Neutral and functionally important genes shed light on phylogeography and the history of high‐altitude colonization in a widespread New World duck

Phylogeographic studies often infer historical demographic processes underlying species distributions based on patterns of neutral genetic variation, but spatial variation in functionally important genes can provide additional insights about biogeographic history allowing for inferences about the potential role of adaptation in geographic range evolution. Integrating data from neutral markers and genes involved in oxygen (O₂)‐transport physiology, we test historical hypotheses about colonization and gene flow across low‐ and high‐altitude regions in the Ruddy Duck (Oxyura jamaicensis), a widely distributed species in the New World. Using multilocus analyses that for the first time include populations from the Colombian Andes, we also examined the hypothesis that Ruddy Duck populations from northern South America are of hybrid origin. We found that neutral and functional genes appear to have moved into the Colombian Andes from both North America and southern South America, and that high‐altitude Colombian populations do not exhibit evidence of adaptation to hypoxia in hemoglobin genes. Therefore, the biogeographic history of Ruddy Ducks is likely more complex than previously inferred. Our new data raise questions about the hypothesis that adaptation via natural selection to high‐altitude conditions through amino acid replacements in the hemoglobin protein allowed Ruddy Ducks to disperse south along the high Andes into southern South America. The existence of shared genetic variation with populations from both North America and southern South America as well as private alleles suggests that the Colombian population of Ruddy Ducks may be of old hybrid origin. This study illustrates the breadth of inferences one can make by combining data from nuclear and functionally important loci in phylogeography, and underscores the importance of complete range‐wide sampling to study species history in complex landscapes.     

Integrating structure,bioinformatics, and enzymology to discover function: BioH,a new carboxylesterase from Escherichia coli

Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.     

Estimation of calcium requirements for optimal productive and reproductive performance,eggshell and tibial quality in egg-type duck breeders

Optimizing the dietary calcium (Ca) level is essential to maximize the eggshell quality, egg production and bone formation in poultry. This study aimed to establish the Ca requirements of egg-type duck breeders from 23 to 57 weeks of age on egg production, eggshell, incubation, tibial, plasma and ovary-related indices, as well as the expression of matrix protein-related genes. Totally, 450 Longyan duck breeders aged 21 weeks of age were allotted randomly into five treatments, each with six replicates of 15 individually caged birds. The data collection started from 23 weeks of age and continued over the following 35 weeks. The five groups corresponded to five dietary treatments containing either 2.8%, 3.2%, 3.6%, 4.0% or 4.4% Ca. The tested dietary Ca levels increased (linear, P <0.01) egg production and egg mass, and linearly improved (P <0.01) the feed conversion ratio (FCR). Increasing the dietary Ca levels from 2.8% to 4.4% increased (P <0.01) the eggshell thickness and eggshell content. The tested Ca levels showed a quadratic effect on eggshell thickness and ovarian weight (P <0.01); the highest values were obtained with the Ca levels 4.0% and 3.6%, respectively. Dietary Ca levels affected the small yellow follicles (SYF) number and SYF weight/ovarian weight, and the linear response (P <0.01) was significant vis-à-vis SYF number. In addition, dietary Ca levels increased (P <0.05) the tibial dry weight, breaking strength, mineral density and ash content. Plasma and tibial phosphorus concentration exhibited a quadratic (P <0.01) response to dietary Ca levels. Plasma calcitonin concentration linearly (P <0.01) increased as dietary Ca levels increased. The relative expression of carbonic anhydrase 2 in the uterus rose (P <0.01) with the increment of dietary Ca levels, and the highest value was obtained with 3.2% Ca. In conclusion, Longyan duck breeders fed a diet with 4.0% Ca had superior eggshell and tibial quality, while those fed a diet with 3.6% Ca had the heaviest ovarian weights. The regression model indicated that the dietary Ca levels 3.86%, 3.48% and 4.00% are optimal levels to obtain maximum eggshell thickness, ovarian weight and tibial mineral density, respectively.     

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

Results obtained from expression profilings of renal cell carcinoma using different “ome”‐based approaches and comprehensive data analysis demonstrated that proteome‐based technologies and cDNA microarray analyses complement each other during the discovery phase for disease‐related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to up‐regulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX‐defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%), and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome‐based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely three candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin α‐1A chain, and ubiquitin carboxyl‐terminal hydrolase L1, the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors.     

Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.