Concurrent knock‐out of at least 20 transporter genes is required to block uptake of hexoses in <i>Saccharomyces cerevisiae</i>

Wieczorke, Roman; Krampe, Stefanie; Weierstall, Thomas; Freidel, Kerstin; Hollenberg, Cornelis P.; Boles, Eckhard

doi:10.1016/s0014-5793(99)01698-1

Cited by 579 publications

(666 citation statements)

References 32 publications

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“…In previous reports in which marker recycling using CRE/loxP was successful (Delneri et al, 2000;Gü ldener et al, 1996;Wieczorke et al, 1999), there was only the short loxP sequence (34 bp) left on the chromosome after marker recycling. The pB3 PGK is 6.3 kb, and most of it is left on the chromosome after the marker has been looped out ( Figure 3C).…”

Section: Discussionmentioning

confidence: 99%

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Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Johansson¹,

Hahn‐Hägerdal²

2002

Yeast

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Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxPzeocin-loxP cassette using pCRE3. The 2m-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counterselection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.

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Section: Discussionmentioning

confidence: 99%

“…A different approach was taken when 20 hexose transporters (Wieczorke et al, 1999) were deleted consecutively using the dominant KanMX marker flanked by loxP repeats (Gü ldener et al, 1996). The CRE recombinase induces a specific recombination between the sites, removing the marker gene.…”

Section: Yeastmentioning

confidence: 99%

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Johansson¹,

Hahn‐Hägerdal²

2002

Yeast

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“…2A). In a gal2Δ strain, the family of hexose transporters [Hxt1-17p or Mal11p, Mph2p, and Mph3p (31)] are likely to be the main transporters of galactose and a likely source of ratio sensing (Fig. 3A).…”

Section: Significancementioning

confidence: 99%

“…3A). The HXT members transport glucose with various affinities (K m from ∼1 mM to 100 mM) (28,29), and some also import galactose, albeit with significantly lower affinity (K m ∼250 mM) (28); Hxt14p can even support growth on galactose in a strain where all other hexose transporters have been deleted (31). Thus, ratio sensing might result from competition between the sugars during uptake.…”

Section: Significancementioning

confidence: 99%

Galactose metabolic genes in yeast respond to a ratio of galactose and glucose

Escalante-Chong

Savir

Carroll

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

125

145

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Natural environments are filled with multiple, often competing, signals. In contrast, biological systems are often studied in "wellcontrolled" environments where only a single input is varied, potentially missing important interactions between signals. Catabolite repression of galactose by glucose is one of the best-studied eukaryotic signal integration systems. In this system, it is believed that galactose metabolic (GAL) genes are induced only when glucose levels drop below a threshold. In contrast, we show that GAL gene induction occurs at a constant external galactose:glucose ratio across a wide range of sugar concentrations. We systematically perturbed the components of the canonical galactose/glucose signaling pathways and found that these components do not account for ratio sensing. Instead we provide evidence that ratio sensing occurs upstream of the canonical signaling pathway and results from the competitive binding of the two sugars to hexose transporters. We show that a mutant that behaves as the classical model expects (i.e., cannot use galactose above a glucose threshold) has a fitness disadvantage compared with wild type. A number of common biological signaling motifs can give rise to ratio sensing, typically through negative interactions between opposing signaling molecules. We therefore suspect that this previously unidentified nutrient sensing paradigm may be common and overlooked in biology.nutrient signaling | signal integration | gene regulation | ratio sensing | yeast

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“…These clones will allow the genes to be employed in other vectors, or in PCR amplification and PCR-based gene disruption, as an alternative to established markers that make use of antibiotics, such as geneticin Gü ldener et al, 1996), hygromycin B, nourseothricin and bialaphos (Goldstein and McCusker, 1999), or heterologous markers, such as Schizosaccharomyces pombe HIS5 (which complements S. cerevisiae HIS3, Wach et al, 1997) and Kluyveromyces lactis URA3 (LangleRouault and Jacobs, 1995;Delneri et al, 1999). This could be of relevance in situations where several members of a gene family must be disrupted before a wide range of phenotypes become apparent (Reifenberger et al, 1995;Wieczorke et al, 1999;Delneri et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

A new family of yeast vectors and S288C‐derived strains for the systematic analysis of gene function

Tomlin¹,

Wixon²,

Bolotin‐Fukuhara³

et al. 2001

Yeast

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The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2D0 ade8D0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.

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Concurrent knock‐out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae

Cited by 579 publications

References 32 publications

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Galactose metabolic genes in yeast respond to a ratio of galactose and glucose

A new family of yeast vectors and S288C‐derived strains for the systematic analysis of gene function

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