Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles

Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequence-dependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has first been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone tail-dependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series.     

Nucleosome Structural Dynamics and Linking Number Paradox

The minireview presents recent results obtained in the experiments with DNA minicircles containing reconstituted nucleosomes. This system allows one to register and to characterize the conformational dynamics of nucleosomes and of subnucleosomal particles containing histone tetramer (H3-H4)₂. In particular, it has revealed an important role of the histone N-terminal tails in this dynamics. Solution of the linking number paradox and the relevance of the results obtained to chromatin structural dynamics are discussed.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Electrostatic contribution to the bending of DNA

A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.     

DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.