Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.     

IRECS: a new algorithm for the selection of most probable ensembles of side-chain conformations in protein models

We introduce a new algorithm, IRECS (Iterative REduction of Conformational Space), for identifying ensembles of most probable side-chain conformations for homology modeling. On the basis of a given rotamer library, IRECS ranks all side-chain rotamers of a protein according to the probability with which each side chain adopts the respective rotamer conformation. This ranking enables the user to select small rotamer sets that are most likely to contain a near-native rotamer for each side chain. IRECS can therefore act as a fast heuristic alternative to the Dead-End-Elimination algorithm (DEE). In contrast to DEE, IRECS allows for the selection of rotamer subsets of arbitrary size, thus being able to define structure ensembles for a protein. We show that the selection of more than one rotamer per side chain is generally meaningful, since the selected rotamers represent the conformational space of flexible side chains. A knowledge-based statistical potential ROTA was constructed for the IRECS algorithm. The potential was optimized to discriminate between side-chain conformations of native and rotameric decoys of protein structures. By restricting the number of rotamers per side chain to one, IRECS can optimize side chains for a single conformation model. The average accuracy of IRECS for the chi1 and chi1+2 dihedral angles amounts to 84.7% and 71.6%, respectively, using a 40 degrees cutoff. When we compared IRECS with SCWRL and SCAP, the performance of IRECS was comparable to that of both methods. IRECS and the ROTA potential are available for download from the URL http://irecs.bioinf.mpi-inf.mpg.de.     

Recovering phylogenetic signal from frog mating calls

Goicoechea, N., De La Riva, I. & Padial, J. M. (2010). Recovering phylogenetic signal from frog mating calls. —Zoologica Scripta, 39, 411–154. Few studies have tried to analyse the phylogenetic information contained in frog mating calls. While some of those studies suggest that sexual selection deletes any phylogenetic signal, others indicate that frog calls do retain phylogenetic informative characters. Discordant results can be the outcome of disparate rates of character evolution and evolutionary plasticity of call characters in different groups of frogs, but also the result of applying different coding methods. No study to date has compared the relative performance of different coding methods in detecting phylogenetic signal in calls, hampering thus potential consilience between previous results. In this study, we analyse the strength of phylogenetic signal in 10 mating call characters of 11 related species of frogs belonging to three genera of Andean and Amazonian frogs (Anura: Terrarana: Strabomantidae). We use six quantitative characters (number of notes per call, note length, call length, number of pulses per note, fundamental frequency and dominant frequency) and four qualitative ones (presence/absence of: pseudopulses, frequency modulation in notes, amplitude modulation in notes and amplitude modulation in pulses). We code quantitative characters using four different coding and scaling methods: (i) gap‐coding, (ii) fixed‐scale, (iii) step‐matrix gap‐weighting with between‐characters scaling, and (iv) step‐matrix gap‐weighting with between‐states scaling. All four coding methods indicate that frog calls contain phylogenetic information. These results suggest that divergent selection on frog mating calls may not always be strong enough to eliminate phylogenetic signal. However, coding methods strongly affect the amount of recoverable information. Step‐matrix gap‐weighting with between‐characters scaling and gap‐coding are suggested as the best methods available for coding quantitative characters of frog calls. Also, our results indicate that the arbitrariness in selecting character states and the method for scaling transitions weights, rather than the number of character states, is what potentially biases phylogenetic analyses with quantitative characters.     

Filtration Methods for Recovering Enteroviruses from Foods

A set of simple, rapid filtration methods has been developed for recovering enteroviruses from foods. The methods employ quite inexpensive equipment, and the resulting extract might be concentrated in any of several ways. Three model enteroviruses were apparently recovered with at least 80% efficiency when inoculated, at levels ranging from thousands to one plaque-forming unit per 20-g sample, into eleven foods representing six major food groups.     

Histone acetyltransferase in chromatin. Extraction of transferase activity from chromatin

Previous work has attempted to localize nuclear histone acetyltransferase activity in the cromatin. Evidence was presented indicating that the transfer of ¹⁴C-acetate from ¹⁴C-acetyl CoA to histones in chromatin was an enzymatic process. We now report on the extraction of part of the histone acetyltransferase activity from rat liver chromatin, employing a procedure originally described for extraction of DNA-dependent RNA polymerase. The K_m of the extracted transferase activity for the substrate acetyl CoA was 5 × 10⁻⁷, the Q₁₀: 1.8 and the optimal pH: 7.1. Serum albumine, protamine and polylysine were poor substrates as compared to histones. Activity of extracted or heated chromatin was not restored upon incubation in the presence of extract. Also the selectivity exhibited by the transferase activity in unextracted chromatin towards arginine-rich histones, was much less pronounced in the extracts prepared from it. It is possible that the influence of steric factors contributing to this specificity in native chromatin is lost upon isolation of the enzyme from it. Alternatively, a less specific isoenzyme may have been extracted.     

