Recovery of pristine boreal forest floor community after selective removal of understorey, ground and humus layers

In boreal spruce forests that rarely experience extensive disturbances, fine-scale vegetation gaps are important for succession dynamics and species diversity. We examined the community implications of fine-scale gap disturbances by selective removal of vegetation layers in a pristine boreal spruce forest in Northern Finland. The aim was to investigate how the speed of recovery depends on the type of disturbance and the species growth form. We also wanted to know if there appeared changes in species composition after disturbance. Five different treatments were applied in the study: Control, removal of the ground layer (bryophytes and lichens), removal of the understorey layer (dwarf shrubs, herbs and graminoids), removal of both the ground and understorey layers, and complete removal of the vegetation and humus layers above the mineral soil. The vegetation recovery was monitored in terms of cover and species numbers over a 5-year period. Understorey layer cover, composed mainly of clonal dwarf shrubs, recovered completely in 4 years in treatments where the humus layer remained intact, whereas ground layer cover did not reach the control level in plots from where bryophytes and lichens were removed. Recovery was faster in terms of species number than species cover. Bryophytes, graminoids and dominant dwarf shrubs appeared in all disturbed plots quickly after disturbance. Seedlings of trees appeared exclusively in disturbed plots. Graminoids dominated after the removal of humus layer. The results indicate that the regeneration of forest floor after small gap disturbance occurs mainly by re-establishment of the dominant species. Although destruction of the humus layer leaves a long-lasting scar to the forest floor, exposing of mineral soil may enhance the sexual reproduction of dominant species and the colonization of weaker competitors.     

Diversifying selection and concerted evolution of a type IV secretion system in Bartonella

We have studied the evolution of a type IV secretion system (T4SS), in Bartonella, which is thought to have changed function from conjugation to erythrocyte adherence following a recent horizontal gene transfer event. The system, called Trw, is unique among T4SSs in that genes encoding both exo- and intracellular components are located within the same duplicated fragment. This provides an opportunity to study the influence of selection on proteins involved in host-pathogen interactions. We sequenced the trw locus from several strains of Bartonella henselae and investigated its evolutionary history by comparisons to other Bartonella species. Several instances of recombination and gene conversion events where detected in the 2- to 5-fold duplicated gene fragments encompassing trwJIH, explaining the homogenization of the anchoring protein TrwI and the divergence of the minor pilus protein TrwJ. A phylogenetic analysis of the 7- to 8-fold duplicated gene coding for the major pilus protein TrwL displayed 2 distinct clades, likely representing a subfunctionalization event. The analyses of the B. henselae strains also identified a recent horizontal transfer event of almost the complete trwL region. We suggest that the switch in function of the T4SS was mediated by the duplication of the genes encoding pilus components and their diversification by combinatorial sequence shuffling within and among genomes. We suggest that the pilus proteins have evolved by diversifying selection to match a divergent set of erythrocyte surface structures, consistent with the trench warfare coevolutionary model.     

Enrichment of phosphoproteins and phosphopeptide derivatization identify universal stress proteins in elicitor-treated Arabidopsis

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO(2)) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO(2)-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding.     

Investigating protein-coding sequence evolution with probabilistic codon substitution models

This review is motivated by the true explosion in the number of recent studies both developing and ameliorating probabilistic models of codon evolution. Traditionally parametric, the first codon models focused on estimating the effects of selective pressure on the protein via an explicit parameter in the maximum likelihood framework. Likelihood ratio tests of nested codon models armed the biologists with powerful tools, which provided unambiguous evidence for positive selection in real data. This, in turn, triggered a new wave of methodological developments. The new generation of models views the codon evolution process in a more sophisticated way, relaxing several mathematical assumptions. These models make a greater use of physicochemical amino acid properties, genetic code machinery, and the large amounts of data from the public domain. The overview of the most recent advances on modeling codon evolution is presented here, and a wide range of their applications to real data is discussed. On the downside, availability of a large variety of models, each accounting for various biological factors, increases the margin for misinterpretation; the biological meaning of certain parameters may vary among models, and model selection procedures also deserve greater attention. Solid understanding of the modeling assumptions and their applicability is essential for successful statistical data analysis.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

Concordant up-regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug–hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2α-, 2β-, 6β-, 16α- and 16β-hydroxytestosterones, while 6α- and 15α-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug–hormone crosstalk.     

Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2μm plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.It is well known that the nuclear genomes of most eukaryotes contain integrated fragments of organellar DNA called NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) (26, 29, 44, 45, 57). These fragments are usually pseudogenes, although some NUMTs and NUPTs have become incorporated into functional nuclear genes (38). The NUMTs present in the nuclear genomes of Saccharomycotina yeast species were recently analyzed by Sacerdot et al. (48).In addition to their mitochondrial genomes, yeast species contain a variety of other extranuclear DNA and RNA elements, including viruses and plasmids. These extrachromosomal elements are usually considered to be autonomous entities that do not interact with nuclear DNA. When our laboratory sequenced the genome of the yeast Vanderwaltozyma polyspora (synonym: Kluyveromyces polysporus) (49), we were therefore surprised to find the genomic region we describe here, which contains integrated fragments of several plasmid- and virus-like sequences. We propose that this region was formed by the capture of plasmid and viral sequences by the same mechanism that captures mitochondrial DNA to form NUMTs (43, 65). In a literature search, we could find only one previous report of a similar finding: Utatsu et al. (59) reported the sequences of two regions of nuclear DNA from Zygosaccharomyces rouxii that were highly similar to parts of the 2μm-like plasmid pSR1 from that species, but rearranged.Before describing the V. polyspora region, and similar regions found in other species, we will first briefly introduce the extrachromosomal RNA and DNA entities that are known to exist in yeasts. Extrachromosomal nucleic acids are relatively uncommon in yeasts: a broad survey of 1,800 strains from 600 species by Fukuhara (14) found that 196 strains (11%) contained some sort of extrachromosomal entity. Among these, 105 strains had a double-stranded RNA (dsRNA), 28 had a linear dsDNA plasmid, and 53 had a circular DNA plasmid of the 2μm family. These elements typically also have a patchy distribution within a species, being found in some individuals or strains but not in others. For instance, Nakayashiki et al. (37) surveyed 70 “wild” strains of Saccharomyces (mostly S. cerevisiae) for the presence of five extrachromosomal elements (2μm DNA plasmid, L-A and L-BC helper RNA viruses, and W and T RNA entities) and found each element to be present in between 1 and 38 of the strains, with 1 strain even containing all five elements simultaneously.