title: 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.
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan.
authors.forename: Tuan Anh
authors.initials: TA
authors.lastname: Pham
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kawai
authors.forename: Emi
authors.initials: E
authors.lastname: Kono
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
mesh_terms: D001583:Benzoxazoles / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism* / Q000648:ultrastructure*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D018014:Gene Transfer Techniques; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014170:Transformation, Genetic
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA