Genetic Improvement of Willow for Bioenergy and Biofuels

Willows (Salix spp.) are a very diverse group of catkin-bearing trees and shrubs that are widely distributed across temperate regions of the globe.Some species respond well to being grown in short rotation coppice (SRC) cycles,which are much shorter than conventional forestry.Coppicing reinvigorates growth and the biomass rapidly accumulated can be used as a source of renewable carbon for bioenergy and biofuels.As SRC willows re-distribute nutrients during the perennial cycle they require only minimal nitro...     

QSAR of multiple mutated antibodies

The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q(2) = 0.74, R(2) = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.     

High Levels of Genetic Diversity in Salix viminalis of the Czech Republic as Revealed by Microsatellite Markers

Willows (Salix spp.) grown as short rotation coppice are recognised as an important bioenergy crop, and breeding programmes are underway in several countries, including the Czech Republic. The basket willow Salix viminalis is one of the few willow species that is widespread in the Czech Republic and thus a potential source of diversity, but the most extensive germplasm collection available shows evidence of redundancy. To investigate levels of variation in natural populations of this species for use in crop improvement programmes, a set of 38 microsatellite markers was used to assess genetic diversity and population structure among 84 S. viminalis individuals collected from seven Czech rivers (the Odra, Be?va, Morava, Dyje, Jihlava, Sázava and Vltava), covering a wide geographic distribution. The markers detected 6.95 alleles per locus on average with 92?% of the sampled individuals having a unique multilocus genotype giving a high clonal richness measure among all samples (R?=?0.952). Three sets of putative clones (with identical genotypes as determined by the markers used here) were also identified. Significant levels of genetic diversity were revealed within all sampling sites. With the exception of sites on the Odra and Morava, pairwise F _ST (0.02?C0.1) values indicated moderate differentiation between sites. Principal coordinates analysis revealed some separation of the Dyje individuals from all others. This was in agreement with the population structure results derived from Bayesian analyses using STRUCTURE software. These results provide the first evidence that potentially useful levels of genotypic variation are present within natural S. viminalis populations in the Czech Republic.     

Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.     

An anchored linkage map for sugar beet based on AFLP,SNP and RAPD markers and QTL mapping of a new source of resistance to <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important sugar-beet disease worldwide and can result in severe losses of root yield and sugar content. We have identified a major QTL for BNYVV resistance from a new source in a segregating population of 158 individuals. The QTL explained an estimated 78% of the observed phenotypic variation and the gene conferring the partial resistance is referred to as Rz4. AFLP was used in combination with bulked segregant analysis (BSA) to develop markers linked to the resistance phenotype. AFLP marker analysis was extended to produce a linkage map that was resolved into nine linkage groups. These were anchored to the nine sugar-beet chromosomes using previously published SNP markers. This represents the first anchored sugar-beet linkage map to be published with non-anonymous markers. The final linkage map comprised 233 markers covering 497.2 cM, with an average interval between markers of 2.1 cM. The Rz4 QTL and an Rz1 RAPD marker were mapped to chromosome III, the known location of the previously identified BNYVV resistance genes Rz1, Rz2 and Rz3. The availability to breeders of new resistance sources such as Rz4 increases the potential for breeding durable disease resistance.     

Proteochemometrics analysis of substrate interactions with dengue virus NS3 proteases

The prime side specificity of dengue protease substrates was investigated by use of proteochemometrics, a technology for drug target interaction analysis. A set of 48 internally quenched peptides were designed using statistical molecular design (SMD) and assayed with proteases of four subtypes of dengue virus (DEN-1-4) for Michaelis (K(m)) and cleavage rate constants (k(cat)). The data were subjected to proteochemometrics modeling, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. Detailed analysis of the models then showed that considerably differing physico-chemical properties of amino acids contribute independently to the K(m) and k(cat) activities. For k(cat), only P1' and P2' prime side residues were important, while for K(m) all four prime side residues, P1'-P4', were important. The models could be used to identify amino acids for each P' substrate position that are favorable for, respectively, high substrate affinity and cleavage rate.     

Leukocyte adhesion deficiency II. Advances and open questions

Leukocyte adhesion deficiency II (LAD II) belongs to a group of human congenital diseases in which the interactions of leukocytes with the vascular endothelium are strongly impaired. LAD II is based on a defect in the synthesis of fucosylated glycostructures. This leads to an immunodeficiency owing to the absence of functional selectin ligands and to strong psychomotor defects, as a result of as-yet unknown reasons. In this review we focused on the current controversies, and open questions that have arisen from recent studies on the genetic defect, therapy and the basis of psychomotor defects in LAD II.