The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

Synthesis and rac-lactide ring-opening polymerisation studies of new alkaline earth tetrahydroborate complexes.

Reaction of K[HC(C(Me)NAr')(2)] (Ar' = 2,6-C(6)H(3)(i)Pr(2)) with Mg(BH(4))(2) afforded the pseudo four-coordinate tetrahydroborate complex Mg{HC(C(Me)NAr')(2)}(BH(4))(THF) (1). The corresponding reaction of Ca(BH(4))(2)(THF)(2) or Sr(BH(4))(2)(THF)(2) gave the pseudo five-coordinate analogues M{HC(C(Me)NAr')(2)}(BH(4))(THF)(2) (M = Ca (2) or Sr (3)). All three compounds 1-3 have been structurally characterised. According to X-ray crystallography and IR spectroscopy all possess κ(3)-bound BH(4) ligands in the solid state. This coordination mode is also maintained in THF solution for 2 and 3, whereas complex 1 appears to form a bis(THF) complex containing a κ(2)-bound BH(4). Reaction of 1 with K[HC(P(Ph(2))NAr')(2)](THF)(2) formed Mg{HC(P(Ph(2))NAr')(2)}(BH(4))(THF)(2) (4) possessing a κ(3)-bound BH(4) ligand in both the solid state and solution. Compounds 1, 2 and 4 are highly active for the ring-opening polymerisation of ε-caprolactone forming dihydroxytelechelic PCL. Compound 1 is also extremely active for the ROP of rac-lactide forming highly heterotactic PLA with good agreement between predicted and measured M(n), in accord with previous studies of alkoxide and amide initiators based on this metal and ligand class. Compounds 2 and 4 were less productive and gave PLA with poorer control of M(n) and negligible heterotactic enrichment. MALDI-ToF MS analysis of the PLA formed with all three catalysts showed a mixture of both -CH(Me)CHO and -CH(Me)CH(2)OH termini arising from the M-BH(4) initiating groups.

A ten-year audit of traditional Chinese medicine and other natural product research published in the Chinese Medical Journal (2000-2009).

BACKGROUND: Clinical research encompasses a wide variety of disciplines. Traditional Chinese medicine (TCM) and natural product research have made great contributions to preventing and treating illness. The number and content of original research reports evaluating TCM and natural products have not previously been described. Information in this area will identify areas of relative strength and weakness in terms of knowledge gaps with respect to clinical conditions and natural product remedies. METHODS: Original research reports (i.e. original articles, brief reports, and research letters) published in the Chinese Medical Journal (CMJ) from January 2000 to December 2009 evaluating TCM and other natural products were reviewed. The United Kingdom Clinical Research Collaboration (UK-CRC) Health Research Classification System was used to analyze the type of health research conducted. Further analysis on the major illnesses addressed and the major herbal components utilized was conducted. RESULTS: One hundred and seventeen original research reports involving TCM or other natural products were identified, comprising 3.82% of the CMJ output in the period covered by this study. Of the different materia medica described in these reports, 74.4% were derived exclusively from plant material, 10.3% from animals, 3.4% from fungi, 1.7% from minerals, and 10.3% were of mixed (plant/animal/fungal/mineral) composition. Twelve herbs were investigated exclusively or were constituents of 66/87 (75.9%) of the plant-based materia medica investigated. Panax ginseng was the most commonly investigated herb or constituent (14/87, 16.1%), followed by Astragalus membranaceus (9/87, 10.3%), Coptis chinensis/Berberis spp. (7/87, 8.0%) and Rheum spp. (7/87, 8.0%). Four UK-CRC health categories accounted for the majority of TCM and other natural product research (cancer, 20.9%; cardiovascular, 19.2%; oral/gastrointestinal, 9.8%; and inflammatory/immune, 9.0%). The most common research activity was "development of treatments and therapeutic interventions", which was undertaken by 103/117 (88.0%) of the research investigations reported. Human clinical trials involving natural products accounted for only 5.31% of the reported studies. CONCLUSIONS: This is a relatively early systematic description of published research from a single journal related to TCM and other natural products. The majority of the research reports described preliminary findings and very few controlled clinical trials in human subjects were reported. Further applied studies will be required to demonstrate the clinical effectiveness, utility and cost-effectiveness of TCM and natural products in clinical practice. The UK-CRC health research classification system is a useful tool for evaluating published research output and could be applied to describe the output from other journals, national and provincial funding bodies, charities, and non-governmental organizations involved in supporting health-related research.

