Shuttle mutagenesis: A method of transposon mutagenesis for Saccharomyces cerevisiae

H. S. Seifert, E. Y. Chen, M. So, F. Heffron

    Research output: Contribution to journalArticle

    210 Scopus citations

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)735-739
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume83
    Issue number3
    DOIs
    StatePublished - Jan 1 1986

    ASJC Scopus subject areas

    • General

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