Calf Thymus Chromatin Research Articles

Non-histone chromosomal proteins easily extractible from chick erythrocytes

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HClO 4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise, non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.

Isolation and characterization of protein A24, a "histone-like" non-histone chromosomal protein.

In earlier studies, the nucleolar levels of protein A24 were found to be markedly decreased in the nucleolar hypertrophy induced by thioacetamide or during liver regeneration (Ballal, N.R., Goldknopf, I.L., Goldberg, D.A., and Busch, H. (1974) Life Sci. 14, 1835-1845; Ballal, N.R., Kang, Y.-J., Olson, M.O.-J., and Busch, H.J. Biol. Chem. 250, 5921-5925). To determine the role of protein A24, methods were developed for its isolation in highly purified form. Milligram quantities of highly purified protein A24 were isolated from the 0.4 N H2SO4-soluble proteins of calf thymus chromatin by exclusion chromatography on Sephadex G-100, followed by preparative polyacrylamide gel electrophoresis. Protein A24 was highly purified as shown by its migration as a single spot on two-dimensional polyacrylamide gel electrophoresis, its single NH2-terminal amino acid, methionine, and the production of approximately 50 peptides by tryptic digestion. Like histones 2A, 2B, 3, and 4. A24 was extractable from chromatin with 0.4 N H2SO4 or 3 M NaCl/7 M urea, but unlike most non-histone proteins or histone 1, protein A24 was not extracted with 0.35 M NaCl, 0.5 M HClO4, or 0.6 M NaCl. Protein A24 was present in only 1.9% of the total amount of histones 2A, 2B, 3 and 4; its molecular weight is 27,000.

Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA.

1. The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. 2. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26 000 was obtained. 3. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated.

Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.

Nucleas action on chromatin: evidence for discrete, repeated nucleoprotein units along chromatin fibrils.

The time course of the fragmentation of calf thymus chromatin by DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6) has been examined by sedimentation of chromatin digests through linear (5-20%) sucrose gradients. The action of nuclease is decidedly nonrandom, ultimately producing roughly equal amounts of acid-soluble oligonucleotides and 11S nucleoprotein particles. The 11S particles contain double-stranded DNA that is approximately 400 A or 120 base-pairs long, as measured by electron microscopic examination of deproteinized samples, and is maintained in a compact conformation within the intact particles. In addition, 15S nucleoprotein particles containing predominantly 800-A lengths of DNA have been isolated from less extensively digested chromatin. Evidence is presented which indicates that the 11S particles are fundamental structural units that are arranged in tandem along certain regions of chromatin fibrils. Preliminary experiments with different nucleases and with chromatin from different mammalian species indicate that these results are a natural consequence of the arrangement of DNA and proteins in mammalian chromatin and are not peculiar to the system described in detail.

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.

Mammalian DNA Polymerases

This chapter states that physiological experiments demonstrating variations of DNA polymerase activities were first done in mammalian systems, whereas the first relevant genetic experiments in bacteria came 12 years after the discovery of Escherichia coli DNA polymerase I. Mammalian DNA polymerase can be isolated and purified without the major problems that arise from associated nucleases. For this reason, it is relatively simple to do mechanistic studies with these enzymes. With bacterial enzymes, on the other hand, there are logical difficulties in proving the nature of the template initiator system required. Purification of mammalian DNA polymerases to homogeneity has been somewhat of a problem, and the first such homogeneous DNA polymerase, the low-molecular-weight DNA polymerase from calf thymus chromatin, is also described in the chapter. The chapter discusses the nature of template initiator systems, the major DNA polymerase activities in cell fractions and the properties of purified mammalian enzymes. Future direction will start with the information presented and eventually proceed into the more complex “replication systems” including the “associated proteins” clearly required for DNA replication. To analyze the participation of the known DNA polymerases in DNA replication, polymerase mutants and selective inhibitors are sorely needed.

The quantitative protein composition of calf thymus chromatin.

The proteins of calf thymus chromatin were analysed quantitatively using a combination of polyacrylamide and cellogel electrophoreses. Quantification was achieved by determining the staining values of each purified histone with amido-black. The results indicate that, on average, 700 molecules histone F1, 850 of F2al, 1440 of F2a2, 1 890 of F2b, 2380 of F3 and 500-1000 non-histone molecules are bound per 10(5) base pairs of DNA. This suggests a moderately dense protein covering of the DNA.

Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.

Comparison of polytene chromatin from Drosophila hydei with rat liver and calf thymus chromatin

1. 1. The polytene chromatin was isolated and purified from salivary glands of third instar larvae of Drosophila hydei. 2. 2. Analysis of the chromatin showed 350 parts protein and 28–33 parts RNA per 100 parts DNA. The deoxyribonucleoprotein is compared to that of rat liver and calf thymus. 3. 3. About half the amount of polytene chromatin proteins are basic; moreover 18% of these basic proteins have extreme pK values greater than 9-3 like the histones have. 4. 4. The ‘ polytene proteins ’ were separated and characterized by two electrophoretic systems. The histones F3, FaAI and possibly F 2A 2 were shown to be identical with the corresponding proteins of calf thymus.

Electron microscopy of chromatin subunit particles

Electron microscope studies of PS-particles obtained by partial nuclease digestion of calf thymus chromatin shows them to be very similar in size and shape to particles which have been detected in native chromatin. Furthermore, measurement of the spacing between pairs of such particles, together with the known length of the DNA coiled in the particles gives an estimate of about 200 base pairs for the chromatin “subunit”.

Relationship between protein and DNA structure in calf thymus chromatin. II. Conformational aspects.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRelation between protein and DNA structure in calf thymus chromatin. II. Conformational aspectsSue Hanlon, Roger S. Johnson, and Aurelia ChanCite this: Biochemistry 1974, 13, 19, 3972–3981Publication Date (Print):September 1, 1974Publication History Published online1 May 2002Published inissue 1 September 1974https://doi.org/10.1021/bi00716a024RIGHTS & PERMISSIONSArticle Views26Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Relationship between protein and DNA structure in calf thymus chromatin. I. Compositional aspects.

Relationship between protein and DNA structure in calf thymus chromatin. I. Compositional aspects.

Distribution of RNA Polymerase Binding Sites in Fractionated Chromatin

Calf-thymus chromatin was fractionated by ion-exchange chromatography on ECTHAM-cellulose and sucrose gradient sedimentation. [ECTHAM-cellulose is a cationic adsorbent prepared by coupling tris(hydroxymethyl)aminomethane to cellulose with epichlorohydrin.] The capacity of these fractionated chromatins to support RNA synthesis by DNA-dependent RNA polymerase of Escherichia coli was examined, using procedures that permit measurement of binding site frequency. Unfractionated calf thymus chromatin has 5-10% as many binding sites as protein-free DNA. By combination of the two fractionation methods, chromatin samples were obtained containing as few as 2% and as many as 47% of the number of binding sites found on protein-free DNA.

Electric dichroism of chromatin

The linear dichroism of sheared calf thymus chromatin, oriented in solution by a pulsed electric field, has been measured. The limiting value of this dichroism is considerably less negative than that of calf thymus DNA, but does not approach the positive values predicted if chromatin were uniformly supercoiled in the manner suggested by X-ray studies. The decay of the dichroism of chromatin, after termination of the electric pulse, is similar to that of DNA, indicating that chromatin retains a high degree of chain flexibility. These data, as well as previous flow dichroism studies (Ohba, 1966; Smart & Bonner, 1971), suggest that chromatin is not predominantly supercoiled in solutions of low ionic strength. Evidence for a structurally heterogeneous model for chromatin is discussed.

Reactive properties of the amino groups of histones in calf thymus chromatin

The reactivity of the five main histones in calf thymus chromatin towards acetic anhydride has been determined using the technique of competitive labelling (Kaplan et al., 1971; Kaplan, 1972). Under trace labelling conditions all the histones incorporate radioactive label from tritiated acetic anhydride, however, only histone IIb2 incorporates the label at a rate which is consistent with a substantial portion of its amino groups being fully exposed. Identification of the reactive functional groups of histone IIb2, revealed that the reactivity could be accounted for entirely by the reactivity of its N-terminal proline. It is concluded that the vast majority if not all the ϵ-amino groups of histones in calf thymus chromatin have abnormally high pK a values and thus appear to be buried, while the N-terminus of histone IIb2 is exposed. This conclusion supports the hypothesis that the ϵ-amino groups of histones form salt linkages with the phosphates of DNA.

