Fission yeast telomeres forecast the end of the crisis

Recent years have placed fission yeast at the forefront of telomere research, as this organism combines a high level of conservation with human telomeres and precise genetic manipulability. Here we highlight some of the latest knowledge of fission yeast telomere maintenance and dysfunction, and illustrate how principles arising from fission yeast research are raising novel questions about telomere plasticity and function in all eukaryotes.     

Mouse Embryonic Lung Culture,A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.Download video file.^{(87M, mp4)}     

Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.     

Regulation of the Ca2+ Sensitivity of Exocytosis by Rab3a

Abstract: Ca²⁺ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca²⁺]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca²⁺ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µ M ) [Ca²⁺], but did not change the maximal activity observed at 10 µ M free [Ca²⁺]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 µ M [Ca²⁺] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca²⁺ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.     

Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile viral gastroenteritis and are responsible for up to 700,000 deaths each year (6, 24). The RV genome consists of 11 segments of double-stranded RNA (dsRNA) which can be separated by polyacrylamide gel electrophoresis (PAGE), resulting in typical dsRNA profiles exhibiting four well-defined size classes of segments. However, some RV show unusual dsRNA profiles in which standard-sized segments are replaced by larger, rearranged forms (for a review, see reference 5). Gene rearrangements were first reported for RV isolated from immunodeficient children with chronic infection (11, 26) and can be obtained experimentally by serial passages at a high multiplicity of infection (MOI) in cell culture (10, 14, 21). We recently showed that rearrangements also occur during acute RV infection (36). Gene rearrangement usually consists of a partial head-to-tail duplication of a segment sequence. In most cases, sequence duplication occurs after the stop codon, leaving the open reading frame (ORF) untouched and the encoded protein unchanged (5, 6). Less frequently, the duplication occurs within the ORF, which thus encodes a modified protein (8, 38).Manipulation at the cDNA level of most positive- and negative-strand RNA viral genomes, followed by rescue of infectious virus, is now well established and has provided a better understanding of RNA virus replication and pathogenesis. In the case of Reoviridae, the development of reverse genetics systems has been hampered by the nature of the genome, which carries 10 to 12 dsRNA segments that are densely packed within the viral particle and are transcribed and replicated within a subviral structure. Recent major advances were obtained in the development of reverse genetics systems for some Reoviridae. For mammalian orthoreo- and orbiviruses, reverse genetics systems based on the transfection of plasmid cDNAs (13) or cDNA-derived mRNAs (2) corresponding to a complete set of 10 RNA segments have been established, allowing the rescue of infectious viral progeny. However, for RV, there is no report indicating that transfection of a complete set of viral mRNAs can result in the rescue of infectious viruses, and it is still unknown whether the RV genome can be infectious. In 2006, Komoto et al. (16) described the first reverse genetics system for RV, based on a model originally developed for influenza viruses by Palese and colleagues (17). In this system, an exogenous RV mRNA synthesized in the cell cytoplasm by the T7 RNA polymerase (T7pol), expressed by a recombinant vaccinia virus, is packaged in place of its homologous counterpart into a helper RV. This reverse genetics system has allowed the recovery of engineered monoreassortant infectious RV (designated recombinant RV) with an incorporated exogenous segment 4 encoding the spike protein VP4. An in vitro-modified cDNA-derived segment 4 was also successfully introduced into a recombinant RV to obtain a virus carrying a VP4 antigenic chimera (15). However, this system is restricted to segments encoding antigenically distinct viral surface proteins (like VP4) because the selection of recombinant viruses requires the use of specific and potent neutralizing antibodies to eliminate wild-type (WT) helper viruses.Results obtained from a preliminary study suggested that rearranged RNA segments might overcome this limitation. Indeed, we first characterized human RV clones containing rearranged segment 7, 11, or both (8) and then compared the fitness levels of rearranged versus WT viruses. We found that viruses with rearranged segment 7 or 11 replicated less well than or equally to WT viruses, as judged by viral growth curve experiments. Surprisingly, in competition growth experiments, rearranged segment 7 or 11 was always selected into the viral progenies, even when mixed infections were performed with a ratio of 1 rearranged to 1,000 WT viruses (4). The absence of a growth advantage conferred on the virus by rearranged segments, combined with their preferential segregation into the viral progenies, suggested that rearranged RNA segments might be packaged preferentially. These observations are in agreement with results of earlier studies showing that rearranged segment 5 or 11 segregated preferentially into viral progenies issued from mixed infections with WT virus (10, 19).We developed a reverse genetics system for RV on the basis of the preferential packaging of rearranged RNAs. We report here the rescue of recombinant viruses carrying cDNA-derived rearranged segment 7 (either unmodified or containing silent mutations introduced by site-directed mutagenesis to generate restriction enzyme sites as markers), with no selection pressure other than serial passage in cell culture. We also report the rescue of a recombinant virus expressing a double-sized recombinant NSP3 protein encoded by an in vitro-modified cDNA-derived rearranged segment 7, showing for the first time that an in vitro-engineered gene encoding a modified nonstructural protein can be introduced into an infectious RV.     

Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway

Tracheal occlusion during lung development accelerates growth in response to increased intraluminal pressure. In order to investigate the role of internal pressure on murine early lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on the first few branch generations and on gene expression. We observed that the elevation of internal pressure induced more than a doubling in branching, associated with increased proliferation, while branch elongation speed increased 3-fold. Analysis by RT-PCR showed that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intraluminal pressure after 48h of culture, suggesting mechanotransduction via internal pressure of these key developmental genes. Tracheal occlusion did not increase the number of branches of Fgfr2b-/- mice lungs nor of wild type lungs cultured with Fgfr2b antisense RNA. Tracheal occlusion of Fgf10(LacZ/-) hypomorphic lungs led to the formation of fewer branches than in wild type. We conclude that internal pressure regulates the FGF10-FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure levels, fixed both by epithelial secretion and boundary conditions, can control or modulate the branching process via FGF10-FGFR2b-Sprouty2.     

Growth pattern and age determination for Cecropia sciadophylla (Urticaceae)

Cecropia species, ranging from Mexico to northern Argentina and the West Indies, are pioneer trees that colonize cleared areas with high light. To determine their ages to help pinpoint the date of the area's disturbance, we need to understand their developmental and architectural changes over time. The simple architecture of Cecropia conforms to the model of Rauh; that is, it has orthotropic axes with lateral flowering and rhythmic branching. The axes are made of a succession of nodes and internodes whose length and associated lateral productions remain measurable for years. Thus, by describing the tree trunk node by node, we can depict the sequence of events involved in tree development. For 25 trees of C. sciadophylla, from two stations in French Guiana and Colombia, we recorded internode length and any presence of branches, and flowers for each node. Using autocorrelation coefficients, we found a high periodicity in flowering and branching, with inflorescences at every 25 nodes, stages of branches spaced by a multiple of 25 nodes, and alternation of long and short nodes every 25 nodes. Considering that flowering is annual for many Cecropia species, the main conclusion of this work is that C. sciadophylla has strong annual growth, branching, and flowering rhythms. In addition, the age of the tree can be estimated retrospectively by observing its adult morphology.