STUDIES ON THE TRANSFORMATION OF INTACT YEAST-CELLS BY THE LIAC/S-DNA/PEG PROCEDURE
STUDIES ON THE TRANSFORMATION OF INTACT YEAST-CELLS BY THE LIAC/S-DNA/PEG PROCEDURE
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DOI：
10.1002/yea.320110408
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发表时间：
1995-04-15
期刊：
影响因子：
2.6
通讯作者：
WOODS, RA
中科院分区：
文献类型：
--
作者：
GIETZ, RD;SCHIESTL, RH;WOODS, RA
使用改进的乙酸锂（LiAc）/单链DNA（SS-DNA）/聚乙二醇（PEG）方案（其产生>1 × 10（6）转化体/μ g质粒DNA）和Schiestl和Gietz（1989）描述的原始方案来研究LiAc/SS-DNA/PEG转化机制的方面。当在50 μ g SS载体DNA存在下用100 ng质粒DNA转化1 × 10（8）个细胞时，观察到最高的转化效率。转化体的产量线性增加，每次转化高达5 μ g质粒。在42摄氏度的20分钟热休克是必要的最大产量。发现PEG将载体DNA和质粒DNA存款沉积到细胞上。SS载体DNA更有效地结合到细胞，并导致更紧密的P-32标记的质粒DNA的结合比双链（DS）载体。LiAc/SS-DNA/PEG转化法未导致细胞融合。在转化反应中，DS载体DNA与DS载体DNA竞争。SS质粒DNA与SS和DS载体DNA的组合转化细胞较差。LiAc/SS-DNA/PEG方法被证明比已知的使细胞可转化的其他处理更有效。讨论了LiAc/SS-DNA/PEG方法转化机理的模型。
An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields >1 x 10(6) transformants/mu g plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 mu g SS carrier DNA. The yield of transformants increased linearly up to 5 mu g plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of P-32-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.