A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species

Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the "noncirculative" mode of transmission dominant in plant viruses (designated "mechanical vector transmission" in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the "helper component" (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.     

Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.     

PAS domains. Common structure and common flexibility

PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved alpha/beta-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.     

Microbial survival of space vacuum and extreme ultraviolet irradiation: strain isolation and analysis during a rocket flight

We have recovered new isolates from hot springs, in Yellowstone National Park and the Kamchatka Peninsula, after gamma-irradiation and exposure to high vacuum (10(-6) Pa) of the water and sediment samples. The resistance to desiccation and ionizing radiation of one of the isolates, Bacillus sp. strain PS3D, was compared to that of the mesophilic bacterium, Deinococcus radiodurans, a species well known for its extraordinary resistance to desiccation and high doses of ionizing radiation. Survival of these two microorganisms was determined in real and simulated space conditions, including exposure to extreme UV radiation (10-100 nm) during a rocket flight. We found that up to 15 days of desiccation alone had little effect on the viability of either bacterium. In contrast, exposure to space vacuum ( approximately 10(-6) Pa) decreased cell survival by two and four orders of magnitude for Bacillus sp. strain PS3D and D. radiodurans, respectively. Simultaneous exposure to space vacuum and extreme UV radiation further decreased the survival of both organisms, compared to unirradiated controls. This is the first report on the isolated effect of extreme UV at 30 nm on cell survival. Extreme UV can only be transmitted through high vacuum, therefore its penetration into the cells may only be superficial, suggesting that in contrast to near UV, membrane proteins rather than DNA were damaged by the radiation.     

Structural divergence of chromosomal segments that arose from successive duplication events in the Arabidopsis genome

Using the extensive segmental duplications of the Arabidopsis thaliana genome, a comparative study of homoeologous segments occurring in chromosomes 1, 2, 4 and 5 was performed. The gene-by-gene BLASTP approach was applied to identify duplicated genes in homoeologues. The levels of synonymous substitutions between duplicated coding sequences suggest that these regions were formed by at least two rounds of duplications. Moreover, remnants of even more ancient duplication events were recognised by a whole-genome study. We describe a subchromosomal organisation of genes, including the tandemly repeated genes, and the distribution of transposable elements (TEs). In certain cases, evidence of the possible mechanisms of structural rearrangements within the segments could be found. We provide a probable scenario of the rearrangements that took place during the evolution of the homoeologous regions. Furthermore, on the basis of the comparative analysis of the chromosomal segments in the Columbia and Landsberg erecta accessions, an additional structural variation in the A.thaliana genome is described. Analysis of the segments, spanning 7 Mb or 5.6% of the genome, permitted us to propose a model of evolution at the subchromosomal level.     

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.     

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double- and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.