Abstract
Background. PIF/Harbinger 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 Tourist-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. The most active DNA transposon identified to date in plants or animals is mPing, a rice Tourist-like MITE that is a deletion derivative of the autonomous Ping element. Ping and the closely related Pong are the only known naturally active PIF/Harbinger elements. Some rice strains accumulate ∼40 new mPing 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 Tourist-MITEs. Results. The ORF1 and TPase proteins encoded by Ping and Pong have been shown to mobilize mPing in rice and in transgenic Arabidopsis. 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 Arabidopsis, the NES mutant protein also catalyzed higher frequencies of mPing excision from the gfp reporter gene. Our yeast assay retains key features of excision and insertion of mPing including precise excision, extended insertion sequence preference, and a requirement for two proteins that can come from either Ping or Pong or both elements. Conclusions. The yeast transposition assay provides a robust platform for analysis of the mechanism underlying transposition catalyzed by the two proteins of PIF/Harbinger elements. It recapitulates all of the features of excision and reinsertion of mPing as seen in plant systems. Furthermore, a mutation of a putative NES in the TPase increased transposition both in yeast and plants.
| Original language | English (US) |
|---|---|
| Article number | 5 |
| Journal | Mobile DNA |
| Volume | 1 |
| Issue number | 1 |
| DOIs | |
| State | Published - Feb 1 2010 |
Bibliographical note
Funding Information:We thank Guojun Yang, Jim Burnette and Eunyoung Cho for their technical assistance and advice. Our thanks are also due to the many undergraduates, including Lee B Sharpe, Lucy Fu, Catherine Bridges and the Spring 09 HHMI Dynamic Genome class, who helped with aspects of the project. We are grateful to Clay Isom and Cody Diehl for their critical review of the manuscript. This research funded by grants from the National Institute of Health, and Howard Hughes Medical Institute Professor award to SRW.