Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules

The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast

Effects of a culturally responsive speech and language intervention for students of Indigenous and non-Indigenous ancestry

This study explored the effectiveness of a speech and language intervention that was designed to be culturally responsive and adapted to provide explicit language instruction. Participants included all 774 kindergarten students from a mid-sized rural school district in British Columbia. Seventy-seven students screened as at risk received the intervention, and the remaining students participated in the regular kindergarten curriculum. Results indicated statistically significant effects of the intervention on language and vocabulary skills. No differential effects were observed between students of Indigenous and non-Indigenous heritage. Results are discussed in terms of culturally responsive and explicit instruction for early language development.Key words: early intervention, language intervention, cultural responsiveness, Aboriginal educationCette étude a examiné l'efficacité d'une intervention orthophonique et linguistique conçue pour être culturellement adaptée et permettre un enseignement explicite des langues. 774 élèves de maternelle d'un district scolaire rural de taille moyenne en Colombie-Britannique ont participé à cette étude. Parmi eux, 77 étudiants, sélectionnés comme étant à risque, ont participé à l'intervention, tandis que les autres étudiants ont participé au programme de maternelle habituel. Les résultats ont montré des effets statistiquement significatifs de l'intervention sur le langage et le vocabulaire. Aucune différence n'a été observée entre les étudiants ayant un patrimoine culturel autochtone ou non autochtone. Ces résultats sont débatus en termes d'enseignement explicite et culturellement adapté pour le développement précoce du langage.Mots clés: intervention précoce, intervention linguistique, sensibilisation à la culturel, éducation des Autochtone

Pendekatan Qspm Sebagai Dasar Perumusan Strategi Peningkatan Pendapatan Asli Daerah Kabupaten Batang, Jawa Tengah

The aim of this research is to analyse of increasing Local Original Income (LOI) strategy and his influence to increasing the regional income. The research was done at Local Government Income of Batang regency. This research also want to know that the LOI strategy was based on the potencies and opportunities. The analyzing use the IFE, EFE, SWOT, and then QSPM to choose strategic formulation; and proportion models. The result of Internal – External analysis show that increasing strategy of LOI have not based on the potencies and opportunities that they have yet. The Local Government Income of Batang Regency needs the intensification strategy for increasing the LOI. By the QSPM analysis, the Local Government Income of Batang Regency needs extensification strategy for LOI acceptance

The mating-specific Gα interacts with a kinesin-14 and regulates pheromone-induced nuclear migration in budding yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating. © 2009 by The American Society for Cell Biology

Synthetic RNA modules for fine-tuning gene expression levels in yeast by modulating RNase III activity

The design of synthetic gene networks requires an extensive genetic toolbox to control the activities and levels of protein components to achieve desired cellular functions. Recently, a novel class of RNA-based control modules, which act through post-transcriptional processing of transcripts by directed RNase III (Rnt1p) cleavage, were shown to provide predictable control over gene expression and unique properties for manipulating biological networks. Here, we increase the regulatory range of the Rnt1p control elements, by modifying a critical region for enzyme binding to its hairpin substrates, the binding stability box (BSB). We used a high throughput, cell-based selection strategy to screen a BSB library for sequences that exhibit low fluorescence and thus high Rnt1p processing efficiencies. Sixteen unique BSBs were identified that cover a range of protein expression levels, due to the ability of the sequences to affect the hairpin cleavage rate and to form active cleavable complexes with Rnt1p. We further demonstrated that the activity of synthetic Rnt1p hairpins can be rationally programmed by combining the synthetic BSBs with a set of sequences located within a different region of the hairpin that directly modulate cleavage rates, providing a modular assembly strategy for this class of RNA-based control elements

The N-terminal intrinsically disordered domain of mgm101p is localized to the mitochondrial nucleoid.

The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted

The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic

Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells

Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations