Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.     

Structure of a classical MHC class I molecule that binds "non-classical" ligands

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β₂-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.     

Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.     

Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.     

RagPools: RNA-As-Graph-Pools--a web server for assisting the design of structured RNA pools for in vitro selection

SUMMARY: Our RNA-As-Graph-Pools (RagPools) web server offers a theoretical companion tool for RNA in vitro selection and related problems. Specifically, it suggests how to construct RNA sequence/structure pools with user-specified properties and assists in analyzing resulting distributions. This utility follows our recently developed approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a 'mixing matrix' approach combined with a graph theory analysis of RNA secondary-structure space; the mixing matrix specifies nucleotide transition rates, and graph theory links sequences to simple graphical objects representing RNA motifs. The companion RagPools web server ('Designer' component) provides optimized starting sequences, mixing matrices and associated weights in response to a user-specified target pool structure distribution. In addition, RagPools ('Analyzer' component) analyzes the motif distribution of pools generated from user-specified starting sequences and mixing matrices. Thus, RagPools serves as a guide to researchers who aim to synthesize RNA pools with desired properties and/or experiment in silico with various designs by our approach. AVAILABILITY: The web server is accessible on the web at http://rubin2.biomath.nyu.edu     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.