Transcriptional and metabolic response in yeast Saccharomyces cerevisiae cells during polyethylene glycol‐dependent transformation

Abstract

Intact cells of the yeast Saccharomyces cerevisiae were transformed with exogenous DNA by incubating the cells with plasmid in the presence of polyethylene glycol (PEG), which has been shown to be required, although the underlying has not been elucidated. In this study, we found that incubation of the S. cerevisiae cells with PEG was not only required for the PEG-dependent transformation but also enhanced transformation, suggesting that PEG might cause an intracellular response. To understand the response, microarray and metabolome analyses were conducted. We found that incubation of the cells without PEG caused up-regulation of several genes, including those which are involved in carbon source metabolisms, e.g. fatty acid metabolism, yielding acetyl-CoA and those involved in stress-response. Contrary to this, incubation of the cells with PEG gave no transcriptional change. These microarray data were supported by the results of metabolome analysis for anionic metabolites, implying that the physical effect of PEG on cell membrane, rather than the effect of PEG itself on the intracellular response, could cause high transformation in the PEG-dependent transformation.

Transformation ofSaccharomyces cerevisiaeand other fungi

“…14,15 As described later in this review, we attempted to clarify the mechanism underlying such transformation. 35,36 electroporation. Electroporation was first used to transform intact S. cerevisiae cells by Hashimoto et al 3 Delorme 16 attempted to establish the optimal conditions for electroporation, by suspending intact S. cerevisiae cells in YPD (yeast extract, bactopeptone and dextrose) medium, performing electroporation and plating them directly on selective medium.…”

“…The mechanism underlying transformation has not been clarified completely even in S. cerevisiae, although it has been proposed by several recent studies. [35][36][37][38] In 2001, Gietz and Woods 39 reviewed the methods of fungal transformation and discussed the mechanisms involved, in the context of S. cerevisiae. However, several improvements have been achieved since then.…”

“…The basic protocols of S. cerevisiae transformation are also provided. We also present a model of the mechanism underlying S. cerevisiae transformation, based on the recent reports [35][36][37][38] and the mechanism underlying transfection in the mammalian system. Although methods requiring special equipments (e.g., electroporation and the biolistic method) are described, we focus on those not requiring special equipments, by concentrating on biological components and events.…”

Ecologically Driven Competence for Exogenous DNA Uptake in Yeast

“…There is additional ambiguous genetic contribution that is classified here as (iii) miscellaneous (genes GSH1, LAP4, TPO2, YGR071C, YNR061C, YDR119 W, NCE103, ATG8, ATX2, SBE2, AXL2, MUB1, YBR267 W, GIS4, YER113C, NSR1; [25,37] ). All these genes were attributed to be important in eukaryotic competence on the basis of three standardized transformation approaches representing different types of EDEC (see Table 1): PEG/Heat shock [25,36,63,82], the so-called yeast transfection [54,66], and AMT [75].…”

High Efficiency DNA Transformation of Saccharomyces cerevisiae with the LiAc/SS-DNA/PEG Method

“…(Gietz RD, unpublished results) to enhance the transformation effi ciency by increasing the permeability of intact cells. However, PEG treatment caused no intracellular response (Kawai et al 2009 ). The model proposes that DNA attaches to the cell wall, passes through and subsequently transits the cell membrane by endocytotic membrane invagination.…”