Transposition of the <it>Tourist</it>-MITE <it>mPing </it>in yeast: an assay that retains key features of catalysis by the class 2 <it>PIF/Harbinger </it>superfamily

• Main Authors: Hancock C Nathan, Zhang Feng, Wessler Susan R
• Format: Article
• Language: English
• Published: BMC 2010-02-01
• Series: Mobile DNA
• Online Access: http://www.mobilednajournal.com/content/1/1/5

<p>Abstract</p> <p>Background</p> <p><it>PIF/Harbinger </it>is the most recently discovered DNA transposon superfamily and is now known to populate genomes from fungi to plants to animals. Mobilization of superfamily members requires two separate element-encoded proteins (ORF1 and TPase). Members of this superfamily also mobilize <it>Tourist</it>-like miniature inverted repeat transposable elements (MITEs), which are the most abundant transposable elements associated with the genes of plants, especially the cereal grasses. The phylogenetic analysis of many plant genomes indicates that MITEs can amplify rapidly from one or a few elements to hundreds or thousands.</p> <p>The most active DNA transposon identified to date in plants or animals is <it>mPing</it>, a rice <it>Tourist</it>-like MITE that is a deletion derivative of the autonomous <it>Ping </it>element. <it>Ping </it>and the closely related <it>Pong </it>are the only known naturally active <it>PIF/Harbinger </it>elements. Some rice strains accumulate ~40 new <it>mPing </it>insertions per plant per generation. In this study we report the development of a yeast transposition assay as a first step in deciphering the mechanism underlying the amplification of <it>Tourist</it>-MITEs.</p> <p>Results</p> <p>The ORF1 and TPase proteins encoded by <it>Ping </it>and <it>Pong </it>have been shown to mobilize <it>mPing </it>in rice and in transgenic <it>Arabidopsis</it>. Initial tests of the native proteins in a yeast assay resulted in very low transposition. Significantly higher activities were obtained by mutation of a putative nuclear export signal (NES) in the TPase that increased the amount of TPase in the nucleus. When introduced into <it>Arabidopsis</it>, the NES mutant protein also catalyzed higher frequencies of <it>mPing </it>excision from the <it>gfp </it>reporter gene. Our yeast assay retains key features of excision and insertion of <it>mPing </it>including precise excision, extended insertion sequence preference, and a requirement for two proteins that can come from either <it>Ping </it>or <it>Pong </it>or both elements.</p> <p>Conclusions</p> <p>The yeast transposition assay provides a robust platform for analysis of the mechanism underlying transposition catalyzed by the two proteins of <it>PIF/Harbinger </it>elements. It recapitulates all of the features of excision and reinsertion of <it>mPing </it>as seen in plant systems. Furthermore, a mutation of a putative NES in the TPase increased transposition both in yeast and plants.</p>