Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.     

The histones of rat testis

Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X₁, X₂, and X₃) were observed. Fraction X₁ had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X₁ were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X₁ had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels.Fraction X₂ migrated between histone fractions F1 and F3 on electrophoresis while X₃ migrated between fractions F2b and F3. Fraction X₃ was isolated with F2b during fractionation by the Johns procedure. Fraction X₂ has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X₂ can be seen in electrophoretic patterns of rat liver histones.The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X₁ and F1, or X₃ and F2b are not attributable to phosphorylation.     

Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

The fractionation of histones isolated from Euglena gracilis.

1. The histones of Euglena gracilis were separated by gel filtration into five fractions. 2. Each fraction was characterized in terms of its electrophoretic, solubility and compositional properties. 3. Euglena gracilis clearly contains histones corresponding to vertebrate H1, H2B, H3 and H4 fractions, although they all differ in containing more lysine. 4. The remaining Euglena histone is considered to be homologous to vertebrate histone H2A, but it differs in having a much higher ratio of lysine to arginine. 5. The Euglena histone H1 appears to be lacking in aspartic acid. 6. Electrophoresis in the presence of sodium dodecyl sulphate indicates that the molecular weights of the Euglena histones are close to those of the homologous vertebrate histones.     

The structural role of histone H1: properties of reconstituted chromatin with various H1 subfractions (H1-1, H1-2, and H1o).

A previous study on the distribution of histone H1 subfractions in chromatin suggested that these proteins differ in the protection they confer to DNA. To elucidate further this suggestion, reconstitution experiments were carried out with purified H1 subfractions (H1-1, H1-2, H1o) and H1-depleted chromatin. We have studied the structural properties of H1o as compared to those of other H1 fractions by electrophoretic analysis of DNA and mononucleosomes obtained after micrococcal nuclease digestion, thermal denaturation, and electron microscopy. The three fractions studied reassociate to H1-depleted chromatin. However, differences in the extent of DNA protection are observed between H1o and the other fractions: H1o induces a more rapid degradation of long oligomers into mononucleosomes; these mononucleosomes bearing H1o only, have a greater electrophoretic mobility; furthermore, thermal denaturation shows that a small fraction of DNA is less efficiently protected by H1o than by the other fractions. Electron microscopy, on the other hand, shows that these differences are not due to areas of chromatin devoid of H1o in the reconstitute and that the reconstituted samples are able, under proper ionic conditions, to refold in a higher-order structure.     

Studies on heterogeneity of the lysine-rich histone from ox pancreas

1. Histone H1 from ox pancrease has been isolated by preparative electrophoresis at pH 2.7 in polyacrylamide slab gel, using the fraction F1 of Oliver et al. (1972, Biochem. J., 129, 349--353) as starting material. 2. The isolated histone H1 showed higher heterogeneity on isoelectric focusing than on polyacrylamide electrophoresis in long gel. The isoelectric points of the main subfractions of histone H1 were at pH 8.0--8.4.     

Metabolism of histones in malignant tissues and liver of the rat and mouse

1. The relative amounts of incorporation in vivo of l-lysine, and in one experiment l-arginine, into different histone fractions from Krebs ascites and a lymphoma ascites cells of mice and a `solid' tumour and liver of rats have been determined. 2. No marked differences in the incorporations of the amino acids into the fractions F1, F2a, F2b and F3 from the tumours were generally observed, although in some experiments there was a greater incorporation into fraction F2b, which could be decreased by further purification. 3. In the tumours the incorporations into all cell protein fractions obtained were approximately the same, indicating that the amount of incorporation was that required for the increase of cell mass. 4. In rat liver, the incorporations into fractions F1, F2a and F3 were not greatly different. That into fraction F2b was variable. The incorporation into the histone fractions was much less than that into the acid-insoluble nuclear residue, indicating that considerable turnover of amino acids in the latter occurs. 5. The decrease in radioactivity of labelled histone and acid-insoluble nuclear protein in vivo during several days confirmed the relatively small turnover of the histone fraction. The time taken for liver whole histone to lose half its radioactivity was about 1 week. A histone fraction of slower metabolism was also detected. 6. It is concluded that no appreciable turnover of protein occurs in any one histone fraction, the somewhat higher values obtained in certain cases being associated with acidic impurities. The apparently high rate of incorporation into histone of resting liver is discussed in relation to recent evidence on DNA metabolism of resting liver.     

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.     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.     

Chromatin proteins from normal,vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Study of the relationship between the subfraction composition of histone F1 and the type of tissue in birds.

The subfraction composition of lysine-rich histone has been studied with the aid of polyacrylamide gel electrophoresis. The subfraction compositions of the histone F1 of several tissues from the chicken, pigeon, and titmouse have been compared. The histone F1 from the tissues investigated consists of four or five subfractions of similar number and electrophoretic mobility (1, 1a, 2, 3, and 4). In the different avian species each subfraction varied its mobility independently of the others. The chicken tissues investigated can be divided into two classes, depending on the relative concentration of subfractions 2 and 3 (A and B): Class A (subfraction 2 is smaller than 3) includes the brain, liver, skeletal muscle, heart, muscular layer of the stomach, and pancreas, and class B (subfraction 2 is larger than 3) includes the intestinal mucosa, thymus, and testes, as well as the liver, heart, and pancreas from a 21-day embryo. Such a division of the tissues corresponds to the varying rate of their cellular renewal. In a parallel examination of the relative concentrations of the individual subfractions in the same tissues from the three avian species it has been found that the relative concentration of subfractions 3 and 2 is increased in the skeletal muscles, heart, brain, and liver, that subfraction 2 is increased in the intestinal mucosa, that subfractions 4 and 3 are increased in the pancreas, and that subfractions 1, 1a, and 4 are increased in the erythrocytes. The results obtained may be interpreted as a consequence of some relationship between the subfraction composition of histone F1 and the type of tissue of the source.     

