Regulation of the galactose pathway in Saccharomyces cerevisiae: induction of uridyl transferase mRNA and dependency on GAL4 gene function

In Saccharomyces cerevisiae, utilization of galactose requires four inducible enzyme activities. Three of these activities (galactose-1-phosphate uridyl transferase, EC 2.7.7.10; uridine diphosphogalactose 4-epimerase, EC 5.1.3.2; and galactokinase, EC 2.7.1.6) are specified by three tightly linked genes (GAL7, GAL10, and GAL1, respectively) on chromosome II, whereas the fourth, galactose transport, is specified by a gene (GAL2) located on chromosome XII. Although classic genetic analysis has revealed both positive and negative regulatory genes that coordinately affect the appearance of all four enzyme activities, neither the basic events leading to the appearance of enzyme activities nor the roles of the regulatory genes have yet been determined. Regulation of inducible enzyme activity could be mediated by events related to transcription, translation, or enzyme activation. For the purpose of studying galactose pathway induction and its regulation, we have developed an immunoprecipitation assay that enables us to detect the GAL7 specified uridyl transferase polypeptide in yeast extracts and among the polypeptides synthesized in an RNA-dependent in vitro translation system. Use of this immunoprecipitation assay in conjunction with in vivo labeling experiments demonstrates the presence of [(3)H]leucine-labeled transferase in extracts prepared from cells grown in galactose but not from cells grown in glucose. This galactose-specific induction of transferase polypeptide is mediated by the de novo appearance of a functional mRNA species whose synthetic capacity is detectable by the combination of in vitro translation and immunoprecipitation. The appearance of functional transferase mRNA depends on wild-type expression of the positive regulatory gene, GAL4. Cells carrying a nonsense (amber) mutation in the GAL4 gene fail to produce the transferase mRNA, whereas a nonsense suppressor of the GAL4 amber mutant regains the galactose-specific mRNA response. Our results establish that the induction of the GAL7 specified uridyl transferase activity is mediated by de novo appearance of a functional mRNA and that this galactose-specific response is dependent on a wild-type GAL4 gene product

Srs2 removes deadly recombination intermediates independently of its interaction with SUMO-modified PCNA

Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair deficient cells, and a second one is to eliminate potentially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon translocation on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar phenotypes, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to displace Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed

The potential psychological benefits of Active Video Games in the rehabilitation of musculoskeletal injuries and deficiencies: A narrative review of the literature

Background: Recent literature suggests that Active Video Games (AVGs) may offer potential psychological benefits during the rehabilitation of musculoskeletal injuries and their corresponding deficiencies. Objectives: To review existing literature regarding the potential psychological benefits of AVGs within the context of rehabilitation from musculoskeletal injury or debilitation. Method: A narrative review of the literature that used the Population, Intervention, Comparison, and Outcomes PICO method was conducted. The literature review included studies that discussed and/or investigated potential psychological benefits of AVGs during musculoskeletal rehabilitation. Of the total 163 papers that were identified, 30 met the inclusion criteria. Results: The Nintendo® WiiTM (Nintendo Co., Ltd, Kyoto, Japan) was the most commonly-used games console that was employed in AVG interventions (15 out of 21), and these studies that investigated potential psychological benefits were typically conducted with elderly populations. These studies reported that using AVGs in musculoskeletal rehabilitation resulted in a number of positive psychological effects (e.g., enjoyment, effects on self). However, most studies lacked a clear theoretical framework, and varied greatly in their designs and methodologies. Conclusion: Despite encouraging findings of AVG use, insufficient evidence exists to reliably verify or refute the potential psychological benefits of AVGs in musculoskeletal rehabilitation. It is recommended that future studies in this area contain a theoretical framework to ensure greater consistency in the methodology used and the execution of the intervention. The potential findings of such investigations may result in the development of optimal, client-tailored rehabilitation programmes

A Bayesian Network Driven Approach to Model the Transcriptional Response to Nitric Oxide in Saccharomyces cerevisiae

The transcriptional response to exogenously supplied nitric oxide in Saccharomyces cerevisiae was modeled using an integrated framework of Bayesian network learning and experimental feedback. A Bayesian network learning algorithm was used to generate network models of transcriptional output, followed by model verification and revision through experimentation. Using this framework, we generated a network model of the yeast transcriptional response to nitric oxide and a panel of other environmental signals. We discovered two environmental triggers, the diauxic shift and glucose repression, that affected the observed transcriptional profile. The computational method predicted the transcriptional control of yeast flavohemoglobin YHB1 by glucose repression, which was subsequently experimentally verified. A freely available software application, ExpressionNet, was developed to derive Bayesian network models from a combination of gene expression profile clusters, genetic information and experimental conditions

Comparative transcriptomic analysis reveals similarities and dissimilarities in saccharomyces cerevisiae wine strains response to nitrogen availability

Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12h, 24h and 96h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape-musts and the development of strategies to optimize yeast performance in industrial fermentations

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts

The concept of "compartment allergy": prilocaine injected into different skin layers

We herein present a patient with delayed-type allergic hypersensitivity against prilocaine leading to spreading eczematous dermatitis after subcutaneous injections for local anesthesia with prilocaine. Prilocaine allergy was proven by positive skin testing and subcutaneous provocation, whereas the evaluation of other local anesthetics - among them lidocaine, articaine and mepivacaine - did not exhibit any evidence for cross-reactivity

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations

Global Transcriptome and Deletome Profiles of Yeast Exposed to Transition Metals

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways