The histones of some human tissues

1. Histones were examined from five human tissues, namely thymus, liver, placenta, bronchial tumour and peripheral leucocytes. 2. The four main histone fractions [F1, F2(b), F2(a), and F3] were isolated and characterized. 3. The amino acid analyses, N-terminal group analyses and electrophoretic patterns were very similar to those of the corresponding fractions of calf thymus. 4. The yields of fractions F1 and F2(b) were high in human thymus, in human bronchial tumour and in some preparations of normal human leucocytes. 5. It is concluded that the pattern of nuclear histones found in lower forms of life was conserved during the evolutionary process leading to man.     

Structure and histone composition of chromatin in Physarum polycephalum]

In Physarum polycephalum several degrees of organisation of deoxyribonucleoprotein fibres were found. The complexes of histones and the DNA duplex seem to "be packed" at first into a 100 A fibre and then into a 200 A fibre of DNP. In Ph. polycephalum the electrophoretic mobilities of histone fractions 4 and 6 are comparable to that of fractions f3/f2b and f2a1 of calf thymus, resp. Histone fractions 3 and 5 move a bit faster than fractions f1 and f2a2, resp. Thus, the myxomycete P. polycephalum is similar to higher eukaryotes as concerns the ultrastructure of chromatin and electrophoretic properties of histones.     

Comparative study of histones from slime mold Physarum polycephalum and calf thymus

The histones from slime mold Physarum polycephalum and calf thymus were characterized in terms of some physico-chemical properties. The molecular weights of six principal histone fractions of Ph. polycephalum were found to be the following: P1--22 700, P3--15 700, P4a--15 000, P4b--14 300, P5--12 800 and P6--10 500. Electrophoretically homogenous histone fractions H1, H2b and H4 of calf thymus and histones P1, P3, P4b and P6 of slime mold were obtained by gel-filtration on Acrylex P-60. These findings suggest that fractions P1, P4a, P4b, P5 and P6 of slime mold Ph. polycephalum are homologus with respect to the histone fractions H1, H3, H2b, H2a and H4 of calf thymus. Only fraction P3 has no corresponding fraction in the calf thymus histones; a fraction corresponding to histone P3 of slime mold was absent.     

In vitro acylation of the ϵ-amino group of l-lysine in calf thymus histones by the carcinogen, β-propiolactone

We had previously reported that the carcinogen, β-propiolactone (BPL) reacted in vitro with histones in whole mouse skin chromatin and that among the histone classes BPL was preferentially bound to the lysine-rich histones H1 and H1°. In order to determine if in vitro reaction of BPL with calf thymus histones resulted in binding of BPL to l-lysine, we synthesized the model compounds ?-N-(3-hydroxypropionyl)lysine (HPL) and ?-N-(2-carboxyethyl)lysine (CEL) from BPL and l-lysine. The α-amino group of l-lysine was protected from reaction with BPL by the formation of a copper chelate.Structures were assigned on the basis of infrared spectra, pKa values and chemical analyses. BPL was reacted in vitro with calf thymus histones and the BPL-reacted calf thymus histones and control calf thymus histones were digested with trypsin followed by pronase. The respective digests were each chromatographed on a column of AA-15 cation-exchange resin. The elution profiles of the two digests were very similar except for the appearance of a new ninhydrin-positive peak (NNPP) in the eluate of the trypsin-pronase digest of BPL-reacted calf thymus histones. When compounds HPL and CEL were added to the trypsin-pronase digest of control calf thymus histones and the mixture chromatographed on AA-15, both compounds were resolved from the other peptide (or amino acid) peaks. HPL was eluted in the same fractions as NNPP, HPL and NNPP exhibited identical R_F values on silica gel TLC with acidic, alkaline and neutral solvents. CEL was not identified as a product of the reaction between BPL and calf thymus histones.     

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.     

Some Researches on Histones

The histone extracted from calf thymus glands is a complex system of proteins, which can be fractionated by chromatography on carboxymethyl cellulose columns into three principal fractions (1) very lysine-rich, (2) moderately lysine-rich, (3) arginine-rich. When examined by starch gel chromatography each of these gives more than one band. Methods have been devised for further separation of the components in some cases. The components show characteristic differences in end groups and certain amino acids as well as in their basic character. Histones extracted from various rat tissues can be separated into similar fractions, of which the amino acid analyses are similar to those derived from calf thymus, within the experimental error. To this extent, no species or tissue specificity of the fractionated histones was observed. Although all the histone fractions contain approximately one basic amino acid to three non-basic amino acids their structure is not regular, as Phillips has shown that in certain fractions the number of non-basic groups between two basic groups may vary from 0 to seven or more. The possible functions of histones are discussed.     

Histones of the fungi, Endomyces magnusii and Neurospora crassa]

Histones of Endomyces magnusii and Neurospora crassa were found to consist of four main fractions similar to calf thymus histones in their electrophoretic mobilities, molecular sizes and chromatographic behaviour on Akrilex P-60. Two of them are homologous to the most conservative histones H3 and H4. Other two fractions correspond to the histones H2A and H2B; however, they have some pecularities. A fraction of N. crassa histones corresponding to the H2B was isolated in a homogeneous state by means of gel filtration. It appeared to be very similar to calf thymus histone H2B in its amino acid composition.     

A method for the selective extraction of histone fractions f2(a)1 and f2(a)2 from calf thymus deoxyribonucleoprotein at pH7

1. A new method has been developed for the specific extraction of histone fraction f2(a) from calf thymus deoxyribonucleoprotein at pH7 by using a mixture of ethanol and guanidinium chloride. 2. Fraction f2(a) has been separated into the subfractions f2(a)1 and f2(a)2 by acetone precipitation from acid solution, and at pH7. 3. Modifications of existing electrophoretic methods are described that enable these fractions to be more easily characterized.     

