How accurately is ncRNA aligned within whole-genome multiple alignments?

Background  

Multiple alignment of homologous DNA sequences is of great interest to biologists since it provides a window into evolutionary processes. At present, the accuracy of whole-genome multiple alignments, particularly in noncoding regions, has not been thoroughly evaluated.     

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.     

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA–PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.     

Multiple Scales of Temporal Variability in Ecosystem Metabolism Rates: Results from 2 Years of Continuous Monitoring in a Forested Headwater Stream

Headwater streams are key sites of nutrient and organic matter processing and retention, but little is known about temporal variability in gross primary production (GPP) and ecosystem respiration (ER) rates as a result of the short duration of most metabolism measurements in lotic ecosystems. We examined temporal variability and controls on ecosystem metabolism by measuring daily rates continuously for 2 years in Walker Branch, a first-order deciduous forest stream. Four important scales of temporal variability in ecosystem metabolism rates were identified: (1) seasonal, (2) day-to-day, (3) episodic (storm-related), and (4) inter-annual. Seasonal patterns were largely controlled by the leaf phenology and productivity of the deciduous riparian forest. Walker Branch was strongly net heterotrophic throughout the year with the exception of the open-canopy spring when GPP and ER rates were co-equal. Day-to-day variability in weather conditions influenced light reaching the streambed, resulting in high day-to-day variability in GPP particularly during spring (daily light levels explained 84% of the variance in daily GPP in April). Episodic storms depressed GPP for several days in spring, but increased GPP in autumn by removing leaves shading the streambed. Storms depressed ER initially, but then stimulated ER to 2–3 times pre-storm levels for several days. Walker Branch was strongly net heterotrophic in both years of the study, with annual GPP being similar (488 and 519 g O₂ m⁻² y⁻¹ or 183 and 195 g C m⁻² y⁻¹) but annual ER being higher in 2004 than 2005 (−1,645 vs. −1,292 g O₂ m⁻² y⁻¹ or −617 and −485 g C m⁻² y⁻¹). Inter-annual variability in ecosystem metabolism (assessed by comparing 2004 and 2005 rates with previous measurements) was the result of the storm frequency and timing and the size of the spring macroalgal bloom. Changes in local climate can have substantial impacts on stream ecosystem metabolism rates and ultimately influence the carbon source and sink properties of these important ecosystems.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

In vivo resolution of multiexponential decays of multiple near-infrared molecular probes by fluorescence lifetime-gated whole-body time-resolved diffuse optical imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.     

Lipopolysaccharide O-Chain Core Region Required for Cellular Cohesion and Compaction of In Vitro and Root Biofilms Developed by Rhizobium leguminosarum

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.