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 study of plant histones: Comparison with vertebrate histones

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.     

典型涡鞭毛虫(Zooxanthella microadriatica)含有多种染色质碱性蛋白

以往的研究都表明涡鞭毛虫类不含真核细胞普遍具有的组蛋白,而仅含1—2种分子量较小、含量较低和碱性较弱的染色质碱性蛋白。但我们采用自己建立的先固定后抽提的方法从典型涡鞭毛虫Zooxanthella microadriatica获得了多种碱性蛋白成分。经SDS-PAGE分析,其中有六条带的迁移率分别十分接近地对应着小牛胸腺的五种组蛋白(H1有两条亚带)。另外迁移率在H1与H3之间的三条互相靠近的电泳带,据其分子量(17—19KDa)分析极可能来自于此细胞中含量极为丰富的叶绿体类核体的染色质,由于碱性性质相似而被一同提取出来。既然我们利用先固定后抽提的方法提取小牛胸腺组蛋白和提取涡鞭毛虫(Crypthecodinium cohnii)的染色质碱性蛋白获得了良好的提取效果和很好地重复了前人的结果,我们认为本工作首次报道了在典型涡鞭毛虫类中也有含有多种染色质碱性蛋白并且很相似于组蛋白(至少在分子量上)的情形,为揭示和澄清组蛋白的起源进化问题提供了新的实验依据。     

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.     

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.     

The isolation of nuclei and basic nucleoproteins from the cellular slime mold Dictyostelium discoideum.

A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X-100. Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum. The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F1. After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.     

Yeast inner histones and the evolutionary conservation of histone-histone interactions.

The inner histones of the yeast, Saccharomyces cerevisiae, have been isolated and identified by their amino acid compositions. H4 appears to be close to its calf and pea counterparts. H2a, H2b, and H3 have diverged. The isolation of the histones was accomplished by consecutive slab-gel fractionation, and a number of novel features of the method are described. These appear to be generally useful for preparing many types of protein. The binding pattern of the yeast inner histones is identical to the binding pattern for calf and for pea histones. Data on interspecies complexing indicate that the surfaces across which the histones interact are very highly conserved.     

The isolation and purification of mouse epidermal nuclei

Calf thymus histones are separated into the five classical components H1, H2A, H2B, H3, and H4 using reversed-phase high-performance liquid chromatography. This method is fast and sensitive; a single run takes 80 min and protein quantities ranging from 3 μg up to 1 mg can be separated. The primary structure of the proteins is not affected, as demonstrated by peptide mapping and gel electrophoresis. Yeast, nematode, and calf thymus histones are compared to each other and have similar retention times for individual peaks, thus demonstrating the evolutionary stability of these proteins by using this different approach.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.     

Constancy of wheat histones during development

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

ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

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.     

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

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

In vivo phosphorylation of histones H1 and H5 in calf thymus and chicken erythrocyte as studied by 31P nuclear magnetic resonance spectroscopy

The 31P NMR method was first applied to characterize in vivo phosphorylation of H1 and H5 in calf thymus and chicken erythrocytes as well as in vitro phosphorylation of H1 and H5 by cAMP-dependent protein kinase. The amino acid residues phosphorylated in vivo in the histones were exclusively serine residues, and the mole fraction of phosphoserine was estimated to be 0.34 and 0.27 per molecule of calf thymus H1 and chicken erythrocyte H5, respectively. Interestingly, chicken erythrocyte H1 was not phosphorylated in vivo. Three H1 subtypes from calf thymus H1 varied in the 31P NMR spectra, and the bisected fragments of calf thymus H1 and chicken erythrocyte H5 exhibited characteristic spectral patterns, indicating that there are considerable diversities of the degree of phosphorylation and phosphorylation sites in very-lysine-rich histones. Furthermore, it was found that the microenvironment of phosphoserine residues phosphorylated in vivo in calf thymus H1 and chicken erythrocyte H5 is quite distinct from that of phosphoserine residues phosphorylated in vitro by bovine heart cAMP-dependent protein kinase.     

Two-dimensional electrophoresis of yeast histones on cellogel strips and polyacrylamide gels

For a quick analytical identification of histones a two-dimensional electrophoretic system has been developed. First the proteins are separated on cellulose acetate strips in alkaline buffer. Then they are reelectrophoresed in a second dimension on polyacrylamide gels either with sodium dodecyl sulfate or in the presence of urea at pH 2.7. The technique is demonstrated with yeast and calf thymus histones.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.