the DNA and associated histones were separated from the free histones by centrifugation in a glycerol gradient. The unbound and reassociated histones were analysed by gel electrophoresis and the histone-DNA complexes characterized by circular dichroism and electron microscopy. We show that H3 and H4 bind first to the DNA between 1.2 M NaCl and 0.85 M NaCl and impose a nucleosome like structure; in a second step histones H2a and H2b are placed around this kernel to complete the nucleosome.

Chapter 33. The Assay of Globin Gene Transcription in Reconstituted Chromatin

Publisher Summary The reconstitution of chromatin from selected components offers a rational approach to the problem of the mechanism of transcriptional control in eukaryotic systems. The complete functional reconstitution of chromatin from all the required components, for example, DNA, histones, and chromatin nonhistone proteins, must be judged by an appropriate assay. This assay should measure the differential synthesis of at least one tissue-specific RNA species when the chromatin, or the reconstituted chromatin, is transcribed in vitro. The combination of procedures fully described in this chapter has allowed evaluating the fidelity of globin gene transcription in reconstituted chromatin. The objective of the chapter is to set out rigorous criteria by which the results of reconstitution attempts should be judged and to describe in detail the experimental procedures and their pitfalls and the technical uncertainties that still remain.

Chapter 32. Methods for Dissociation, Fractionation, and Selective Reconstitution of Chromatin

Publisher Summary Chromatin reconstitution provides a direct approach for assaying the contribution of various genome-associated macromolecules to defining the availability of specific genetic sequences for transcription. While there are several modifications of the technique, chromatin is generally dissociated in the presence of high salt-urea and then reconstituted by gradually removing the salt, followed by removal of the urea. The procedures employed for the isolation of chromatin have a very direct bearing on the final product. Chromatin could be appropriately defined by the isolation protocol. It therefore follows that the validity of chromatin as representative of the genome found in the nucleus of intact cells and the feasibility of dissociating, fractionating, and reconstituting chromatin are dependent on the method used for chromatin isolation. The chapter discusses the method used in laboratories for chromatin dissociation and reconstitution and illustrates evidence for the level of fidelity attained by the procedure.

Hydroxyapatite thermal chromatography of chromatin. Elution pattern of mouse satellite dna in partially dehistonized and reconstituted chromatin

1. 1. It has previously been found that during hydroxyapatite thermal chromatography of mouse-liver chromatin and DNA, mouse-satellite DNA eluted at higher temperature from chromatin, than it did from purified DNA. 2. 2. The elution profile of mouse-satellite DNA in chromatin depleted of histone H1 was shifted to lower temperature. After the removal of all lysine-rich histones (HI + H2A + H2B) a further shift occurred and the elution profile coincided with that obtained upon the melting of purified DNA. 3. 3. The reconstitution of chromatin restored the higher temperature elution profile of satellite DNA.

Effect of proteolysis on transcriptional fidelity of reconstituted chromatin

The effect of proteolysis on the transcriptional properties of reconstituted rat liver chromatin was studied. Within the sensitivity of currently available methods, proteolysis of chromosomal proteins by chromatin-bound protease during chromatin reconstitution has no apparent effect on: (1) number of initiation sites, (2) proportion of reiterating and unique sequences of DNA transcribed, (3) size of the RNA transcribed, and (4) transcription of DNA sequences complementary to poly(A) containing messenger RNA.

Analysis and reconstruction of Xenopus ribosomal chromatin nucleosomes.

The double-stranded DNA from staphylococcal(micrococcal)-nuclease-derived chromatin sub-units (nucleosomes) from transcriptionally active somatic cells of heterozygous mutant (0/+ nu) embryos of the amphibian Xenopus laevis has been analyzed. To determine whether the transcriptionally active ribosomal genes of these 0/+ nu embryos are selectively hydrolyzed by micrococcal nuclease cleavage and, more generally to determine whether any particular frequency class of DNA sequences was differentially removed by nuclease treatment of chromatin, DNA reassociation (cot) curves of heterozygote (0/+ nu) mutant subunit (nucleosome) DNA were compared with the cot curves obtained from whole 0/+ nu DNA. The results of these experiments clearly demonstrated that cleavage of the chromatin by micrococcal nuclease, to the point where about 75% of the DNA is present as fragments about 200 base pairs long, does not result in any preferential removal (hydrolysis) of either the ribosomal cistrons or any particular frequency class of gene sequences. However, these same reassociation curves also show that 0/+ nu DNA has a noticable decrease in sequence complexity in all of its frequency classes relative to that of whole 0/+ nu DNA. Further experiments demonstrated that this decreased complexity is probably due to two factors: (a) a change in the nucleotide base composition of the nucleosome DNA relative to that of whole DNA (base composition analyses indicate that the subunit DNA has about 10% fewer dA + dT residues than whole DNA); and (b) the cleavage by micrococcal nuclease of the base sequences between adjacent chromatin subunits within each of the frequency classes of DNA. Further evidence supporting the similarity of nucleosome and whole cellular DNA comes from DNA-driven cot reassociation experiments involving the renaturation of individually isolated frequency classes of DNA from nucleosomes (i.e., unique sequences and intermediate repetitive sequences) with an excess of whole (sheared) cellular DNA, which showed that the frequency classes of DNA within nucleosomes are indistinguishable, on kinetic grounds, from those of non-nuclease-treated DNA. Addition experiments involving solution hybridization reactions followed by hydroxyapatite analysis of nucleic acid duplex formation have indicated that nucleosome DNA derived from transcriptionally active cells contains fragments of DNA that anneal to both ribosomal RNAs and poly(A)-containing messenger RNAs. And, finally, the results from chromatin reconstitution experiments using isolated ribosomal DNA (rDNA) are presented that indicate that the association of nucleosomes with DNA in the formation of chromatin subunits is probably not base-sequence specific. All of these experiments are consistent with (but do not prove) the idea that transcriptionally active somatic cell ribosomal and unique-sequence genes can be partially, or perhaps transiently, associated with histones in the form of nucleosomes or chromatin subunits. These results are then discussed in terms of a dynamic model for the association of nucleosomes with transcriptionally active chromatin.

