Transfer RNA gene-targeted retrotransposition of Dictyostelium TRE5-A into a chromosomal UMP synthase gene trap.
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.
Beck
Peter
P
Institut für Pharmazeutische Biologie, Universität Frankfurt/M. (Biozentrum), Marie-Curie-Strasse 9 D-60439 Frankfurt am Main, Germany.
Dingermann
Theodor
T
Winckler
Thomas
T
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D018390:Gene Targeting; D017125:Genes, Protozoan; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D016053:RNA, Protozoan / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D018626:Retroelements / Q000235:genetics*
D016054:DNA, Protozoan; D009097:Multienzyme Complexes; D016053:RNA, Protozoan; D018626:Retroelements; D009963:Orotic Acid; C027157:uridine 5'-monophosphate synthase; C001242:5-fluoroorotic acid; D012343:RNA, Transfer; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase