The Role of Cell Wall Revealed by the Visualization of Saccharomyces cerevisiae Transformation

Abstract

Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.

Modified Cre- loxP Recombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue

“…Using fluorescence microscopy and scanning electron microscopy of S. cerevisiae, Murata et al recently reported that DNA targeted for transformation enters to the cell through endocytotic membrane invagination (37). Moreover, PEG mediates the binding of negatively charged DNA to the negatively charged cell surface (5,12,21).…”

Ecologically Driven Competence for Exogenous DNA Uptake in Yeast

“…Accordingly, the earlier reports of cation-and/or cation/heat shock-aided transformation in yeast [7,40] may also represent cases [8,63,87]. Proper DNA attachment to the cell surface seems ubiquitous in overall competence (except biolistic and mediated) since DNA in solution cannot enter the cell unless adhered to its surface first [64]. This shows that if properly attached to the surface, the exogenous DNA needs little or no other outer factor(s) to transform the yeast cell since competence is possible solely by PEG [25].…”

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

“…This clearly suggests that some labeled internalized DNA seems to co-localize to the endosome compartment labeled with GFP. Pham et al ( 2011a ) showed that the 42°C heat shock was important only to intact cell transformation as spheroplasts did not respond to it. These authors also used negatively charged Nanogold particles in an attempt to study the uptake of DNA into the cell.…”