Large-scale mutagenesis of the yeast genome using a Tn7-derived multipurpose transposon.
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 approximately 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.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA. anujk@umich.edu
Anuj
A
Kumar
Michael
M
Seringhaus
Matthew C
MC
Biery
Robert J
RJ
Sarnovsky
Lara
L
Umansky
Stacy
S
Piccirillo
Matthew
M
Heidtman
Kei-Hoi
KH
Cheung
Craig J
CJ
Dobry
Mark B
MB
Gerstein
Nancy L
NL
Craig
Michael
M
Snyder
D000595:Amino Acid Sequence; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology; D017931:DNA Primers; D004251:DNA Transposable Elements; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics
D017931:DNA Primers; D004251:DNA Transposable Elements; D002945:Cisplatin