Systems for applied gene control in Saccharomyces cerevisiae

Maya, Douglas; Quintero, Marı́a José; Muñoz-Centeno, Mari Cruz; Chávez, Sebastián

doi:10.1007/s10529-008-9647-z

Cited by 43 publications

(32 citation statements)

References 46 publications

(52 reference statements)

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“…For this purpose, ORF P1192R was cloned in the yeast expression plasmid pYES2, under the control of the yeast GAL1 promoter. Transcription from this promoter is strongly repressed in the presence of glucose, but in the presence of galactose and in the absence of glucose expression can be up-regulated up to 2000-fold (Maya et al, 2008). This plasmid was then introduced in S. cerevisiae cells of strain JCW26 (Wasserman et al, 1993), which contain a thermosensitive mutation in TOP2 (top2 ts) that allows them to grow at 25 1C (permissive temperature), but makes Top2p non-functional at 35 1C (restrictive temperature).…”

Section: Pp1192r Is a Cytoplasmic Proteinmentioning

confidence: 99%

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

Coelho¹,

Martins²,

Ferreira³

et al. 2015

Virology

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Topoisomerases modulate the topological state of DNA during processes, such as replication and transcription, that cause overwinding and/or underwinding of the DNA. African swine fever virus (ASFV) is a nucleo-cytoplasmic double-stranded DNA virus shown to contain an OFR (P1192R) with homology to type II topoisomerases. Here we observed that pP1192R is highly conserved among ASFV isolates but dissimilar from other viral, prokaryotic or eukaryotic type II topoisomerases. In both ASFV/Ba71V-infected Vero cells and ASFV/L60-infected pig macrophages we detected pP1192R at intermediate and late phases of infection, cytoplasmically localized and accumulating in the viral factories. Finally, we used a Saccharomyces cerevisiae temperature-sensitive strain in order to demonstrate, through complementation and in vitro decatenation assays, the functionality of P1192R, which we further confirmed by mutating its predicted catalytic residue. Overall, this work strengthens the idea that P1192R constitutes a target for studying, and possibly controlling, ASFV transcription and replication.

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Section: Pp1192r Is a Cytoplasmic Proteinmentioning

confidence: 99%

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

Coelho¹,

Martins²,

Ferreira³

et al. 2015

Virology

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“…Nutritionally modulated promoters such as MET3 or GAL1 (for review, see Maya et al 2008) are not ideal for regulated gene expression studies as the changes in growth medium are likely to result in pleiotropic effects on cell metabolism. In particular, both a galactose to glucose shift and the presence or absence of methionine were reported to impact on RNA turnover in yeast (Dichtl et al 1997;Bousquet-Antonelli et al 2000).…”

Section: Introductionmentioning

confidence: 99%

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae

Alexander¹,

Barrass²,

Dichtl³

et al. 2010

RNA

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We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 39-end cleavage and polyadenylation, that is, cotranscriptionally.

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“…Moreover, if these systems mediate only an "on/off" situation, expression at a defined, user-specified level is not permitted. Therefore, tunable and metabolism-independent promoter systems have been developed for yeast, plant, insect, and mammalian systems, allowing more flexibility in experimental approaches (23,24,33,46).…”

mentioning

confidence: 99%

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger

Meyer

Wanka

Gent

et al. 2011

Appl Environ Microbiol

161

165

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Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracyclinedependent transactivator rtTA2 S -M2, and one module harbors the rtTA2 S -M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined.

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Systems for applied gene control in Saccharomyces cerevisiae

Cited by 43 publications

References 46 publications

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger

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