Prediction of identity by descent probabilities from marker-haplotypes

The prediction of identity by descent (IBD) probabilities is essential for all methods that map quantitative trait loci (QTL). The IBD probabilities may be predicted from marker genotypes and/or pedigree information. Here, a method is presented that predicts IBD probabilities at a given chromosomal location given data on a haplotype of markers spanning that position. The method is based on a simplification of the coalescence process, and assumes that the number of generations since the base population and effective population size is known, although effective size may be estimated from the data. The probability that two gametes are IBD at a particular locus increases as the number of markers surrounding the locus with identical alleles increases. This effect is more pronounced when effective population size is high. Hence as effective population size increases, the IBD probabilities become more sensitive to the marker data which should favour finer scale mapping of the QTL. The IBD probability prediction method was developed for the situation where the pedigree of the animals was unknown (i.e. all information came from the marker genotypes), and the situation where, say T, generations of unknown pedigree are followed by some generations where pedigree and marker genotypes are known.     

Selection and flexible optimization of binding modes from conformation ensembles

This paper describes a new semi-flexible docking approach named Fado (flexible alignment and docking), which incorporates flexibility by using an ensemble of precomputed ligand conformers. A primary ligand is defined as a linear combination over all input conformers. An optimization with regard to the linear coefficients makes the ligand flexible. Initially, a point matching problem utilizing the Merck Molecular Force Field (MMFF) is modeled in order to compute the correct orientation of the ligand with respect to the target. The problem is then solved through a local optimization approach (RPROP). This is done for 20 randomized ligand orientations, yielding 20 binding modes per complex. Evaluating these modes illustrates that our method is able to reproduce the binding modes of molecules within a few minutes of CPU time. A representative dataset of diverse protein-ligand complexes could be reproduced with 78% accuracy below 2A RMSD distance to the reference crystal structure. Fado is available upon request to the authors (see also http://www.zib.eu/Numerik/projects/docking/projectlong.en.html).     

Characterizing RNA ensembles from NMR data with kinematic models

Functional mechanisms of biomolecules often manifest themselves precisely in transient conformational substates. Researchers have long sought to structurally characterize dynamic processes in non-coding RNA, combining experimental data with computer algorithms. However, adequate exploration of conformational space for these highly dynamic molecules, starting from static crystal structures, remains challenging. Here, we report a new conformational sampling procedure, KGSrna, which can efficiently probe the native ensemble of RNA molecules in solution. We found that KGSrna ensembles accurately represent the conformational landscapes of 3D RNA encoded by NMR proton chemical shifts. KGSrna resolves motionally averaged NMR data into structural contributions; when coupled with residual dipolar coupling data, a KGSrna ensemble revealed a previously uncharacterized transient excited state of the HIV-1 trans-activation response element stem–loop. Ensemble-based interpretations of averaged data can aid in formulating and testing dynamic, motion-based hypotheses of functional mechanisms in RNAs with broad implications for RNA engineering and therapeutic intervention.     

Recovering normal networks from shortest inter-taxa distance information

Phylogenetic networks are a type of leaf-labelled, acyclic, directed graph used by biologists to represent the evolutionary history of species whose past includes reticulation events. A phylogenetic network is tree–child if each non-leaf vertex is the parent of a tree vertex or a leaf. Up to a certain equivalence, it has been recently shown that, under two different types of weightings, edge-weighted tree–child networks are determined by their collection of distances between each pair of taxa. However, the size of these collections can be exponential in the size of the taxa set. In this paper, we show that, if we have no “shortcuts”, that is, the networks are normal, the same results are obtained with only a quadratic number of inter-taxa distances by using the shortest distance between each pair of taxa. The proofs are constructive and give cubic-time algorithms in the size of the taxa sets for building such weighted networks.

     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

回收琼脂糖凝胶中DNA的简便方法

多年来国内分子生物学实验室所采用的几种从琼脂糖凝胶中回收DNA的方法不同程度地存在着各种问题。现介绍一种新的从琼脂糖凝胶上分离和提取DNA的简易方法,采用实验室常用的吸附柱中的吸附膜对DNA进行拦截、纯化和回收。实验证明这种方法回收DNA具有简便快捷、成本低和效率高等优点,是一种切实可行的方法。     

Transcription of chromatin from mouse fibroblasts

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended «light and a compact «heavy component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II).Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.