Nuclease digestion of reconstituted chromatin

Nuclease digestion experiments have recently provided evidence for the existence of a periodic structure in chromatin [l-4]. It is not clear, whether and to what extent the periodic structure re-forms when dissociated chromatin is reconstituted. The procedures for dissociation and reconstitution of chromatin may be divided into two groups according to the presence [5-91 or absence [IO-l 21 of urea. In this work we study the degradation products with micrococcal nuclease (EC 3.1.4.7) and DNAase I (EC 3.1.4.5) of chromatin reconstituted in the presence and in the absence of urea. Chromatin reconstituted by a salt step gradient only give discrete fragmentation patterns, suggesting that these preparations possess a secondary structure similar to that of untreated chromatin. The presence of urea during the reconstitution procedure abolishes the specificity of enzyme fragmentation of chromatin DNA.

Effects of estrogen on gene expression in the chick oviduct. Control of ovalbumin gene expression by non-histone proteins.

Chromatin was isolated from the oviducts of chicks which had either received diethylstilbestrol stimulation for 14 days (stimulated chromatin) or been treated and then withdrawn from hormone administration (withdrawn chromatin). These chromatin preparations were dissociated and then reconstituted in the presence of various non-histone protein fractions. cDNAov was used as a probe for measuring the level of mRNAov sequences synthesized in vitro from various reconstituted chromatins. Our results indicate that extractable non-histone proteins from stimulated chromatin are capable of activating the in vitro transcription of the ovalbumin gene when included in the reconstitution of withdrawn chromatin. On the other hand, addition of extractable non-histone proteins from withdrawn chromatin to stimulated chromatin does not affect the synthesis of mRNAov sequences. Therefore, the extractable non-histone proteins from stimulated chromatin contain a positive regulator(s) which controls the in vitro expression of the ovalbumin gene.

Mode of reconstitution of chicken erythrocyte and reticulocyte chromatin

The mode of reconstitution of chicken erythrocyte and reticulocyte chromatin has been investigated. Chromatin was dissociated in 2 M NaCl, 5 M urea, and 0.01 M potassium phosphate (pH 7.2) and was dialyzed against various NaCl concentrations in 5 M urea and 0.01 M potassium phosphate (pH 7.2). Histone reassociation to DNA occurs with the binding of histone H5 at 0.5 M NaCl in 5 M urea, followed by histone H1 at 0.4 M NaCl in 5 M urea. All the classes of histones are reassociated with DNA at 0.2 M NaCl in 5 M urea and binding of all classes of histones is complete in 0.1 M NaCl and 5 M urea. Nonhistone proteins reassociate with DNA before and at the same time that histones reassociate with DNA. Binding of nonhistone proteins to DNA appears to be complete in 5 M urea and 0.01 M potassium phosphate (pH 7.2). There is also found in both erythrocyte and reticulocyte chromatin a nonhistone protein present in relatively high concentrations, which remains associated with DNA in 2 M NaCl and 5 M urea. This tightly bound protein appears as one major band when chromatographed on sodium dodecyl sulfate-polyacrylamide gels, with a molecular weight of 95 000. This protein is soluble in phenol and sodium dodecyl sulfate but is insoluble in 5 M urea or 4 M guanidine hydrochloride. A fraction of reticulocyte nonhistone proteins was found to bind to DNA-cellulose in 5 M urea. The majority of these proteins elute at 0.15 M NaCl in 5 M urea but a significant fraction elutes at NaCl concentrations at which the bulk of the histones do not bind to DNA. The proteins that bind to free DNA have low molecular weights and do not show species speciificity. Approximatley 50% of the reticulocyte nonhistone protein does not bind to a DNA-cellulose column in 5 M urea and may require histones for complete reassociation.