Bruschi C V, Comer A R, Howe G A
Department of Microbiology and Immunology, School of Medicine, East Carolina University, Greenville, North Carolina 27858-4354.
Yeast. 1987 Jun;3(2):131-7. doi: 10.1002/yea.320030209.
We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake. Two new aspects of the process have been investigated in order to evaluate its specificity. Such aspects are: (i) effect of monovalent vs. divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake. We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension. This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs. divalent cations. While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG. In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.
我们研究了酿酒酵母全细胞DNA转化的机制，特别强调细胞壁复合物在DNA摄取中的作用。为了评估该过程的特异性，对该过程的两个新方面进行了研究。这些方面是：(i) 与转化DNA孵育期间单价阳离子与二价阳离子的作用，以及 (ii) DNA吸附和摄取的时间。我们发现阳离子需求的特异性是一种菌株依赖性特征，受细胞悬浮液中转化DNA的存在影响。这一发现得到了几个实验室报告的支持，即一些酵母菌株在单价阳离子与二价阳离子之间表现出相互排斥的可转化性。虽然热休克和聚乙二醇 (PEG) 都能诱导质粒DNA分子的不可逆吸附，但DNA摄取似乎仅在去除PEG后才会发生。在这项研究过程中，我们开发了一种用氯化钙进行全细胞DNA转化的新的替代方法，该方法能够转化对其他方法无反应的菌株。