Contribution à l'étude de la structure de la chromatine: I - Etude physico-chimique de la stabilité de la chromatine

Physicochemical studies of calf thymus chromatin were performed on micromicellar suspensions by thermal denaturation. These diluted suspensions were obtained, by a controlled shearing method, from a compact gel chromatin. Sedimentation and free-flow electrophoresis determined the size distribution of these particles. The most important result is a new transition on the melting profiles corresponding to a sudden increase of solution turbidity. This chromatin solution transition occurs at a higher temperature than usual DNA transition. The degree of « turbidity transitiondiminishes with micelle size but disappears when they are very mildly degraded by DNAases and when F1 histone fraction is removed. This transition is not only size dependent but also depends on the micellar structure. This phenomenon is interpreted as an excluded volume effect by contact between compact and native regions of nucleoprotein micelles and denatured coils of DNA. Our study tried to show that the degree of turbidity transition can be a criterion of chromatin native structure. Une étude physico-chimique de la dénaturation thermique de la chromatine extraite du thymus de veau a été effectuée sur des suspensions micromicellaires préparées par cisaillement contrôlé d'un gel concentré. La stabilité thermique des suspensions a été étudiée, d'une part sur les particules natives en fonction de leur taille dont la distribution a été déterminée par sédimentation et électrophorèse en veine liquide, et d'autre part, sur des particules dont la structure a été perturbée par l'action des DNAases et des sels. Le résultat essentiel est l'observation d'une séparation de phase sous forme d'un fin précipité. Elle se traduit par l'apparition, sur les courbes de fusion, d'une augmentation brusque de la turbidité formant une nouvelle transition. Celle-ci est caractérisée par une température supérieure à la température de fusion du DNA dans la chromatine. L'amplitude de cette transition décroit en fonction de la taille des micelles mais elle est complètement supprimée après une dégradation modérée par les DNAases ou après élimination de la fraction d'histone F1; par conséquent cette séparation de phase dépend, à la fois, de la taille et de la structure interne des micelles. Cette transition peut-être expliquée par l'existence d'effets de volume exclu dans un microgel par contact de parties condensées et de parties partiellement dissociées, par élévation de température. Cette transition prend la valeur de critère physico-chimique caractérisant l'état structural natif des préparations de nucléoprotéines.

The Effect of Trypsin on Nuclease-resistant Chromatin Fragments

Abstract Fragments of calf thymus chromatin that selectively resist micrococcal nuclease digestion are found to be highly compact nucleoprotein particles, each containing a single piece of tightly folded or supercoiled DNA. These particles exhibit an anomalous CD spectrum, and melt in the upper end of the chromatin melting range. Upon trypsin digestion, only about 20% of the protein is removed, but the properties of the particles are greatly modified. They unfold, as judged from hydrodynamic studies, the CD spectrum becomes very much like that of DNA, and the melting point drops by about 20°. We conclude that proteins in the particles serve to maintain the compact structure.

Optical studies of complexes of quinacrine with DNA and chromatin: implications for the fluorescence of cytological chromosome preparations.

The fluorescence and circular dichroism of quinacrine complexed with nucleic acids and chromatin were measured to estimate the relative magnitudes of factors influencing the fluorescence banding patterns of chromosomes stained with quinacrine or quinacrine mustard. DNA base composition can influence quinacrine fluorescence in at least two ways. The major effect, evident at low ratios of quinacrine to DNA, is a quenching of dye fluorescence, correlating with G-C composition. This may occur largely prior to relaxation of excited dye molecules. At higher dye/DNA saturations, which might exist in cytological chromosome preparations stained with high concentrations of quinacrine, energy transfer between dye molecules converts dyes bound near G-C base pairs into energy sinks. In contrast to its influence on quinacrine fluorescence, DNA base composition has very little effect on either quinacrine binding affinity or the circular dichroism of bound quinacrine molecules. The synthetic polynucleotides poly(dA-dT) and poly(dA)-poly(dT) have a similar effect on quinacrine fluorescence, but differ markedly in their affinity for quinacrine and in the circular dichroism changes associated with quinacrine binding. Quinacrine fluorescence intensity and lifetime are slightly less when bound to calf thymus chromatin than when bound to calf thymus DNA, and minor differences in circular dichroism between these complexes are observed. Chromosomal proteins probably affect the fluorescence of chromosomes stained with quinacrine, although this effect appears to be much less than that due to variations in DNA base composition. The fluorescence of cytological chromosome preparations may also be influenced by fixation effects and macroscopic variations in chromosome coiling.