Salt-Induced Conformation and Interaction Changes of Nucleosome Core Particles

Small angle x-ray scattering was used to follow changes in the conformation and interactions of nucleosome core particles (NCP) as a function of the monovalent salt concentration C_s. The maximal extension (D_max) of the NCP (145 ± 3-bp DNA) increases from 137 ± 5 Å to 165 ± 5 Å when C_s rises from 10 to 50 mM and remains constant with further increases of C_s up to 200 mM. In view of the very weak increase of the R_g value in the same C_s range, we attribute this D_max variation to tail extension, a proposal confirmed by simulations of the entire I(q) curves, considering an ideal solution of particles with tails either condensed or extended. This tail extension is observed at higher salt values when particles contain longer DNA fragments (165 ± 10 bp). The maximal extension of the tails always coincides with the screening of repulsive interactions between particles. The second virial coefficient becomes smaller than the hard sphere virial coefficient and eventually becomes negative (net attractive interactions) for NCP₁₄₅. Addition of salt simultaneously screens Coulombic repulsive interactions between NCP and Coulombic attractive interactions between tails and DNA inside the NCP. We discuss how the coupling of these two phenomena may be of biological relevance.     

Nucleosome Interactions and Stability in an Ordered Nucleosome Array Model System

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.     

Electrostatic Interactions between Arginines and the Minor Groove in the Nucleosome

Abstract

Proteins rely on a variety of readout mechanisms to preferentially bind specific DNA sequences. The nucleosome offers a prominent example of a shape readout mechanism where arginines insert into narrow minor groove regions that face the histone core. Here we compare DNA shape and arginine recognition of three nucleosome core particle structures, expanding on our previous study by characterizing two additional structures, one with a different protein sequence and one with a different DNA sequence. The electrostatic potential in the minor groove is shown to be largely independent of the underlying sequence but is, however, dominated by groove geometry. Our results extend and generalize our previous observation that the interaction of arginines with narrow minor grooves plays an important role in stabilizing the deformed DNA in the nucleosome.     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

Salt-Induced L-Forms of Staphylococcus aureus

When growing cultures of a salt-sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 m NaCl and 0.5% bovine serum albumin, typical colonies of L-form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L-form induction.     

Sequence-directed Mapping of Nucleosome Positions

Abstract

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.     

Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.     

盐胁迫下苜蓿中盐蛋白的诱导产生

盐胁迫下苜蓿叶片中蛋白质的合成受到抑制,而其离体叶绿体中蛋白质合成增强,ABA阻碍了后者的蛋白质合成。NaCl胁迫下,“松江”和“肇东”两品种的根和叶中均无新多肽出现。在盐敏感的“松江”品种离体叶绿体中,NaGl诱导70,65,60和43kD4种多肽产生,ABA诱导60和17kD两种多肽产生;在较抗盐的“肇东”品种离体叶绿体中,NaGl诱导83,80kD和43kD3种多肽产生,但100mmol/L NaCl并不诱导83kD多肽出现,ABA无明显作用。两品种的43kD多肽和肇东品种的80kD多肽都存在于类囊体膜上,而松江品种的60kD多肽则存在于叶绿体间质中。     

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.     

X-Ray Structure of the Nucleosome Core Particle

Abstract

Two monoclinic crystal forms (P2₁,C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2₁ crystal form has been solved to 15 Å resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2₁ asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity.     

Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5′-end to the 3′-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.     

Hidden Markov Analysis of Nucleosome Unwrapping Under Force

Transient conformational changes of DNA-protein complexes play an important role in the DNA metabolism but are generally difficult to resolve. Single molecule force spectroscopy has the unique capability to follow such reactions but Brownian fluctuations in the end-to-end distance of a DNA tether can obscure these events. Here we measured the force-induced unwrapping of DNA from a single nucleosome and show that hidden Markov analysis, adopted for the nonlinear force-extension of DNA, can readily resolve unwrapping events that are significantly smaller than the Brownian fluctuations. The resulting probability distributions of the tether length are used to accurately resolve small changes in contour length and persistence length. The latter is shown to be directly related to the DNA bending angle of the complex. The wormlike chain-adapted hidden Markov analysis can be used for any transient DNA-protein complex and provides a robust method for the investigation of these transient events.     

Comparison of Salt-Induced Osmotic Adjustment and Trigonelline Accumulation in Two Soybean Cultivars

21-d-old seedlings of the soybean (Glycine max) cvs. Essex and Forrest were treated with NaCl in a step-wise manner over 9 d (3 d 30 mM, 3 d 70 mM, and 3 d 100 mM) and maintained under 100 mM NaCl for an additional 14 d. During salt treatment, osmotic potential decreased more in cv. Forrest relative to cv. Essex. In non-stressed leaf tissue, cv. Forrest contained more trigonelline (TRG) relative to cv. Essex. During salt treatment, TRG amounts increased in cv. Forrest but were unchanged in cv. Essex. Both cvs. osmotically adjusted in response to salt stress; the maximal osmotic adjustment was 0.80 and 0.18 MPa in cv. Forrest and cv. Essex, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Catalysis of Dialanine Formation by Glycine in the Salt-Induced Peptide Formation Reaction.

Mutual catalysis of amino acids in the salt-induced peptide formation (SIPF) reaction is demonstrated for the case of glycine/alanine. The presence of glycine enhances dialanine formation by a factor up to 50 and enables dialanine formation at much lower alanine concentrations. The actual amounts of glycine play an important role for this catalytic effect, the optimal glycine concentration is 1/8 of the alanine concentration. The mechanism appears to be based on the formation of the intermediate Gly-Ala-Ala tripeptide, connected to one coordination site of copper(II) ion, and subsequent hydrolysis to dialanine and glycine.     

Controls of Nucleosome Positioning in the Human Genome

Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase–seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase–seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned—in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.