Transfer RNA Gene-targeted Retrotransposition of Dictyostelium TRE5-A into a Chromosomal UMP Synthase Gene Trap

Abstract

The genome of the eukaryotic microorganism Dictyostelium discoideum hosts a family of seven non-long terminal repeat retrotransposons (TREs) that show remarkable insertion preferences near tRNA genes. We developed an in vivo assay to detect tRNA gene-targeted retrotransposition of endogenous TREs in a reporter strain of D. discoideum . A tRNA gene positioned within an artificial intron was placed into the D. discoideum UMP synthase gene. This construct was inserted into the D. discoideum genome and presented as a landmark for de novo TRE insertions. We show that the tRNA gene-tagged UMP synthase gene was frequently disrupted by de novo insertions of endogenous TRE5-A copies, thus rendering the resulting mutants resistant to 5-fluoroorotic acid selection. Approximately 96% of all isolated 5-FOA-resistant clones contained TRE5-A insertions, whereas the remaining 4% resulted from transposition-independent mutations. The inserted TRE5-As showed complex structural variations and were found about 50 bp upstream of the reporter tRNA gene, similar to previously analysed genomic copies of TRE5-A. No integration by other members of the TRE family was observed. We found that only 51% of the de novo insertions were derived from autonomous TRE5-A.1 copies. The remaining 49% of new insertions were due to TRE5-A.2 elements, which lack the proteins required for reverse transcription and integration, but retain functional promoter sequences.