Toward a realistic model of mutations affecting fitness

Analysis of a recent mutation accumulation (MA) experiment has led to the suggestion that as many as one-half of spontaneous mutations in Arabidopsis are advantageous for fitness. We evaluate this in the light of data from other MA experiments, along with molecular evidence, that suggest the vast majority of new mutations are deleterious.     

Identification of HMG-5 as a double-stranded telomeric DNA-binding protein in the nematode Caenorhabditis elegans

Many protein components of telomeres, the multifunctional DNA-protein complexes at the ends of eukaryotic chromosomes, have been identified in diverse species ranging from yeast to humans. In Caenorhabditis elegans, CEH-37 has been identified by a yeast one hybrid screen to be a double-stranded telomere-binding protein. However, the role of CEH-37 in telomere function is unclear because a deletion mutation in this gene does not cause severe telomere defects. This observation raises the possibility of the presence of genetic redundancy. To identify additional double-stranded telomere-binding proteins in C. elegans, we used a different approach, namely, a proteomic approach. Affinity chromatography followed by Finnigan LCQ ion trap mass spectrometer analysis allowed us to identify several candidate proteins. We further characterized one of these, HMG-5, which is encoded by F45E4.9. HMG-5 bound to double-stranded telomere in vitro as shown by competition assays. At least two telomeric DNA repeats were needed for this binding. HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells, but not in the hatched larvae or adults. HMG-5 mainly localized to the chromosomal ends, indicating that HMG-5 also binds to telomeres in vivo. These observations suggest that HMG-5 may participate, together with CEH-37, in early embryogenesis by acting at the telomeres.     

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.     

Aspergillus melleus protease-catalyzed peptide synthesis using the carbamoylmethyl ester as an acyl donor in 1,1,1,3,3,3-hexafluoro- 2-propanol/N,N-dimethylformamide

The coupling between the carbamoylmethyl ester of an N-protected amino acid or dipeptide (at 25 mM) and an amino acid amide (at 100 mM) was achieved using Aspergillus melleus protease in 1,1,1,3,3,3-hexafluoro-2-propanol/N,N-dimethylformamide (1:1, v/v); the coupling efficiencies were dependent largely on the combination of amino acid residues: e.g. the dipeptide yields after 48 h were for l-Ala + Gly, 100% and for l-Leu + l-Leu, 16%.     

A Systematic Analysis of Buried Polar Side Chains and Their Interactions in α-Helical Proteins

Examination of 80 -helical proteins and domains demonstrates that they contain from 1 to more than 20 completely buried (water-inaccessible) polar side chains. As a rule the latter have partners for H-bonding but the resulting H-bond system is often not saturating. Basing on statistical analysis, we determined the optimal number of H-bonds for every type of polar side chain, and discuss the structural role of vacant donors and acceptors. About half of the H-bonds formed by buried side chains pertain to interhelix contacts of the (side chain)–(side chain) and (side chain)–(main chain) types. Such interactions appear to be a most important factor determining the mutual arrangement of -helices in proteins. Analysis of the frequency of occurrence of various interacting pairs reveals that these interactions are selective.     

SWORDS: A statistical tool for analysing large DNA sequences

In this article, we present some simple yet effective statistical techniques for analysing and comparing large DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain databases housed in the Internet, we demonstrate how SWORDS can be conveniently used by molecular biologists and geneticists to unmask biologically important features hidden in large sequences and assess their statistical significance.     

Ordering in aqueous polysaccharide solutions. II. Optical rotation and heat capacity of aqueous solutions of a triple-helical polysaccharide schizophyllan

Deuterium oxide solutions of schizophyllan, a triple-helical polysaccharide, undergoing an order-disorder transition centered at 17 degrees C, were studied by optical rotation (OR) and heat capacity (C(p)) to elucidate the molecular mechanism of the transition and water structure in the solution and frozen states. The ordered structure at low temperature consisted of the side chains and water in the vicinity forming an ordered hydrogen-bonded network surrounding the helix core and was disordered at higher temperature. In the solution state appeared clearly defined transition curves in both the OR and C(p) data. The results for three samples of different molecular weights were analyzed theoretically, treating this transition as a typical linear cooperative transition from the ordered to disordered states and explained quantitatively if the molecular weight polydispersity of the sample was considered. The excess heat capacity C(EX)(p) defined as the C(p) minus the contributions from schizophyllan and D(2)O was estimated. In the frozen state it increased with raising temperature above 150 K until the mixture melted. This was compared with the dielectric increment observed in this temperature range and ascribed to unfreezable water. From the heat capacity and dielectric data, unfreezable water is mobile but more ordered than free water. In the solution state, the excess heat capacity originates from the interactions of D(2)O molecules as bound water and structured water, and so forth. Thus the schizophyllan triple helix molds water into various structures of differing orders in solution and in the solid state.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Unblocked statistical-coil tetrapeptides in aqueous solution: quantum-chemical computation of the carbon-13 NMR chemical shifts

We recently reported a theoretical characterization of representative ensembles of statistical-coil conformations for tetrapeptides with unblocked termini in aqueous solution, at pH 7. The results showed good agreement between the computed Boltzmann-averaged and experimentally-determined values for both the vicinal coupling constants ³J_NH and the -proton chemical shifts. Here, we carry out a cluster analysis of the ensembles of conformations generated in that study, and use them to compute the Boltzmann-averaged values of the quantum-chemical ¹³C chemical shifts for different amino acids in the unblocked tetrapeptides GGXA (where X stands for Phe, Arg, His, Glu, Ile, Lys, Gln, Tyr, Leu, Thr, Ala, Gly and Val). The values of the ¹³C chemical shifts in these thirteen amino acids (for which experimental data are available) were computed by using Density Functional Theory with a 6–311+G(2d,p) basis set. Good agreement is found in terms of both the correlation coefficient (R) and standard deviations of the difference between the computed Bolztmann-averaged and the NMR-determined values for the ¹³C chemical shifts. These results suggest that it may be possible to build a reliable theoretically-derived database of chemical shifts for statistical-coil residues. The results of the current study contribute to our understanding of the relations between chemical shifts, dihedral angles and vicinal coupling constants, ³J_NH. In addition, they can shed light as to how the statistical-coilconformation is related to the conformational preference of more structured states, such as the -helical conformation.     

Scoring functions in protein folding and design

We present an analysis of the assumptions behind some of the most commonly used methods for evaluating the goodness of the fit between a sequence and a structure. Our studies on a lattice model show that methods based on statistical considerations are easy to use and can capture some of the features of protein-like sequences and their corresponding native states, but unfortunately are incapable of recognizing, with certainty, the native-like conformation of a sequence among a set of decoys. Meanwhile, an optimization method, entailing the determination of the parameters of an effective free energy of interaction, is much more reliable in recognizing the native state of a sequence. However, the statistical method is shown to perform quite well in tests of protein design.