A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast

Mannhaupt, Gertrud; Pilz, Ursula; Feldmann, Horst

doi:10.1016/0378-1119(88)90405-2

Cited by 17 publications

(11 citation statements)

References 21 publications

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“…In order to facilitate the expression analysis, a plasmid in which the promoter region of GIT1 was fused to the bacterial cat reporter gene in vector yCp7-32cat was constructed (13). CAT activity was determined for wild-type, ino1⌬, pho86⌬, and ino1⌬ pho86⌬ strains which were grown initially in high-P i , I ϩ medium and then transferred to four different media which varied in inositol and phosphate concentrations (Fig.…”

Section: Vol 2 2003mentioning

confidence: 99%

Inositol and Phosphate Regulate GIT1 Transcription and Glycerophosphoinositol Incorporation in Saccharomyces cerevisiae

et al. 2003

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Glycerophosphoinositol is produced through deacylation of the essential phospholipid phosphatidylinositol. In Saccharomyces cerevisiae, the glycerophosphoinositol produced is excreted from the cell but is recycled for phosphatidylinositol synthesis when inositol is limiting. To be recycled, glycerophosphoinositol enters the cell through the permease encoded by GIT1. The transport of exogenous glycerophosphoinositol through Git1p is sufficiently robust to support the growth of an inositol auxotroph (ino1⌬). We now report that S. cerevisiae also uses exogenous phosphatidylinositol as an inositol source. Evidence suggests that phosphatidylinositol is deacylated to glycerophosphoinositol extracellularly before being transported across the plasma membrane by Git1p. A genetic screen identified Pho86p, which is required for targeting of the major phosphate transporter (Pho84p) to the plasma membrane, as affecting the utilization of phosphatidylinositol and glycerophosphoinositol. Deletion of PHO86 in an ino1⌬ strain resulted in faster growth when either phosphatidylinositol or glycerophosphoinositol was supplied as the sole inositol source. The incorporation of radiolabeled glycerophosphoinositol into an ino1⌬ pho86⌬ mutant was higher than that into wild-type, ino1⌬, and pho86⌬ strains. All strains accumulated the most GIT1 transcript when incubated in media limited for inositol and phosphate in combination. However, the ino1⌬ pho86⌬ mutant accumulated approximately threefold more GIT1 transcript than did the other strains when incubated in inositol-free media containing either high or low concentrations of P i . Deletion of PHO4 abolished GIT1 transcription in a wild-type strain. These results indicate that the transport of glycerophosphoinositol by Git1p is regulated by factors affecting both inositol and phosphate availabilities and suggest a regulatory connection between phosphate metabolism and phospholipid metabolism.

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Section: Vol 2 2003mentioning

confidence: 99%

Inositol and Phosphate Regulate GIT1 Transcription and Glycerophosphoinositol Incorporation in Saccharomyces cerevisiae

et al. 2003

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“…The functional expression of the cat gene placed under the control of a yeast promoter was verified by using the regulatory region from the PH05 gene in yeast. It was demonstrated that in this construct the cat expression was induced in phosphate-free medium in the same manner as observed for the endogenous PH05 gene (13). Different detection procedures have been described for CAT (for references see (14)), among which the fluor diffusion CAT assay is widely accepted (14,15).…”

mentioning

confidence: 94%

“…This activity is often encoded within transposons or plasmids (11,12) that make bacteria resistant to chloramphenicol, a potent inhibitor of translational elongation in prokaryotic cells. A number of yeast shuttle vectors have been constructed that use cat isolated from the transposon Tncam204 or Tn9 as the reporter gene (2,13). The functional expression of the cat gene placed under the control of a yeast promoter was verified by using the regulatory region from the PH05 gene in yeast.…”

mentioning

confidence: 99%

Quantitative Aspects of the Use of Bacterial Chloramphenicol Acetyltransferase as a Reporter System in the YeastSaccharomyces cerevisiae

Alipour

Eriksson

Norbeck

et al. 1999

Analytical Biochemistry

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“…A variety of intracellular reporter proteins are available for use in mammalian cells such as (i) specific enzymes (bgalactosidase, thymidine kinase or chloramphenicol acetyltransferase) Gorman et al, 1982;Mannhaupt et al, 1988;Searle et al, 1985), (ii) fluorescent reporter proteins represented by the pioneering green fluorescent protein (e.g. GFP, YFP or RFP) (Hadjantonakis et al, 2003;Yang et al, 2000), or (iii) bioluminescent proteins of the luciferase type (de Wet et al, 1985;Lorenz et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Fluri

Kelm

Lesage

et al. 2007

Biotech & Bioengineering

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Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.

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A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast

Cited by 17 publications

References 21 publications

Inositol and Phosphate Regulate GIT1 Transcription and Glycerophosphoinositol Incorporation in Saccharomyces cerevisiae

Inositol and Phosphate Regulate GIT1 Transcription and Glycerophosphoinositol Incorporation in Saccharomyces cerevisiae

Quantitative Aspects of the Use of Bacterial Chloramphenicol Acetyltransferase as a Reporter System in the YeastSaccharomyces cerevisiae

InXy and SeXy, compact heterologous reporter proteins for mammalian cells

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