An electrophoretic comparison of the histones of various strains of Tetrahymena pyriformis

Histones were extracted from isolated macronuclei of several strains of the ciliated protozoan Tetrahymena pyriformis and compared by electrophoresis on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gels. High resolution urea-acrylamide-gel electrophoresis resolves Tetrahymena histones into five main classes. The lysine-rich histone H1 exhibits microheterogeneity within each strain, mostly due to phosphorylation, and varies extensively in electrophoretic mobility and apparent molecular weight among the strains. Both H3 and H4 are constant among Tetrahymena strains and consist of several secondarily modified subspecies. However, while the electrophoretic constancy of H4, observed in higher organisms, extends to this lower eukaryote, H3 of each Tetrahymena strain migrates faster than calf thymus H3 in both gel systems. This suggests that H3 is not as rigidly conserved as H4. Fraction HX has no electrophoretic counterpart in calf thymus histone. It consists of five subfractions, each of which displays a remarkably constant electrophoretic mobility among the various strains. H2B is electrophoretically variable among Tetrahymena strains. The intersyngen and interphenoset diversity of Tetrahymena histone is shown to be comparable to that found among vertebrate classes.     

Interactions of calf-thymus histone fractions in aqueous solution with 8-anilinonaphthalene-1-sulphonic acid

1. The interactions of histone fractions with 8-anilinonaphthalene-1-sulphonic acid were investigated by fluorimetry and spectrofluorimetry and the results were interpreted with the aid of equilibrium-dialysis techniques. 2. Characteristic differences were found between the various histone fractions, and with fractions F3 and F2a the binding was found to be salt-dependent. 3. Evidence was obtained indicating a slow change of the physical state of fractions F3 and F2a in the presence of salt, and the binding by these two fractions in the presence of salt was greater by an order of magnitude than by fractions F1 and F2b. 4. Conditions favouring binding were also those favouring histone aggregation; SO(4) (2-) ions activated binding at a lower concentration than Cl(-) ions; urea, guanidinium ions and high concentrations of I(-) ions were inhibitory to binding. 5. After histones had been kept in the presence of salt for a long time the reversal of interaction on decreasing the salt concentration was incomplete. 6. The inhibition of binding by fraction F2a in the presence of urea or fraction F2b depended on the time sequence of addition of the reagents. 7. Artificial nucleoproteins made by precipitating DNA with the histone fractions in neutral 0.14m-sodium chloride showed the same order of interaction as was found for the fractions in solution. 8. Comparison of the binding by fraction F2a with that by bovine plasma albumin showed that in both cases there were a large number of weakly binding sites but that fraction F2a lacked the small number of strongly binding sites found in albumin. No slow change of binding in the presence of salt was found for albumin. 9. Binding by fraction F2b increased the affinity of the protein for further molecules of the adsorbate. 10. The results are discussed in relation to the close relationship between binding and aggregation and the possible role of non-polar interactions as determined by the balance between polar and non-polar amino acids in the histone fractions.     

The stepwise removal of histones from chicken erythrocyte nucleoprotein

1. A fractionation of chicken erythrocyte histones was achieved simultaneously with their extraction from saline-washed nuclei by stepwise titrations to progressively lower pH values. 2. Different acids and dilute buffer solutions of comparable pH behaved similarly in stepwise extractions of histones. 3. The histone preparations so obtained were characterized by their amino acid composition and behaviour on zone electrophoresis in starch gels. 4. The fractionation by titration was quite sharp at appropriate pH ranges, and the histone fraction that is apparently unique to avian erythrocytes was obtained without contamination by other histone fractions. 5. Histones prepared by stepwise titration were fractionated further by cation-exchange and exclusion chromatography. The chromatographic behaviour and amino acid composition of the components permitted comparison with histones prepared by other methods. 6. Histone fraction IIb was resolved into its subfractions IIb(1) and IIb(2) by exclusion chromatography on Bio-Gel P-60. 7. Histone fractions III and IV, previously reported to be absent from chicken erythrocyte nuclei, were found in extracts made at pH1.     

An evolutionary comparison of plant histones.

Histones were extracted from chromatin of the following: a moss (Polytrichum juniperinum); the primitive vascular plants Psilotum nudum and Equisetum arvense; a fern (Polypodium vulgare); the gymnosperms fir (Abies concolor), yew (Taxus canadensis) and Gingko biloba; the dicotyledonous angiosperms tobacco (Nicotiana tabacum) and maple (Acer saccharinum); and the monocotyledonous angiosperms corn (Zea mays) and lily (Lilium longiflorum). The histones were subjected to polyacrylamide gel electrophoresis and compared to standard histones of pea (Pisum sativum) and cow (Bos taurus). All species have histones of the exact electrophoretic mobility of histones F2a1 and F3 of cow and pea. All species have histones of low electrophoretic mobility assumed to be F1 histones. None of the plant histones displayed electrophoretic mobility between F3 and F2a1 while animal histone fractions F2b and F2a2 do migrate to this position. No animal histone fraction was found to migrate between F3 and F1 while a major plant fraction, designated "F2b-like" was found to migrate to this position in all plant species studied except for the moss and Psilotum. A band of similar mobility was strikingly absent from the histones of these two species.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.