Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol.
The effects of Li(+) and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li(+) could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li(+) to protoplasts. In the present work, the effects of Li(+) and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li(+) could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.
Chen
Ping
P
College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China.
Liu
Hui-Hui
HH
Cui
Ran
R
Zhang
Zhi-Ling
ZL
Pang
Dai-Wen
DW
Xie
Zhi-Xiong
ZX
Zheng
Hu-Zhi
HZ
Lu
Zhe-Xue
ZX
Tong
Hua
H
D002412:Cations / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D008094:Lithium / Q000737:chemistry / Q000494:pharmacology*; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011092:Polyethylene Glycols / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure*; D014162:Transfection / Q000379:methods*
D002412:Cations; D011092:Polyethylene Glycols; D004247:DNA; D008094:Lithium