Identification and fractionation of the total histone of the cotton plant

In order to isolate individual fractions of the histones of the cotton plant of variety 108-F and to investigate them further, the total histone has been chromatographed on Acrylex P-60 and Bio-Gel P-30. It has been shown that the most complete fractionation is achieved on a Acrylex P-60. By electrophoresis of the fractions in 15% PAAG in the presence of 6.25 M urea and on the basis of amino acid compositions, the following order of elution of the histones has been established: H1, H2B + H2A, H3, and H4. In a comparison of the amino acid composition of the H3 and H4 histones of the cotton plant with histones enriched with arginine from other species, a somewhat higher lysine : arginine ratio in these fractions has been found. On comparing the electrophoretic mobilities of the histone fractions from the cotton plant with the histones of calf thymus, some difference is observed which is apparently connected with a difference in their molecular weights.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the TadzhSSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 832–838, November–December, 1979.     

Study of the total histone of the cotton plant of variety 108-F

From two-day etiolated shoots of the cotton plant of variety 108-F we have isolated the total histone and purified it on a column of CM-cellulose. It has been shown by gel electrophoresis and amino acid analysis that the total histone of the cotton plant, like the total histone of wheat, consists of five main fractions, the amount of the lysine-rich fraction A₁ being greater in the cotton plant than in wheat (23% and 19%, respectively), and the amount of arginine-rich fraction A₄ being less (19.2 and 24.3%, respectively). The total histone of the cotton plant is less basic (ratio of basic amino acids to acidic amino acids in the cotton-plant histone 1.24, in wheat histone 1.9, and in calf thymus histone 1.78) but richer in lysine as compared with arginine than the histones of wheat and calf thymus.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 109–112, January–February 1980.     

Electrophoretic investigation of the histones of the cotton plant in polyacrylamide gel containing sodium dodecylsulfate

In order to study the fractional compositions of the histones isolated from the cotton plant, they have been subjected to electrophoretic separation in polyacrylamide gels (PAAGs) containing sodium dodecylsulfate (NA-DDS). It has been established that the rate and sequence of migration of the histones under consideration in PAAGs differ from the corresponding values of the histones of the calf thymus. A comparative evaluation of the histones of the cotton plant and of the calf thymus has been made by two-dimensional electrophoresis. The molecular weights of fractions of the cotton-plant histone were determined from their electrophoretic mobilities in PAAGs with Na-DDS:H1 - 23,500;H2A - 14,600; H2B - 14,960; H3 - 14,300; and H4 - 11,300.     

A comparative study of the histones of some varieties of cotton plant

Tetraploid varieties of the cotton plant belonging to the medium- and thin-fibered types have been studied with respect to their histone/DNA ratio, their content of histone fractions, and the amino acid composition of the total histones. It has been shown that these varieties differ with respect to their contents of the lysine-rich histone fraction Hl, and also by the amounts of certain amino acids in the total histone.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the TadzhSSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 99–102, January–February, 1985.     

Histones of the cotton plant. Molecular characteristics of histone H1

A characterization of the histones of two varieties of the cotton plant,Gossypium hirsutum andG. barbadense, has been given with the aid of various electrophoretic systems. Their molecular weights have been determined. An analysis has been made of the cleavage of histone H1 at tyrosine residues. The positive charge and the molecular length of histone H1 have been determined by the method of incomplete succinylation.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 491–497, July–August, 1986.     

Histones of the cotton plant. Molecular characteristics of histone H1

A characterization of the histones of two varieties of the cotton plant,Gossypium hirsutum andG. barbadense, has been given with the aid of various electrophoretic systems. Their molecular weights have been determined. An analysis has been made of the cleavage of histone H1 at tyrosine residues. The positive charge and the molecular length of histone H1 have been determined by the method of incomplete succinylation.     

Competitive bindings of histones with DNA in formation of plant nucleohistones

The nature of the relative affinity of histones for DNA has been studied with the aid of the electrophoretic analysis of the DNP complexes obtained by the addition of the total histone to a nuclear histone with a DNA:histone ratio of 1:1. It has been established that, on the addition of histone, complexes are formed the composition of which changes according to the degree of competition of the histone fractions. Some differences have been detected in the nature of the formation of nuclear histones of plant and animal origin due to features of the primary structure of the histones. The results obtained confirm a hypothesis put forward previously about the different strengths of the bonds of histones with DNA and the role of dissociation in the functioning of the genetic apparatus of the cells of higher organisms.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 865–868, November–December, 1988.     

基于ProteinGoggle 2.0的组蛋白H4蛋白质变体的自上而下表征

由于大量可能蛋白质变体以及每一个翻译后修饰大量可能位点的存在,核心组蛋白上密集的组合式翻译后修饰的自上而下表征一直是一个巨大的分析挑战。结合高分辨串级质谱,基于同位素质荷比和轮廓指纹比对的整体蛋白质数据库搜索引擎ProteinGoggle 2.0在组蛋白翻译后修饰的自上而下鉴定方面拥有诸多独特的优势。该文报道ProteinGoggle 2.0对HeLa核心组蛋白H4的数据库搜索及蛋白质变体的鉴定结果。基于从UniProt网站下载的人类核心组蛋白H4的纯文本文件和“鸟枪法”注释,ProteinGoggle 2.0首先创建包含所有可能蛋白质变体的理论数据库;从纯文本文件中提取的信息主要是氨基酸序列、可能的翻译后修饰（单甲基化、二甲基化、三甲基化、乙酰化和磷酸化）及氨基酸变异（A77→P）。在控制质谱水平假阳性率低于1%的前提下,共鉴定到426个蛋白质变体,这是目前为止H4蛋白质变体的最全报道。这些ProteinGoggle 2.0鉴定到的H4蛋白质变体也与之前报道的ProSightPC 2.0的鉴定结果进行了肩并肩比较。总而言之,ProteinGoggle 2.0可以对具有复杂组合修饰及氨基酸变异的蛋白质组进行数据库搜索和蛋白质变体鉴定。     

Identification of ADP-ribosylated histones by the combined use of high-performance liquid chromatography and electrophoresis

Reversed-phase high-performance liquid chromatography (HPLC) was employed for analysing mono- and oligo(ADP-ribosyl)ated histones. Under the chromatographic conditions described, the ADP-ribosylated histones showed similar retention times to the unmodified histones, although the molecular weight and the charge of the proteins are significantly altered by their modification. The simultaneous elution of unmodified and labelled modified histones was detected by two types of gel electrophoresis and by autoradiography. In addition, the HPLC fractions did not display overlapping ladders of the multiply modified histones, as is commonly seen in one-dimensional electrophoretic analyses of unfractionated material. Hence individual bands could be unambiguously assigned. After in vitro labelling of isolated rat liver nuclei, the following ADP-ribosylated and unmodified histones were identified by HPLC and gel electrophoresis: histone H1(0), four histone H1 subfractions, histone H2A.1, histone H2A.2, oxidized histone H2A.2, histone H2A.X, histone H2A.Z, histone H2B, three histone H3 variants and histone H4.     

Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylbenz[a]anthracene, with chromatin histone proteins

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3 greater than H1 greater than H2b greater than H4 greater than H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.     

Electrophoretic heterogeneity of maize histones

Histones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate-containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X-100, used for the dimension in two-dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X-100 resolved subfractions represent oxidation forms.     

Tritium-labeled 5-oxo-Pro-Arg-Pro

Conditions of tritium introduction into 5-oxo-Pro-Arg-Pro have been developed. Labelled 5-oxo-Pro-Arg-Pro has been obtained in 75% yield and molar radioactivity of 60 Ci/mmol. Tritium distribution over amino acid residues in the peptide has been determined. 5-Oxo-Pro : Arg : Pro ratio in the labelled peptide has been found to be 1 : 5.2 : 2.8. Nonterminal amino acid has been found to contain the major portion of tritium, about 35 Ci/mmol; i.e., arginine includes more than half of the label. Such a feature can be explained by the effect of readily protonated guanidine group of arginine. The influence of charged guanidine group of arginine seems to increase the shift of electron density in the neighboring C–H bonds of arginine and proton mobility, thus increasing protium–tritium exchange at these carbon atoms.     

Mapping Post-translational Modifications of Histones H2A, H2B and H4 in Schizosaccharomyces pombe

Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC-nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe (S. pombe), the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism.     

Polysaccharides of the cell walls of fungi pathogenic for the cotton plant

The dynamics of the secretion of polysaccharides by fungi pathogenic for the cotton plant — Verticillium dahliae and Fusarium oxysporum — have been studied. An alkaline hydrolysate of the fungal cell walls has been obtained. Total fractions of the polysaccharides have been separated on Sephadex G-50 and Acrilex P-4, and their monosaccharide compositions have been determined.Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 198–201, March–April, 1995. Original article submitted October 19, 1994.     

Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an <Emphasis Type="Italic">ADA2</Emphasis>-independent Gcn5p activity

Background  

The acetylation of the core histone NH₂-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.     

In vitro determination of the release kinetics of peptides and free amino acids during the digestion of food proteins

The kinetics of peptide release during in vitro digestion of 4 protein sources (casein, cod protein, soy protein, and gluten) were investigated. Samples were sequentially hydrolyzed with pepsin (30 min) and pancreatin (2, 4, or 6 h) in a dialysis cell with continuous removal of digestion products. Nondialyzed digests were fractionated by ion-exchange chromatography and ultrafiltration. Animal proteins were digested at a greater rate than plant proteins. Target amino acids of specific enzymes appeared more rapidly in the dialyzed fractions when compared to other amino acids. Throughout the hydrolysis, nondialyzed digests contained a higher proportion of peptide mixtures with basic-neutral properties. Except for gluten, peptide fractions with molecular weights that exceeded 10 kDa (basic-neutral, BN > 10) were rapidly hydrolyzed during the first 2 h of pancreatin digestion. The kinetics of release and the composition of peptide fractions were different when the protein sources were compared. The analysis of amino acids revealed that threonine and proline proportions were relatively high in BN > 10 and in peptide fractions with molecular weight between 10-1 kDa (BN 10-1), while tyrosine, phenylalanine, lysine, and arginine predominated in the low molecular weight (<1 kDa) fractions. More resistant peptides were generally rich in proline and glutamic acid. The role of in vitro digestion assays in dietary protein quality evaluation is discussed.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.