Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

Association of nucleosome core particle DNA with different histone oligomers. Transfer of histones between DNA-(H2A,H2B) and DNA-(H3,H4) complexes

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.     

Fractionation and characterization of histones from barley (Hordeum vulgare) leaves

Histones were extracted from purified nuclei isolated from four cereals,viz. barley (Hordeum vulgare), wheat(Triticum aestivum), Aegilops squarrosa and corn (Zea mais), and from tobacco (Nicotiana tabacum). Analysis of the histones on SDS gels showed complex electrophoretic patterns with one species of both H3 and H4, one to three species of H1 and two to five species of H2. Judged from the electrophoretic patterns, the histones from barley, wheat and Aegilops are identical but different from those of corn with respect to H2. Like tobacco, corn showed two H2 components, whereas barley, wheat and Aegilops showed five H2 components. SDS gel electrophoresis of histones extracted from buds and roots of germinating seeds at different steps of germination and from different parts of ten-day-old leaves revealed that the existence in barley of multiple histone 2 variants is not restricted to any particular stage of differentiation of barley. Histones from barley leaves were resolved into four fractions by Biogel P-100 gel filtration and histones 2 were further fractionated by their differential solubility in HCl-ethanol. Each of these five fractions (H1, H3, H4, H2A and H2B, respectively) were characterized by electrophoresis on SDS or Triton-acid-urea gels and by their amino acid compositions as compared with the homologous histones of calf thymus and chicken erythrocytes. This revealed the following:

1. H3 and H4 are strictly analogous to their animal counterparts. However, H4 has an unexplained lower electrophoretic mobility in Triton-containing acid-urea gels.
2. H1 contains three components with lower electrophoretic mobilities than H1 from erythrocytes, contains more alanine than lysine and has a lower ratio of basic to acidic residues.
3. Both H2A and H2B contain at least four variants each, with higher molecular weights than in animals and higher lysine to arginine ratios. H2A variants comigrate in acid-urea-Triton gels with chicken erythrocytes H2A, whereas H2B migrate much slower.

It was concluded that the presence of multiple major variants of H2A and H2B is a frequent but not universal feature in cereals. The existence of these variants is not restricted to the embryonic stage as previously suggested for wheat (31).     

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA.

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.     

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.     

The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.

This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones.     

Major high mobility group like proteins of Drosophila melanogaster embryonic nuclei

Nuclei from Drosophila melanogaster embryos contain three major proteins which are extracted by 0.35 M NaCl and by 2% perchloric acid. One of these is histone H1, and we refer to the other two as A63 and A13 in accordance with their molecular weights determined by electrophoresis on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (63,000 and 13,000, respectively). The molecular weight of A13, based on its amino acid composition, is approximately 10,000. The amino acid analyses of A63 and A13 show that both of these proteins have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group proteins isolated from vertebrate tissues. While A13 comigrates with histone H2A on NaDodSO4-polyacrylamide gels and with H2B on acid/urea gels, it can be readily resolved from the histones by Triton/acid/urea-Na DodSO4 two-dimensional electrophoresis.     

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.     

Comparative aspects of basic chromatin proteins in dinoflagellates

Previous work on histone-like proteins in dinoflagellates is summarized, together with some new data to give an overview of basic proteins in these algae. The first two dinoflagellates studied were both found to contain one major acid-soluble protein that migrated to the same position in acidic-urea gels. When several other genera were studied however, it became apparent that the histone-like proteins from different dinoflagellates were similar but not identical. In view of the great diversity of living dinoflagellates it is speculated that further differences in dinoflagellate basic chromatin proteins will be revealed. Electrophoretic data from the eukaryotic (endosymbiont) nucleus of Peridinium balticum showed the presence of five major components. It is speculated that two of these proteins represent an H1-like doublet and two others correspond to the highly conserved histones H3 and H4. The fifth component is a new histone that may substitute for H2A and H2B in the nucleosome. Because histones and nucleosomes are present in all higher organisms but completely lacking in procaryotes, studies on basic proteins in dinoflagellates will provides insights into the evolution of histones and eucaryotic chromatin organization.     

Isoelectric focusing and isoelectric points of bovine liver histones

A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of histones.     

Analysis of Histones from the Endosymbiont Nucleus of a Binucleate Dinoflagellate1,2

Histones were prepared from chromatin of the eukaryotic (endosymbiont) nucleus of Peridinium balticum (Levander) Lemmermann. The amino acid composition of whole histone was rich in lysine and similar to that of Olisthodiscus luteus and Euglena gracilis. Electropheretic analysis of these proteins in acidic-urea disc gels revealed four major bands: one with a mobility slightly lower than that of calf thymus HI; and three others which comigrated with calf H2B, H2A, and H4, respectively. The low mobility band was soluble in 5% perchloric acid and was sensitive to FeCl₃ destaining. Electrophoresis in slab gels containing 0.1% SDS revealed five major components, with approximate molecular weights of 23,000, 20,000, 15,000, 13,000, and 11,000, respectively. The 15,000 and 11,000 dalton histones had mobilities identical to those of calf H3 and H4, respectively. The two highest molecular weight components were soluble in 5% perchloric acid. No bands were found to comigrate with calf H2A or H2B but a band was present that migrated to a position intermediate between calf H2A and H4 (13,000 dalton histone). Two-dimensional gels consisting of acidic-urea gels in the first dimension and SDS gels in the second dimension revealed that the 20,000 dalton component and the 13,000 dalton component are not resolved in the acidic-urea gel. As a working hypothesis, it is suggested that two of the five bands seen in SDS gels represent an H1-like doublet, and two are analagous to H3 and H4, respectively. The remaining histone may replace H2A and H2B.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

A complex pattern of H2A phosphorylation in the mouse testis

Phosphorylation of H2A histones in mouse testis was examined using testis tubule cultures labeled with 32PO4. Histones were analyzed by two systems of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography of the gels. Of the 32PO4 detected in histones, 95% was incorporated by certain modified forms of the H2A variants H2A.1 and H2A.X. Phosphorylation sites were mapped to N- and C-terminal regions of the modified variants by SDS gel electrophoresis and autoradiography of peptides generated by cleavage of in vitro-labeled proteins with N-bromosuccinimide. Incorporation rates differed for N- and C-terminal regions from different modified forms, demonstrating a complex pattern of H2A phosphorylation in the mouse testis.     

Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.     

Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.