High-efficiency electroporation by freezing intact yeast cells with addition of calcium

Suga, Minoru; Hatakeyama, Tatsuko

doi:10.1007/s00294-003-0385-4

Cited by 39 publications

(29 citation statements)

References 36 publications

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“…The different versions of V5-clr2 were amplified from the pREP41PkN (V5) vector by PCR using primers D80 and D81. The PCR products were then introduced into strain PJ1085 ( clr2::ura4 + ) by electroporation using a Bio-Rad gene pulser (Bio-Rad) according to [33]. Cells were plated onto YEA plates and after an overnight incubation replica plated onto Fluoro-orotic acid (FOA) plates.…”

Section: Methodsmentioning

confidence: 99%

Silencing Motifs in the Clr2 Protein from Fission Yeast, Schizosaccharomyces pombe

et al. 2014

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The fission yeast, Schizosaccharomyces pombe, is a well-established model for heterochromatin formation, but the exact sequence of events for initiation remains to be elucidated. The essential factors involved include RNA transcribed from repeated sequences together with the methyltransferase Clr4. In addition, histone deacetylases, like Clr3, found in the SHREC complex are also necessary for transcriptional silencing. Clr2 is another crucial factor required for heterochromatin formation found in the SHREC complex. The function of Clr2 has been difficult to establish due to the lack of conserved domains or homology to proteins of known molecular function. Using a bioinformatics approach, three conserved motifs in Clr2 were identified, which contained amino acids important for transcriptional repression. Analysis of clr2 mutant strains revealed a major role for Clr2 in mating-type and rDNA silencing, and weaker effects on centromeric silencing. The effect on mating-type silencing showed variegation in several of the strains with mutated versions of Clr2 indicating an establishment or maintenance defect. Moreover, the critical amino acids in Clr2 were also necessary for transcriptional repression in a minimal system, by the tethering of Clr4 upstream of a reporter gene, inserted into the euchromatic part of the genome. Finally, in silico modeling suggested that the mutations in Clr2 cause disruption of secondary structures in the Clr2 protein. Identification of these critical amino acids in the protein provides a useful tool to explore the molecular mechanism behind the role of Clr2 in heterochromatin formation.

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Section: Methodsmentioning

confidence: 99%

Silencing Motifs in the Clr2 Protein from Fission Yeast, Schizosaccharomyces pombe

et al. 2014

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“…Electrocompetent cells of the reporter strain were prepared following a protocol based on the methods described by Suga and Hatakeyama [58], [59]. Yeast cells were grown overnight in YPDA liquid medium (20 g/L Difco peptone, 10 g/L yeast extract, 0.009% adenine hemisulphate).…”

Section: Methodsmentioning

confidence: 99%

Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro

Chow

Shahravan

et al. 2008

PLoS ONE

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Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

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“…S. cerevisiae strain CENPK2-1D (MATα; his3D1; leu2-3_112; ura3-52; trp1-289; MAL2-8c; SUC2) was used for testing the citrate transporter candidates. Preparation of CENPK2-1D yeast electro-competent cells and transformation was performed according to Suga and Hatakeyama [35]. Yeast transformed cells were selected in synthetic media (SD) with 2% (w/v) dextrose, 0.67% (w/v) Yeast Nitrogen Base without amino acids (BD), 0.14% (w/v) Yeast Synthetic Drop-out Medium supplement without uracil, tryptophan, histidine and leucine (Sigma-Aldrich), 0.0076% (w/v) histidine, 0.0076% (w/v) tryptophan and 0.038% (w/v) leucine.…”

Section: Methodsmentioning

confidence: 99%

Aspergillus nigercitrate exporter revealed by comparison of two alternative citrate producing conditions

Odoni

Laothanachareon

Vázquez‐Vilar

et al. 2018

Preprint

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23Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate 24 biosynthesis and secretion, although it is amongst the most studied biotechnological production 25 processes. Carbon excess relative to various other medium constituents is key, but the complex 26 interplay between the limiting factors required for extracellular citrate accumulation remains 27 elusive. It is thought that one of the industrial bottlenecks for citrate production is citrate export, 28 however, no A. niger citrate exporter has yet been identified. Here, we show that the phenotype 29 of increased extracellular citrate accumulation can have fundamentally different underlying 30 mechanisms, depending on how this response is triggered, and that combining gene expression 31 analyses of the different conditions can lead to the compilation of a shortlist of the most 32 promising citrate exporter candidates. Specifically, we found that varying the amount and type 33 of supplement of an arginine auxotrophic A. niger strain shows down-regulation of citrate 34 metabolising enzymes in the condition in which more citrate is accumulated extracellularly. 35This contrasts with the transcriptional adaptations triggered by iron limitation, which also 36 induces increased A. niger citrate production. By combining data obtained from these two 37 manners of inducing comparatively high extracellular citrate accumulation, we were able to 38 compile a shortlist of the most likely citrate transporter candidates. Two of the most promising 39 candidates were tested in the yeast Saccharomyces cerevisiae, one of which showed the ability 40 to secrete citrate. Deletion of the endogenous A. niger gene encoding the corresponding 41 transporter abolished the ability of this fungus to secrete citrate. Instead, under conditions that 42 usually favour A. niger citrate production, we found increased accumulation of extracellular 43 oxalate. Our findings provide steps in untangling the complex interplay of different 44 mechanisms underlying A. niger citrate accumulation, and we identify, for the first time, a 45 3 fungal citrate exporter, offering a valuable tool for improvement of A. niger as biotechnological 46 cell-factory for organic acid production. 47 Author Summary 48Citrate is widely applied as acidifier, flavouring and chelating agent. Industrial citrate 49 production currently relies on the filamentous fungus Aspergillus niger. Although the industrial 50 production process using A. niger has vastly improved since initiated almost 100 years ago, 51 citrate export remains a bottleneck. Here, we studied the gene expression pattern of A. niger 52 under various citrate producing conditions. Using these expression patterns and different 53 computational approaches, we compiled a shortlist of putative citrate exporter candidates. In 54 this way, we were able to identify a gene encoding a transporter protein capable of citrate 55 export. We show that the yeast Saccharomyces cerevisiae, normally a citrate non-producer, 56 secretes detectab...

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High-efficiency electroporation by freezing intact yeast cells with addition of calcium

Cited by 39 publications

References 36 publications

Silencing Motifs in the Clr2 Protein from Fission Yeast, Schizosaccharomyces pombe

Silencing Motifs in the Clr2 Protein from Fission Yeast, Schizosaccharomyces pombe

Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro

Aspergillus nigercitrate exporter revealed by comparison of two alternative citrate producing conditions

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