A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.     

Effects of growth-light quantity,growth-light quality and CO2 concentration on Rubisco deactivation during low PFD or darkness

The effects of CO₂ concentration and the effects of growth-light conditions on Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation were examined for Spinacea oleracea (spinach). Rubisco deactivation kinetics and the degree that Rubisco activation limited the rise in photosynthesis following an increase in photon flux density (PFD) were determined from gas-exchange time courses. There were no significant differences in the apparent relaxation time for Rubisco deactivation among leaves exposed to high or low CO₂ (50 or 1000 mol mol^-1) and low PFD (170 mol m^-2 s^-1) or darkness. However, when PFD was increased to 1700 mol m^-2 s^-1 following a period of low PFD or darkness, leaves exposed to low CO₂ × low PFD showed a lower contribution to the photosynthetic induction process by the activation of Rubisco than leaves exposed to the other treatments. For the growth-light experiments, spinach was grown under high PFD × high red:far-red ratio (R:FR), low PFD × high R:FR, or low PFD × low R:FR light environments. Leaves that matured under the low PFD × low R:FR treatment showed a lower percent change in photosynthesis due to Rubisco activation than leaves exposed to the other growth-light treatments. However, there were no significant differences among the growth-light treatments in the maximum contribution of Rubisco activation to the induction response or in the apparent relaxation time for Rubisco deactivation during shade events.     

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.     

Finding the molecular basis of quantitative traits: successes and pitfalls

Understanding the molecular basis of quantitative genetic variation is a principal goal for biomedicine. Although the complex genetic architecture of quantitative traits has so far largely frustrated attempts to identify genes in humans by standard linkage methodologies, quantitative trait loci (QTL) have been mapped in plants, insects and rodents. However, identifying the molecular bases of QTL remains a challenge. Here, we discuss why this is and how new experimental strategies and analytical techniques, combined with the fruits of the genome projects, are beginning to identify candidate genes for QTL studies in several model organisms.     

Quantifying the similarities within fold space

We have used GRATH, a graph-based structure comparison algorithm, to map the similarities between the different folds observed in the CATH domain structure database. Statistical analysis of the distributions of the fold similarities has allowed us to assess the significance for any similarity. Therefore we have examined whether it is best to represent folds as discrete entities or whether, in fact, a more accurate model would be a continuum wherein folds overlap via common motifs. To do this we have introduced a new statistical measure of fold similarity, termed gregariousness. For a particular fold, gregariousness measures how many other folds have a significant structural overlap with that fold, typically comprising 40% or more of the larger structure. Gregarious folds often contain commonly occurring super-secondary structural motifs, such as beta-meanders, greek keys, alpha-beta plait motifs or alpha-hairpins, which are matching similar motifs in other folds. Apart from one example, all the most gregarious folds matching 20% or more of the other folds in the database, are alpha-beta proteins. They also occur in highly populated architectural regions of fold space, adopting sandwich-like arrangements containing two or more layers of alpha-helices and beta-strands.Domains that exhibit a low gregariousness, are those that have very distinctive folds, with few common motifs or motifs that are packed in unusual arrangements. Most of the superhelices exhibit low gregariousness despite containing some commonly occurring super-secondary structural motifs. In these folds, these common motifs are combined in an unusual way and represent a small proportion of the fold (<10%). Our results suggest that fold space may be considered as continuous for some architectural arrangements (e.g. alpha-beta sandwiches), in that super-secondary motifs can be used to link neighbouring fold groups. However, in other regions of fold space much more discrete topologies are observed with little similarity between folds.     

Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum

The fungal pathogens Fusarium graminearum and F. culmorum cause ear blight disease on cereal crops worldwide. The disease lowers both grain quality and grain safety. Disease prevalence is increasing due to changes in cropping practices and the difficulties encountered by plant breeders when trying to introgress the polygene-based resistance. The molecular basis of resistance to Fusarium ear blight in cereal species is poorly understood. This is primarily due to the large size of cereal genomes and the expensive resources required to undertake gene function studies in cereals. We therefore explored the possibility of developing various model floral infection systems that would be more amenable to experimental manipulation and high-throughput gene function studies. The floral tissues of tobacco, tomato, soybean and Arabidopsis were inoculated with Fusarium conidia and this resulted in disease symptoms on anthers, anther filaments and petals in each plant species. However, only in Arabidopsis did this initial infection then spread into the developing siliques and seeds. A survey of 236 Arabidopsis ecotypes failed to identify a single genotype that was extremely resistant or susceptible to Fusarium floral infections. Three Arabidopsis floral mutants that failed to develop anthers and/or functional pollen (i.e. agamous-1, apetala1-3 and dad1) were significantly less susceptible to Fusarium floral infection than wild type. Deoxynivalenol (DON) mycotoxin production was also detected in Fusarium-infected flowers at >1 ppm. This novel floral pathosystem for Arabidopsis appears to be highly representative of a serious cereal crop disease.