Immunofluorescent study of the basic chromosomal protein set of green algae and Euglena]

Using antisera to fractions H1, H2a, H3 and H4 of the calf thymus histones, a comparative immunofluorescent investigation of these proteins in the nuclei of Chlamydomonas reinhardii, Haematococcus pluvialis, Dunaliella salina and Euglena gracilis was carried out. It has been shown that according to the immunofluorescent test, the nuclei of these algae contain proteins close to fractions H2a, H3 and H4 of the calf thymus histones. H1 fraction in these algae is either absent or can be considered as a protein immunochemically non-related to H1 fraction of the calf thymus histone. For quantitative evaluation (in units of the immunological distance) of the difference between histones of the algae and of the calf thymus in situ by indirect immunofluorescence, it was suggested to use the ultimate dilutions of antisera to histones. It was shown that the ultimate dilutions were correlated with titres of antisera in the reaction of microcomplement fixation. Such an approach and the data obtained are of interest for studying into the evolution of nucleosome histones in unicellular and multicellular eukaryotes.     

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.     

Antisera to individual histone fractions of the calf thymus. II. A study of the immunochemical specificity of the histones by an indirect immunofluorescence method]

Immune antisera to 5 fractions (H1, H2a, H2b, H3, H4) of calf thymus histone were assayed using indirect immunofluorescence (IIF). The analysis of such sera by this technique, as well as the data on complement fixation obtained previously, show that these antisera are highly active and specific for various test-objects: thymys, liver nuclei of rat, chicken, and calf, chicken erythrocytes, metaphase chromosomes of mouse fibroblasts. These antisera are of importance for the evaluation of species- and tissue-specificity of different histone fractions. Using the IIF reaction, a comparison was made between the nucleosome fraction H3, which is evolutionary stable, and fraction HI from calf thymus, rat and chicken liver, and chicken erythrocytes.     

Homology of histones H2B from calf thymus and P4B from slime mold Physarum polycephalum

A comparative study of the amino acid composition of histone fractions P4b from slime mold Physarum polycephalum and H2B from calf thymus was carried out using peptide mapping. It was shown that 75% of peptides are common for both proteins. The slime mold histones contain two fractions (P4B and P3), which are homologous to the H2B histone fraction of calf thymus. The data of amino acid analysis, peptide mapping and some physico-chemical properties of the histones revealed the following correlation of the two types of histone fractions: P1--H1, P4a--H3, P4b and P3--H2B, P5-H2A, P6--H4.     

A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Constancy of wheat histones during development

Summary Histones were extracted from leaves of winter- and spring-wheat seedlings, flowering shoots of spring wheat, shoots of vernalized and control winter wheat, and roots and shoots of winter wheat, and were compared by polyacrylamide-gel electrophoresis. No differences were found either in the electrophoretic mobility or relative quantity of the various fractions. Wheat histones contained fractions of the exact electrophoretic mobility as F2a1 and F3 of calf thymus and pea histones. Other major fractions of the wheat histones had electrophoretic mobilities similar to those of F1, F2b and F2a2 of peas.     

Interaction of quinacrine mustard with calf thymus histone fractions

1. Interactions of histone fractions with quinacrine mustard were investigated by fluorimetry and spectrophotometry and the results were interpreted with the aid of thinlayer chromatography. 2. Characteristic differences were found between the various histone fractions of calf thymus. The conditions that favoured histone conformational changes and aggregation, also favoured interaction between histones and the dye; low concentrations of SO(4) (2-) brought about more interaction than did Cl(-); urea, guanidinium and iodide ions were inhibitory to binding. 3. Changes in the physical state of all the quinacrine mustard-protein complexes occurred as a function of ionic strength and pH. The most salt-dependent interaction was found in the arginine-rich histone fraction. 4. The interaction of the calf thymus histone fractions with quinacrine mustard was compared with the interaction of bovine plasma albumin and protamine with quinacrine mustard. 5. The relationship between dye-binding and the aggregation of histone fractions was discussed.     

STUDIES ON HISTONE FRACTION F2A1 IN MACRO- AND MICRONUCLEI OF TETRAHYMENA PYRIFORMIS

Histone fraction F2A1 has been isolated and purified from macronuclei of the ciliate Tetrahymena pyriformis. It migrates as a single species on sodium dodecyl sulphate-acrylamide gel electrophoresis, with a molecular weight indistinguishable from that of calf thymus F2A1. The solubility properties of Tetrahymena F2A1 are also similar to those of calf thymus F2A1. Electrophoretic analyses on urea-acrylamide gels indicate that Tetrahymena F2A1 consists of four or five subspecies, the two fastest having electrophoretic mobilities identical with those of the two major electrophoretically separable forms of calf thymus F2A1. High resolution (long gel) electrophoresis coupled with incorporation of radioactive acetate both in vivo and in vitro suggest that, as in the case of calf thymus F2A1, differentical acetylation of a parent molecule can explain the observed electrophoretic heterogeneity of Tetrahymena F2A1. Electrophoretic analysis of histones isolated from the micronucleus, which is genetically less active than the macronucleus, indicates that it contains largely the relatively unacetylated (parent) form of histone F2A1.     

Presence of histones in Aspergillus nidulans

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea- starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.     

Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro

This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages. Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column. SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones. Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo. Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation. Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation. Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation. In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation.     

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.