Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Marschalek, Rolf; Kalpaxis, Dimitrios L.; Dingermann, Theo

doi:10.1002/j.1460-2075.1990.tb08233.x

Cited by 7 publications

(6 citation statements)

References 49 publications

(41 reference statements)

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“…It was of particular interest to use mutants lacking a regulated PFK for the expression of DdPFK, as this may allow us for the first time to investigate the effect of substituting a non-allosteric PFK for an allosteric one on cell physiology. Yeasts are known to be excellent host organisms for the production of foreign proteins [13][14][15][16], including genes from D. discoideum [17][18][19]. More specifically, PFK isozymes from Escherichia coli and muscle have been successfully expressed in Saccharomyces cerevisiae [20].…”

Section: Introductionmentioning

confidence: 99%

Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

1995

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Phosphofructokinase (PFK) from yeast has been replaced by the non-allosteric isozyme from the slime mold Dictyostelium discoideum. This has been achieved by overexpression of the latter in a PFK-deficient strain of Saccharomyces cerevisiae under the control of the PFK2 promoter. Transformants complemented the glucose-negative growth phenotype exhibiting generation times on glucose-containing media similar to those of an untransformed strain being wild-type for yeast PFK genes. The PFK produced reacted with an antibody against D. discoideum PFK. It exhibited the same subunit size, quaternary structure and kinetic parameters than those of the wild-type enzyme, and was also devoid of specific regulatory properties.

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Section: Introductionmentioning

confidence: 99%

Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

1995

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“…tRNAGU(amber) gene derivatives from D.discoideum with tetO insertions in their 5'-flanking regions produce active suppressor tRNAs in vivo in yeast A tRNAGiU(CUA) suppressor gene from D.discoideutn is functional in S.cerevisiae Marschalek et al, 1990). Two derivatives of this gene, obtained after Bal31 deletion of 5'-flanking nucleotides up to positions -46 and -7, were chosen for insertion of a synthetic oligonucleotide containing the Tet operator 01 of transposon Tn 10 ( Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…Cloning of the tRNAGlU(UUC) gene from the cellular slime mould D.discoideum (Dingermann et al, 1989) and modification of the gene to an amber suppressor tRNA gene has been described (Marschalek et al, 1990). The 5'-flanking region of this gene was partially deleted by Bal3 1 digestion.…”

Section: Plasmid Dnasmentioning

confidence: 99%

“…Insertion of the tetO, operator in front of a tRNAGIUII(amber) derivative from D.discoideum. The glu2(Su CUA) gene was derived from a D.discoideum tRNAGIu(UUC) gene by primer-directed mutagenesis (Marschalek et al, 1990). The 5'-flanking region of this gene was partially deleted by Bal31 up to nucleotides -7 and -46 and was replaced by a multiple cloning site.…”

Section: Introductionmentioning

confidence: 99%

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RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

et al. 1992

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We have investigated whether the RNA polymerase III‐driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator‐repressor system. The bacterial tet operator (tetO) was inserted at two different positions (−7 and −46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8‐1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose‐‐but not on glucose‐‐containing media. Regulation of the su‐tRNA gene with the tetO fragment inserted at position −7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met‐ phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su‐tRNA gene with the tetO fragment inserted at position −46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su‐tRNA at least 50‐fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5′‐flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.

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“…Here we developed a temperature-sensitive allele of trnW UCA by investigating four different mutations predicted to affect the secondary structure of the tRNA. The first mutation was based on work by Marschalek et al in 1990 [ 36 ], who developed a temperature-sensitive version of a Dictylostelium discoideum glutamic acid tRNA and tested it in Saccharomyces cerevisiae . The authors showed that their C to U transition in the acceptor stem led to a less stable secondary structure, so the pre-tRNA could be processed to a mature tRNA at 22 °C but not at 37 °C.…”

Section: Introductionmentioning

confidence: 99%

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

Young

Purton

2018

Microb Cell Fact

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BackgroundThe chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnWUCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.ResultsA mutated version of our trnWUCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.ConclusionsCITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1033-5) contains supplementary material, which is available to authorized users.

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Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Cited by 7 publications

References 49 publications

Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

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