Two polymorphic linker histone loci in Guinea fowl erythrocytes

A variable migration of linker histone H1.b and H1.c spots in two-dimensional polyacrylamide gel patterns of total erythrocyte histone H1 has been detected during population screening in two differently plumaged Guinea fowl strains. Alloforms, H1.b1 and H1.b2 as well as H1.c1 and H1.c2, differing in apparent molecular weights tended to form only phenotypes b1 and b2 or c1 and c2 in a white-feathered strain while all phenotypes (b1, b2 and b1b2 or c1, c2 and c1c2, respectively) were present in a black-feathered population. Accordingly, the white-feathered population significantly deviated from the Hardy-Weinberg principle (chi-square test, d.f=1, p<0.001) due to a lack of heterozygotes while the black-feathered population conformed to the Hardy-Weinberg equilibrium (p>0.05) at both H1.b and H1.c loci. Differential electrophoretic mobilities of the C-peptides from a partial chemical cleavage (N-bromosuccinimide) or limited enzymatic digestion (α-chymotrypsin and protease V8) of the histone H1.b and H1.c alloforms seem to indicate that altered amino acid sequence segments might be located either at the C-terminal end of globular domain or in the C-terminal domain itself.     

Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.     

The fractionation of histones isolated from Euglena gracilis.

1. The histones of Euglena gracilis were separated by gel filtration into five fractions. 2. Each fraction was characterized in terms of its electrophoretic, solubility and compositional properties. 3. Euglena gracilis clearly contains histones corresponding to vertebrate H1, H2B, H3 and H4 fractions, although they all differ in containing more lysine. 4. The remaining Euglena histone is considered to be homologous to vertebrate histone H2A, but it differs in having a much higher ratio of lysine to arginine. 5. The Euglena histone H1 appears to be lacking in aspartic acid. 6. Electrophoresis in the presence of sodium dodecyl sulphate indicates that the molecular weights of the Euglena histones are close to those of the homologous vertebrate histones.     

Natural allelic variation of duck erythrocyte histone H1b

In our previous work (J. Palyga, Genetic polymorphisms of histone H1. b in duck erythrocytes. Hereditas 114, 85-89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b(1) and b(3) were common in all populations screened, the allele b(2), with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines.The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1. b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Peptide mapping of basic proteins by proteolysis in acetic acid/urea-minislab polyacrylamide gels

A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications.     

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.     

H1° histones of normal and cancer human cells

Summary H1° histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1° histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1° histones showed that: (a) all the H1° preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; (b) H1° is distinguishable from other H1 histones by the presence of methionine and histidine; (c) H1° histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1° histone is not inversely related with cell growth rate nor with the expression of the -fetoprotein gene.     

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.     

The isolation and identification of a novel intermediate in ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

The reaction product obtained from HeLa cell nuclei incubated with [³H]NAD was specifically hydrolyzed with snake venom phosphodiesterase. Analysis of the hydrolyzed product revealed that it is a homopolymer consisting of 4–5 repetition of ADP-ribose units. The [³H]poly ADP-ribosylated histone fraction was anslyzed by urea-acetic acid polyacrylamide gel electrophoresis. The radioactive peak was clearly separated from the stained histone H1 band, while a slight overlap was observed. When chromatographed on a SP-Sephadex C-50 column, more than 90% of the radioactivity of [³H]poly(ADP-ribose) was eluted in accordance with histones but not with nonhistone contaminants. On a sodium dodecyl sulfate polyacrylamide gel electrophoresis, a major radioactive peak appeared at a position very close to the histone Hl band, which disappeared by the treatment with alkali prior to electrophoresis. A selective extraction of histone Hl with 5% perchloric acid showed that histone Hl contained about 85% of the radioactivity incorporated into whole histones.     

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.     

Gel Electrophoretic Analysis of Histones in Lens Epithelium, Lens Fiber, Liver, Brain, and Erythrocytes of Late Chick Embryos

Histones from 19-day-old chick embryo lens epithelium, lens fibers, liver, brain, and erythrocytes were electrophoresed in polyacrylamide gels using buffers containing sodium dodecylsulfate, acetic acid urea, or mixtures of Triton X-100 acetic acid urea. In the last two buffer systems, histone bands were characterized by their apparent molecular weights determined by electrophoresis in the second dimension in sodium dodecylsulfate containing polyacrylamide gels. From the densitograms of the stained gels, the relative proportion of protein in different histone bands was estimated. With the exception of the erythrocyte-specific histone H5, all histones from different tissues examined in any of the gel systems migrated with the same mobilities. In lens epithelium and lens fibers, all histones were present in identical proportions. As compared to liver and brain, the total amount of histone H1 was significantly lower in lens cells and erythrocytes, possibly reflecting differences between the differentiated states. However, no tissue-specific differences were found in the relative distribution of histone H1 I and H1 II among lens epithelium, lens fiber, liver and, brain, but a threefold higher H1 I: H1 II ratio (0.5–0.7) was found in erythrocytes.     

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus.

The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.     

Distribution of Allelic Forms of Erythrocyte H1 Histones in Japanese Quail Populations Divergently Selected for Amount of Weight Loss After Transient Starvation

Three polymorphic subtypes of erythrocytehistone H1 (H1.a, H1.b, and H1.z) were analyzed using asodium dodecyl sulfate polyacrylamide gel in quailpopulations divergently selected for a high (line 1) or low (line 2) reduction in body massfollowing temporary food withdrawal. Both H1.b and H1.zhistone alleles were found to be differently distributedin these populations during the selection period. The frequency of b¹ in line 2 wasapproximately 1.9-2.8 times lower than in line 1 andapproached the values in line 1 when the selection wassuspended. Similarly, the frequency of allelez² at locus H1.z increased significantly (about 1.6-2.3 times)in line 2 during selection and returned to the initialvalues when selection was stopped. On the other hand,allele a⁰ at locus H1.a was kept atrelatively low levels (usually below 0.05) in both linesduring selection. At that time its level wasapproximately three to four times lower than in a randommating control population. When selection was suspended, the frequency of a⁰ in line 1increased significantly, approaching the values in thecontrol line, and remained essentially unchanged in line2. Thus, all three polymorphic histone H1 loci in quailresponded through changes in allele frequencies to thebreeding selection, which was directed at the amount ofbody weight loss upon transient starvation. It seemsthat either H1 histone locus could be linked to loci controlling the rate of body weightreduction following starvation or weight loss duringfasting might be influenced by a panel of H1 histonealleles that can contribute to functional differences in avian chromatin.     

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.     

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.     

Histone H1 kinase from mouse plasmacytoma. Further characterization and molecular structure

A cAMP-independent protein kinase which phosphorylates histone H1 to a high level and which may correspond to the mitotic H1 kinase has been partially purified and characterized from mouse plasmacytoma microsomes [Quirin-Stricker, C., and Schmitt, M. (1981) Eur. J. Biochem. 118, 165-172]. The present study compares the microsome-associated and the chromatin-associated histone H1 kinases isolated from mouse plasmacytoma cells. The results indicate that the two H1 kinases are indistinguishable by several criteria. The molecular structure of the microsome-associated histone H1 kinase has been determined (a) by exclusion chromatography on Ultrogel, (b) by electrophoresis in non-denaturing polyacrylamide gels of graded porosity and (c) by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the H1 kinase activity peak from an AcA-34 Ultrogel column. All these techniques gave the same result: H1 kinase may exist in a native form as a monomeric enzyme with an apparent relative molecular mass of 90 000 +/- 8000.     

Purification and amino acid sequence of chicken liver cathepsin L

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.