SHUTTLE MUTAGENESIS - A METHOD OF TRANSPOSON MUTAGENESIS FOR SACCHAROMYCES-CEREVISIAE
SHUTTLE MUTAGENESIS - A METHOD OF TRANSPOSON MUTAGENESIS FOR SACCHAROMYCES-CEREVISIAE
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DOI：
10.1073/pnas.83.3.735
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发表时间：
1986-02-01
影响因子：
11.1
通讯作者：
HEFFRON, F
中科院分区：
文献类型：
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作者：
SEIFERT, HS;CHEN, EY;HEFFRON, F
我们已经将转座子诱变的方法推广到了真核生物酿酒酵母。含有选择性酵母基因的细菌转座子可以在大肠杆菌中转座到酵母DNA的克隆片段中，转座子的插入可以通过同源重组返回到酵母基因组中。首先，将待诱变的克隆酵母DNA片段转化到含有携带转座元件的F因子衍生物的大肠杆菌中。培养物被培养成允许转座和共整合形成，并且在接合时，选择含有转座子插入的酵母序列的受体。用限制性内切酶从载体上去除酵母DNA，并将转座子插入酵母中。该过程需要最少的操作次数，并且每个接合菌落都包含一个独立的插入。我们描述了用于这一过程的12个转座子Tn3衍生物以及几个简化该方法的克隆载体。
We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.