Single-step selection for Ty1 element retrotransposition.

Abstract

The yeast retrotransposon Ty) has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty) element such that the intron is on the sense strand of the Ty) element; therefore, splicing and retrotransposition of marked Tyl transcripts can give rise to His' cells. Fusion of the Tyl-H3mHIS3AI element to the inducible GAL] promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymfIIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty) transposition rate to be between 3 x 10-7 and 1 x 10-5 transpositions per Ty) element per generation.Variations in the transposition rate of individual Ty) elements are correlated with the relative abundance of their transcripts.

T-body formation precedes virus-like particle maturation inS. cerevisiae

“…To be able to follow the distribution of specific Ty1 RNA variants, we employed wild type (wt) S. cerevisiae strains of the BY background transformed with the galactose-inducible Ty1H3his3AI element (hereafter denoted tagTy1), widely used as a tool to study the biology of Ty1 and other viruses 12,13 (Fig. 1A).…”

“…In agreement with this, while the transposition level of endogenous Ty1 elements are very low, 26 ectopic expression of Ty1 stimulates T-body formation as well as VLP accumulation and transposition efficiency. 2,12,26 Alternatively, or in addition to this, VLP assembly into T-bodies may restrict the actions of host VLP destabilizing factors, such as Fus3p. 27 In this scenario, the exit of mature VLPs from the T-bodies, or the dissolution of T-bodies altogether, may be required for the release of the preintegration complex (Int plus …”

“…Individual Ty1 copies vary with respect to sequence, expression level and transposition activity, 3,10,11 making precise analysis of Ty1 biology rather cumbersome. In this paper, we use the PgalTy1H3 his3AI element 12 as a model system to show that an undisrupted TYA ORF is both necessary and sufficient to form T-bodies. Our data support a role of the T-body in the initial assembly of immature VLPs during the transposition cycle.…”

Host genome surveillance for retrotransposons by transposon-derived proteins

“…Quantitative PCR (qPCR) expression analysis was done using a two-step real-time RT-PCR. Mobility of a Tf1 integrant was assessed using Tf1-neo strains carrying an artificial intron (AI) 48,49 inserted in the opposite orientation of neo, thereby inactivating neo. Retrotransposition rate was determined by fluctuation analysis 50 …”

Isw2 regulates gene silencing at the ribosomal DNA locus in Saccharomyces cerevisiae

“…When rDNA silencing is disrupted, Ty1his3AI-236 is transcribed by Pol II leading to a higher rate of retrotransposition. Ty1his3AI-236 contains a modified his3 allele that, after retrotransposition, can be converted to a functional HIS3 gene [32]. Simple plate assays and quantitative experiments measuring His + colony formation provide an indication of the rate of retrotransposition of the genetically marked Ty1 element.…”