Glycine betaine loses its osmoprotective activity in a bspA strain of Erwinia chrysanthemi

Erwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high-salt media. Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase. Initial GB uptake is not affected by the bspA mutation. However, in high-salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability. BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel. We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high-salt media containing this osmoprotectant.     

Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep

In this study, we demonstrate the use of a genome-wide association mapping together with RNA-seq in a reduced number of samples, as an efficient approach to detect the causal mutation for a Mendelian disease. Junctional epidermolysis bullosa is a recessive genodermatosis that manifests with neonatal mechanical fragility of the skin, blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions. In Spanish Churra sheep, junctional epidermolysis bullosa (JEB) has been detected in two commercial flocks. The JEB locus was mapped to Ovis aries chromosome 11 by GWAS and subsequently fine-mapped to an 868-kb homozygous segment using the identical-by-descent method. The ITGB4, which is located within this region, was identified as the best positional and functional candidate gene. The RNA-seq variant analysis enabled us to discover a 4-bp deletion within exon 33 of the ITGB4 gene (c.4412_4415del). The c.4412_4415del mutation causes a frameshift resulting in a premature stop codon at position 1472 of the integrin β4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB skin samples and the absence of integrin β4 labeling in immunohistochemical assays. Genotyping of c.4412_4415del showed perfect concordance with the recessive mode of the disease phenotype. Selection against this causal mutation will now be used to solve the problem of JEB in flocks of Churra sheep. Furthermore, the identification of the ITGB4 mutation means that affected sheep can be used as a large mammal animal model for the human form of epidermolysis bullosa with aplasia cutis. Our approach evidences that RNA-seq offers cost-effective alternative to identify variants in the species in which high resolution exome-sequencing is not straightforward.     

Functional properties of muscle-derived cells related to morphological characteristics

Satellite cells represent a specific lineage of myogenic progenitors that allow skeletal muscle postnatal growth and repair. They have been described as being heterogeneous in nature, a characteristic associated with functional disparities. Here, we aimed at determining whether the morphometric characteristics of freshly extracted turkey muscle-derived cells (MDC) could represent a distinctive criterion between them and could also be associated with their behavioural features. Morphometric analysis showed that MDC displayed wide cell size diversity, from 4 to 10 μm. Lineage marker analysis was performed on MDC sorted by their size using counterflow centrifugal elutriation and showed that the cell size was associated with the specific expression of myogenic markers, revealing different commitment levels. In vitro, the smallest MDC exhibited limited myogenic activity while larger MDC displayed a myogenic potential that increased with their size. Ultrastructural analysis revealed that the smallest MDC shared quiescent cell features, whereas the other cells displayed metabolic activity that also increased as a function of their size. Collectively, our results demonstrate that the size of freshly extracted MDC is indicative of their respective progression towards myogenic differentiation lineage. This criterion could be useful for the early separation of more or less committed cells in the myogenic programme.Gregory Jouvion and Karl Rouger contributed equally to this work.     

Natural variation reveals relationships between pre-stress carbohydrate nutritional status and subsequent responses to xenobiotic and oxidative stress in Arabidopsis thaliana

Background

Soluble sugars are involved in responses to stress, and act as signalling molecules that activate specific or hormone cross-talk transduction pathways. Thus, exogenous sucrose treatment efficiently induces tolerance to the herbicide atrazine in Arabidopsis thaliana plantlets, at least partially through large-scale modifications of expression of stress-related genes.

Methods

Availability of sugars in planta for stress responses is likely to depend on complex dynamics of soluble sugar accumulation, sucrose–starch partition and organ allocation. The question of potential relationships between endogenous sugar levels and stress responses to atrazine treatment was investigated through analysis of natural genetic accessions of A. thaliana. Parallel quantitative and statistical analysis of biochemical parameters and of stress-sensitive physiological traits was carried out on a set of 11 accessions.

Key Results

Important natural variation was found between accessions of A. thaliana in pre-stress shoot endogenous sugar levels and responses of plantlets to subsequent atrazine stress. Moreover, consistent trends and statistically significant correlations were detected between specific endogenous sugar parameters, such as the pre-stress end of day sucrose level in shoots, and physiological markers of atrazine tolerance.

Conclusions

These significant relationships between endogenous carbohydrate metabolism and stress response therefore point to an important integration of carbon nutritional status and induction of stress tolerance in plants. The specific correlation between pre-stress sucrose level and greater atrazine tolerance may reflect adaptive mechanisms that link sucrose accumulation, photosynthesis-related stress and sucrose induction of stress defences.Key words: Arabidopsis thaliana, natural variation, atrazine sensitivity, carbon nutrition, endogenous soluble sugars, oxidative stress, sucrose, xenobiotic stress     

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.     

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs.     

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa)

The GENETPIG database has been established for storing and disseminating the results of the European project: 'GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpig/.