Transposon tagging using Ty elements in yeast.

Abstract

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.

RNAi in Budding Yeast

“…5D). To examine whether RNAi can suppress retrotransposition, we ectopically expressed a Ty1 element marked with HIS3, which enabled transposition to be detected as plasmid-independent complementation of histidine auxotrophy (33). Consistent with our molecular findings for endogenous elements, the RNAi-competent strain permitted much less transposition (Fig.…”

“…Galactose-induced S. cerevisiae strains expressing the indicated S. castellii genes were tested for transposition by growth on plates with 5-FOA and lacking histidine (5-FOA-His). Cells grow without histidine and are resistant to 5-FOA when the HIS3-marked Ty1 element has transposed into the genome and the URA3-marked plasmid carrying the original HIS3-marked element has been lost (33). Also shown is growth on media selective for plasmid loss but not transposition (5-FOA).…”

Genetic Characterization of Three Distinct Mechanisms Supporting RNA-Driven DNA Repair and Modification Reveals Major Role of DNA Polymerase ζ

“…Integrated Ty overexpression strains were constructed via the delitto perfetto approach. The CORE cassette was inserted into the CAN1 locus (CM-1099 and CM-1100) and removed by PCR amplified fragments containing either pGAL1 from BDG102 plasmid or pGAL1-Ty1 from pGTyClaI plasmid, which were kindly provided by Dr. David Garfinkel (Garfinkel et al, 1988), to construct CM-1093, CM-1095, CM-1099 and CM1100, respectively. Deletion mutants derived from these strains were constructed by replacement of the open reading frame of the gene of choice with either the kanMX4, hygMX4, natMX4 and/or KlURA3.…”

The PLB2 Gene of Saccharomyces cerevisiae Confers Resistance to Lysophosphatidylcholine and Encodes a Phospholipase B/Lysophospholipase

“…Yeast Strains and Growth Conditions. The S. cereVisae strain DG338 (MatR his3∆200 ura3-167 GAL) and the closely related strain DG340 (Mata lys2 his3∆200 ura3-52 trp1) were a kind gift of David Garfinkel (See Table 1) (28). All gene deletions were created through homologous recombination with a PCR-generated fragment for targeted replacement of the wild-type allele with a deletion allele containing a selectable marker (29).…”