Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans

The histones of Caenorhabditis elegans (Nematoda) have been identified by correlating criteria of electrophoresis and amino acid composition with the five main histones from calf thymus. C. elegans H1(1) consists of at least two subtypes with approximate molecular weights of 20,000 and 18,500 daltons as resolved by SDS polyacrylamide gel electrophoresis. They are some 10% smaller than the two subtypes of calf histone H1. The differences are also corrobated by the amino acid composition of the nematode and calf H1 complements. Nematode H2A resembles calf H2A in chromatographic and electrophoretic properties and in the amino acid composition, although it lacks histidine, which seems to be replaced by lysine. Like calf H2A, it is dimorphic as shown by Triton/acid/urea polyacrylamide gel electrophoresis. The H2B complement from C. elegans consists of two proteins with a molecular weight of approximately 12,500. They can be separated by ion-exchange chromatography, but they are very analogous to each other and to calf H2B in amino acid composition. Each form is also resolved into two more subtypes by Triton/acid/urea polyacrylamide gel electrophoresis. Nematode H3 resembles calf thymus H3 in its electrophoretic behaviour; three subfractions can be distinguished in Triton/acid/urea gels. C. elegans H4 is very similar to calf H4 in its chromatographic, electrophoretic and solubility properties, but differs significantly in composition. The meaning of this difference is discussed with regard to the generally observed stringent conservation of H4 sequences between distantly related species.     

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.     

The histones of the insect trypanosomatid, Crithidia fasciculata

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.     

Electrophoretic separation of histones and high-mobility-group proteins on acid-urea-Triton gels

The electrophoretic mobilities of calf thymus histones and high-mobility-group (HMG) nonhistone proteins were studied on a newly modified polyacrylamide gel containing acetic acid, urea, and the nonionic detergent Triton X-100 in combination with glycine in the electrode buffer. This gel system avoids stacking gel, photopolymerization of acrylamide, and preelectrophoresis. Under extremely low Triton concentrations some H3 variant forms (H3.1) were preferentially separated by their slower migration from bulk H3. Under increasing concentrations of Triton in the gel in the presence of 3 or 6 M urea, the mobilities of H2A.1, H3.2, H2A.2, H4, and H2B were sequentially retarded. The mobilities of H1 and HMGs remained virtually unchanged under all conditions. This gel system is able to resolve charge-modified histones.     

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.     

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.     

Histones from Trypanosoma lewisi nuclei]

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.     

Etude des proteines an cours de la germination des graiues: Isolement et analyse des histories de radis Par

Histories of radish seedlings liave been characterised by comparison with pea histones extracted and purified under the same conditions. Submitted to acrylamide gel analysis the radish histones are resolved into main protein bands comparable to those of pea or calf thymus histones. However, protein bands of lesser importance which are not to be found in electrophoretic profils of pea or calf thymus histones, appear together with the main bands of radish histones. Study of these supplementary proteins through isoelectric focusing makes it possible to distinguish two groups: contaminating acid proteins and weakly basic proteins characterised by their high amide content.     

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.     

Comparative electrophoretic properties of histones from trout and chicken erythrocytes and calf thymus at different concentrations of EDTA]

An introduction of EDTA into an electrophoretic system was found to cause specific changes in the histone distribution patterns. The electrophoretic mobility of histones H3, H2b and H2a from three evolutionally unrelated sources (trout and chicken erythrocytes and calf thymus) is increased and that for histones H1 and H5 is decreased with respect to histone H4. In general the decrease of electrophoretic mobility of the histones in the presence of EDTA is correlated with the content of basic amino acids in these histones. The effect observed can be used from electrophoretic analysis of histones.     

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.     

Histones from gonads of the star-fish Asterias rubens.

1. Histones were isolated from gonads of the star-fish Asterias rubens and characterized by their amino acid composition and their electrophoretic migration. 2. Comparative studies with calf thymus homologous histones show the highly conservative structure of the histones H3 and H4, and the variability of the other histones namely H1 and H2B.     

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.     

Histone H5 in nucleated erythrocytes of fish as determined by radioimmunoassay

Tissue-specific histone H5 in the nucleated erythrocytes of dogfish, scup, skate, tautog, sea robin and toad fish was studied. The presence of this histone was inferred by its electrophoretic mocility on polyacrylamide gels containing either acid-urea or sodium dodecyl sulfate. By radioimmunoprecipitation assays, cross reaction was observed between fish histones and an anti-H5chicken antibody. The antibody was specific to chicken histone H5; purified chicken histone H1 and calf thymus total histones did not cross react. It is concluded that fish histone H5 shares common antigenic determinants with the chicken H5 histone.     

Affinity chromatography of cysteine-containing histone.

Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.     

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.     

Isolation and characterization of histones from Anopheles albimanus Weidemann

1. Histones from Anopheles albimanus adults were prepared by a combination of techniques including chromatin isolation and selective extractions. 2. The anopheline histones were identified on acid urea gels by comparing their electrophoretic profile with that of calf thymus histones and histones isolated from other tissue. 3. Excellent separation of histones was obtained after the extractions by a single electrophoretic run. 4. In addition to the five major classes of histones found in eukaryotes, a sixth class was detected and tentatively identified as histone H5. 5. This is the first report of histone H5 and its function in insects.     

The histones of rainbow trout erythrocytes include an erythrocyte-specific histone.

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erythrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation. To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to extablish identities of corresponding histone fractions, An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.     

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.