Heterologous high yield expression and purification of neurotensin and its functional fragment in Escherichia coli.

Peptide synthesis is widely used for the production of small proteins and peptides, but producing uniformly isotopically labelled peptides for NMR and other biophysical studies could be limited for economic reasons. Here, we propose a use of a modified pGEV-1 plasmid to express neurotensin (NT(1-13)), pGlu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(10)-Tyr(11)-Ile(12)-Leu(13)-OH, as a C-terminal fusion protein with the GB1 domain of streptococcal protein G. The free carboxyl-terminus is important for the function of several peptide hormones, including neurotensin. Therefore, for the pGEV-NT(1-13) construct, the C-terminal pGEV-encoded 6xHis tag was removed and an N-terminal 8xHis tag was introduced for affinity purification. To facilitate removal of tags using CNBr cleavage, a methionine was introduced at the N-terminal of the peptide. Furthermore, this pGEV-NT(1-13) plasmid was used as a template to include a Pro-7 to Met mutation for CNBr cleavage, giving NT(8-13), the sub-fragment crucial for the biological activity of this peptide. These two constructs are being used to produce uniformly labelled NT(1-13) and NT(8-13) in high yield and in a cost effective way, using cheap (15)N and/or (13)C source. The modification proposed here using the pGEV-1 plasmid could be an alternative option for the high expression of other isotopically labelled and unlabelled short peptides, including hormones and hydrophobic membrane peptides.

Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans.

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Improved yield of a ligand-binding GPCR expressed in E. coli for structural studies.

The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in Escherichia coli as a functional, ligand-binding receptor, but yields of purified receptor are still low for comprehensive structural studies. Here, several approaches have been examined to optimize the yields of active, ligand-binding receptor. Optimisation of E. coli strain and induction protocol yielded a significant improvement in expression of active receptor. Expression of the receptor in BL21(DE3) cells, in combination with autoinduction improved expression 10-fold when compared with previously reported expression protocols using IPTG-mediated induction in DH5alpha cells. Optimization of the purification protocol revealed that supplementation of buffers with phospholipids enhanced recovery of active receptor. The methods examined are potentially applicable to other GPCRs expressed in E. coli.

Topography, energy and the global distribution of bird species richness.

A major goal of ecology is to determine the causes of the latitudinal gradient in global distribution of species richness. Current evidence points to either energy availability or habitat heterogeneity as the most likely environmental drivers in terrestrial systems, but their relative importance is controversial in the absence of analyses of global (rather than continental or regional) extent. Here we use data on the global distribution of extant continental and continental island bird species to test the explanatory power of energy availability and habitat heterogeneity while simultaneously addressing issues of spatial resolution, spatial autocorrelation, geometric constraints upon species' range dynamics, and the impact of human populations and historical glacial ice-cover. At the finest resolution (1 degree), topographical variability and temperature are identified as the most important global predictors of avian species richness in multi-predictor models. Topographical variability is most important in single-predictor models, followed by productive energy. Adjusting for null expectations based on geometric constraints on species richness improves overall model fit but has negligible impact on tests of environmental predictors. Conclusions concerning the relative importance of environmental predictors of species richness cannot be extrapolated from one biogeographic realm to others or the globe. Rather a global perspective confirms the primary importance of mountain ranges in high-energy areas.

Human impacts and the global distribution of extinction risk.

Understanding the global geographical distribution of extinction risk is a key challenge in conservation biology. It remains controversial, however, to what extent areas become threat hotspots simply because of high human impacts or due to predisposing ecological conditions. Limits to the taxonomic and geographical extent, resolution and quality of previously available data have precluded a full global assessment of the relative roles of these factors. Here, we use a new global database on the geographical distributions of birds on continents and continental islands to show that, after controlling for species richness, the best predictors of the global pattern of extinction risk are measures of human impact. Ecological gradients are of secondary importance at a global scale. The converse is true for individual biogeographic realms, within which variation in human impact is reduced and its influence on extinction risk globally is therefore underestimated. These results underline the importance of a global perspective on the mechanisms driving spatial patterns of extinction risk, and the key role of anthropogenic factors in driving the current extinction crisis.

Functional and developmental impact of cytosolic protein N-terminal methionine excision in Arabidopsis.

Protein N-terminal methionine (Met) excision (NME) is carried out by two types of Met aminopeptidases (MAPs), MAP1 and MAP2, in eukaryotes. Three enzymes, MAP1A, MAP2A, and MAP2B, have been identified in the cytoplasm of Arabidopsis (Arabidopsis thaliana). MAP transcript quantification revealed a predominance of MAP2B and developmental and organ-specific regulation of both MAP1A and MAP2s. By combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic NME in Arabidopsis and of the respective contributions of the two types of cytoplasmic MAPs throughout development. dsRNA interference and knockout (KO) plant lines targeting either MAP1A alone or both MAP2s simultaneously were constructed and shown to display wild-type phenotypes. In the MAP1A KO context, modulating MAP2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development, with particularly strong effects on the root system. Reciprocally, complete MAP2 inhibition in various MAP1A knocked-down genetic backgrounds also generated a gradient of developmentally abnormal plants, but the effects on the root system were milder than in the KO context. In the absence of MAP2 activity, the severity of the phenotype in the MAP1A knocked-down lines was correlated to the extent of MAP1A mRNA accumulation. Complete cytoplasmic NME inactivation blocked development after plant germination. Thus, in plants, (1) cytoplasmic NME is essential; (2) MAP1A and MAP2s are functionally interchangeable, which is not the case in fungi and animals, as a complete block of either MAP-type activity does not cause any visible molecular or phenotypic effect; and (3) a minimal level of cytoplasmic MAP is required for normal development.

Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome.

The LMR1 5.2 kb interspersed repeat of Leptosphaeria maculans was described by Taylor and Borgmann [Mol. Plant Microbe Interact. 7 (1994) 181] as an uncharacterized repeated element sharing homologies with both LINEs and SINEs. Here, we used the LMR1 sequence as a template to identify the full-length element within a 184-kb genomic sequence corresponding to the pericentromeric region of the 2.80 Mb chromosome of isolate v23.1.3. This region comprises (i) one 6980-bp full-sized Pholy element bordered by two 275- to 280-bp long terminal repeats (LTRs), (ii) five Pholy-related sequences, usually truncated at their 3' ends, and (iii) five solo-LTRs. Structural features strongly suggested that Pholy corresponds to an ancient copia-like retrotransposon, sharing strong homologies with the Elsa retrotransposon of Stagonospora nodorum. Pholy was also suggested to be specific to pericentromeric regions. Comparative analysis of the structure of the Pholy-like sequences occurring in the 184-kb contig and in other parts of the genome showed that this family of repeats is highly degenerated following extensive repeat induced point mutation (RIP).