Ty1 Retrotransposition Frequency Assay Using a Chromosomal Ty1his3AI or Ty1kanMXAI Element

Background

Ty1 is a long terminal repeat (LTR) retrotransposon that is structurally and evolutionarily related to retroviruses. Retrotransposition of Ty1 occurs when Ty1 RNA is reverse transcribed within cytoplasmic virus-like particles, resulting in synthesis of a cDNA that is transported back to the nucleus and integrated into the genome of its host (Boeke et al., 1985; Garfinkel et al., 1985). The integrated Ty1 element consists two LTRs in the same orientation that flank two open reading frames: GAG, which encodes a structural protein that forms the virus-like particle and binds Ty1 RNA, and POL, which encodes three enzymatic proteins- protease, reverse transcriptase and integrase (Figure 1). Ty1 elements are the most active and abundantly transcribed of all five families of LTR-retrotransposons in S. cerevisiae. Ty1 has been shown to be regulated by hundreds of genes, several different environmental conditions and various DNA damaging agents (Curcio et al., 2015). To measure the relative frequencies of Ty1 retrotransposition in different genetic backgrounds and under different environmental conditions, a chromosomal Ty1 element under the control of the native promoter was marked with a retrotransposition indicator gene (Figure 1). A retrotransposition indicator gene is a selectable marker gene whose coding sequence is interrupted by an intron in the opposite orientation to transcription (Figure 1). When placed within the Ty1 retrotransposon such that Ty1 and the marker gene are in opposite transcriptional orientations, the intron is spliced out of the Ty1 transcript but cannot be spliced out of the marker gene transcript. When the spliced Ty1 transcript undergoes retrotransposition, however, it recreates a functional marker gene in the transposed copy of the element. The fraction of viable cells in which the selectable marker gene is detected phenotypically is the retrotransposition frequency. This assay is a modification of that first described by Curcio and Garfinkel in 1991.

The specific assay conditions provided here are for derivatives of strain BY4741 containing a chromosomal Ty1 element that is marked with the retrotransposition indicator gene, his3AI or kanMXAI (Curcio and Garfinkel, 1991, Bryk et al., 2002). Cells that sustain retrotransposition of Ty1his3AI harbor a Ty1HIS3 element and are His⁺ prototrophs (i.e., capable of growth on medium lacking histidine; Figure 1). Cells that sustain retrotransposition of Ty1kanMXAI harbor a Ty1kanMX element and are resistant to G418.


Figure 1. Genetic assay for detection of cells that sustain a retrotransposition event of the chromosomal Ty1his3AI element. The Ty1 element consists of long terminal repeats (boxed arrowheads) flanking a GAG and a POL open reading frame. The his3AI indicator gene, which consists of the HIS3 marker gene interrupted by an intron in the antisense orientation, is inserted downstream of POL. Ty1his3AI RNA is transcribed, and the intron within the HIS3 open reading frame is spliced out. The spliced RNA is then subject to reverse transcription during the process of retrotransposition. The resulting Ty1HIS3 cDNA lacks the intron and contains functional HIS3 gene sequences. Integration of the cDNA into the host genome renders the cell His⁺. (Adapted from Curcio et al., 2015)