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.     

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.     

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.     

The histones of the endosymbiont alga of Peridinium balticum (Dinophyceae)

The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively.     

Chromatin structure from the marine sponge Geodia cydonium

The histones isolated from the siliceous sponge Geodia cydonium have been separated using two electrophoretic techniques. A comparison of their mobilities with those of calf thymus and rat liver show that some Geodia histone species (H3, H1 and H1(0) exhibit electrophoretic variance. The results show, that as in other eukaryotic systems the sponge chromatin contains the core histones (H2A, H2B, H3 and H4) and the linker histone (H1). ADP-ribosylation of Geodia histones and separation of the individual histones by electrophoresis resulted in four histones being radiolabeled. Digestion of Geodia chromatin with endogenous endonuclease is shown to result in the formation of nucleosome particles containing approximately 200 base pairs of DNA. A major product of endogenous endonuclease digestion is a relatively stable 110 base pair intermediate. Incubation of chromatin with DNase II and separation of the products under denaturing conditions reveals 20 bands migrating at 10 base intervals.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

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.     

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.     

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 from spermatozoa of the horseshoe crab

It is shown that histones are the nuclear proteins present in spermatozoa of the horseshoe crab Limmulus polyphemus, an arthropod which is considered a living fossil. They have been characterized and found to be closely related to calf thymus histones. The only difference is the presence of an additional histone in small amounts (2?3% of the whole histones) which has intermediate properties between H1 and H2b.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

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.     

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.     

Histones H3 and H2a are homologous to the lambda repressor and cro proteins in 22 residue segments implicated in DNA binding

The histones H3 and H2a from calf thymus are homologous to the repressor and cro repressor proteins of bacteriophage lambda in a 22-residue segment that has been implicated by mutational and model-building studies in DNA binding. In the lambda proteins this segment is folded into a helix-turn-helix unit of supersecondary structure, and we propose that the homologous regions in the histones possess the same fold. Homology was quantified with a unified procedure based on criteria of identity of key residues, primary structural homology and similarity of secondary structural potential. It has previously been shown that a set of other prokaryotic DNA-binding proteins have primary structural homology with the two lambda proteins. Homologies detected between the histones H4 and H2b and members of this set suggest that these histones also contain the putative DNA-binding fold.     

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.     

Differences among subfractions of H1 histone in retention of linear and superhelical DNA on filters

Four kinds of rabbit thymus H1 histone differ among themselves in their ability to retain DNA on nitrocellulose filters. This is true for linear, or superhelical DNA, but the order of effectiveness of the different H1 histones depends on the physical conformation of the DNA. For linear DNA the binding efficiencies of the H1 histones are: RTL2 = RTL3 greater than RTL4 greater than RTL1. This order of effectiveness parallels the effectiveness of the H1 histones previously found for the condensation of linear DNA as observed by circular dichroism and viscosity. The binding efficiencies of the various histones toward superhelical DNA were: RTL4 greater than RTL3 greater than RTL1 greater than RTL2. The variation in amino acid sequence between different rabbit thymus H1 histones might thus introduce structural variations in nucleohistone fibers and perhaps in chromatin.     

Analysis of the binding of C-reactive protein to histones and chromatin

C-reactive protein (CRP) is an acute phase serum protein in man which binds to phosphocholine (PC) in a calcium-dependent manner. CRP has been shown to bind to chromatin and nucleosome core particles. However, CRP does not bind to DNA and there is conflicting evidence regarding the binding of CRP to histones. In the present study, binding of CRP to chromatin was confirmed by ELISA using chromatin bound to microtiter wells. When chromatin depleted of histone H1 was used in the same assay, no CRP binding was detected. Similar results were observed using a competitive inhibition ELISA. These results indicate an important role for H1 in the binding of CRP to chromatin. Further studies were done to characterize the binding of CRP to purified individual histones. CRP binding to histones was demonstrated first by blotting. Calf thymus histones were separated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with 125I-CRP. CRP bound to H1 and H2A and to a lesser extent to H2B. Non-specific binding to H3 was seen and no binding to H4 was observed. CRP binding to purified individual histones was tested by ELISA. Essentially identical results were seen to those obtained by blotting. CRP binding to the H2A-H2B complex was observed as well as reactivity with trypsin-resistant fragments of H2A, H2B, and H3. By blotting and by ELISA all CRP reactions were blocked by PC and EDTA indicating binding through the calcium-dependent PC-binding site on CRP. These studies further characterize the nature of the binding of CRP to chromatin and histones and show that the presence of H1 on chromatin is required for CRP binding.     

Displacement of histones by sperm-specific proteins at different stages of spermatogenesis of squid

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B. A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343-350]. Neither the fractional nor subfractional composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2-1 and I2-2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol). Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two-stage order: histones----I1----I2.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].