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Large-Scale Mutagenesis of the Yeast Genome Using a Tn7-Derived Multipurpose Transposon

¹ Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA ² Department of Molecular, Cellular, and Developmental Biology and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216 ³ Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA ⁴ Center for Medical Informatics, Department of Anesthesiology, Yale University, School of Medicine, New Haven, Connecticut 06510, USA

We present here an unbiased and extremely versatile insertional library of yeast genomic DNA generated by in vitro mutagenesis with a multipurpose element derived from the bacterial transposon Tn7. This mini-Tn7 element has been engineered such that a single insertion can be used to generate a lacZ fusion, gene disruption, and epitope-tagged gene product. Using this transposon, we generated a plasmid-based library of 300,000 mutant alleles; by high-throughput screening in yeast, we identified and sequenced 9032 insertions affecting 2613 genes (45% of the genome). From analysis of 7176 insertions, we found little bias in Tn7 target-site selection in vitro. In contrast, we also sequenced 10,174 Tn3 insertions and found a markedly stronger preference for an AT-rich 5-base pair target sequence. We further screened 1327 insertion alleles in yeast for hypersensitivity to the chemotherapeutic cisplatin. Fifty-one genes were identified, including four functionally uncharacterized genes and 25 genes involved in DNA repair, replication, transcription, and chromatin structure. In total, the collection reported here constitutes the largest plasmid-based set of sequenced yeast mutant alleles to date and, as such, should be singularly useful for gene and genome-wide functional analysis.

[Supplemental material is available online at www.genome.org.]

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2875304.

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