Use of ribozyme cleavage kinetics to measure salt-induced changes in solution pH.

Several ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid-base catalysis. Interpreting the apparent pK(a) values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS ribozyme as a "biological pH meter" to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.

Additional roles of a peripheral loop-loop interaction in the Neurospora VS ribozyme.

Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS ribozyme, a tertiary loop-loop kissing interaction involving stem-loops I and V is also required to rearrange the secondary structure of stem-loop I such that nucleotides at the base of stem I, which contains the cleavage-ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ~1000-fold enhancement to the rates of cleavage and ligation. Large Mg(2+)-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg(2+); whereas ligation is favored >10-fold at millimolar concentrations of Mg(2+). In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg(2+). These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage-ligation equilibrium of the ribozyme.

Evidence for the phenotypic neutrality of the Neurospora Varkud (V) and Varkud satellite (VS) plasmids.

The Varkud satellite (VS) plasmid, which requires the Varkud (V) plasmid for replication, is found in the mitochondria of several natural isolates of Neurospora. The VS transcript is sufficiently abundant that it might be expected to alter the function of mitochondria; however, previous limited characterization revealed no effect. In this work we have used genetic, biochemical and proteomic approaches to search for effects of the V and VS plasmids. We observed differences in the relative abundance of several mitochondrial proteins between plasmid-containing and plasmid-lacking natural isolates, but subsequently found these not to be due to the plasmids. We constructed a pair of iso-nuclear and iso-mitochondrial strains that differed only by the presence or absence of V and VS, and observed only subtle differences in the abundance of several mitochondrial proteins. We further attempted to detect a cryptic plasmid-related phenotype by growing this pair of strains in the presence of a variety of inhibitors of mitochondrial function or other stress conditions: this also revealed no effect of the plasmids. These observations suggest that, despite the high concentration of VS RNA in the mitochondrion, the V and VS plasmids do not cause substantial changes in the host.

Gel-based mass spectrometric and computational approaches to the mitochondrial proteome of Neurospora.

We have used gel electrophoretic techniques including isoelectric focusing, blue native, acid-urea, 16-benzyldimethyl-n-hexadecylammonium chloride or SDS first dimensions and SDS Laemmli or tricine second dimensions to separate the proteins from highly-purified Neurospora mitochondria and sub-mitochondrial fractions (membrane, soluble, protein complexes and ribonucleoproteins). The products of 260 genes, many of them in multiple processed or modified forms, were identified by MALDI-TOF mass spectrometry. This work confirms the existence, expression, and mitochondrial localization of the products of 55 Neurospora genes previously annotated only as predicted or hypothetical, and of 101 genes not identified in previous mass spectrometry studies. Combined with previous mass spectrometry studies, and re-evaluation of genome annotations, we have compiled a curated list of 358 proteins identified in proteomic studies that are likely to be bona fide mitochondrial proteins plus 80 other identified proteins that may be mitochondrial. Literature data mining and computational predictions suggest that Neurospora mitochondria also contain another 299 proteins not yet identified in proteomics projects. Taken together, these data suggest that the products of at least 738 genes comprise the Neurospora mitochondrial proteome.

Data variance and statistical significance in 2D-gel electrophoresis and DIGE experiments: comparison of the effects of normalization methods.

Identifying changes in the relative abundance of proteins between different biological samples is often confounded by technical noise. In this work, we compared eight normalization methods commonly used in two-dimensional gel electrophoresis and difference gel electrophoresis (DIGE) experiments for their ability to reduce noise and for their influence on the list of proteins whose difference in abundance between two samples is determined to be statistically significant. With respect to reducing noise we find that, while all methods improve upon unnormalized data, cyclic linear normalization is the least well suited to gel-based proteomics and the performances of the other methods are similar. We also find in DIGE data that the choice of normalization method has less of an impact on the noise than does the decision to use an internal reference in the experimental design and that both normalization and standardization using the internal reference are required to maximally reduce variance. Despite the similar noise reduction achieved by most normalization methods, the list of proteins whose abundance was determined to differ significantly between biological groups differed depending on the choice of normalization method. This work provides a direct comparison of the impact of normalization methods in the context of common experimental designs.