Searching for promoter activity in RIME/Ingi retrotransposons from Trypanosoma brucei: binding of a nuclear protein to their 5' extremity.
In Trypanosoma brucei only two promoters for protein-encoding genes have been characterized so far. The RIME and Ingi elements of T. brucei are similar in structure to the non-long terminal repeat retrotransposons. Internal promoters usually located at their 5' end drive transcription of several of the latter elements. During a search for promoter activity within RIME and Ingi we focused on a region at the 5' end of both elements, which we termed rime5. A 50 kDa nuclear protein was found to specifically bind to the double strand and single strand sense of rime5 DNA. However, constructs containing several rime5 fragments inserted upstream of a chloramphenicol acetyltransferase (CAT) reporter gene failed to promote both transcription and expression of this gene in transient transfection assays. Finally, we have analyzed the expression of the Ingi elements and despite the high level of transcripts detectable in the cytoplasm, antibodies raised against two different domains of the single open reading frame did not detect any component in total extracts from T. brucei, suggesting that few Ingi copies, if any, are actually active.
García-Salcedo
José A
JA
Laboratory of Molecular Parasitology, IBMM, University of Brussels, 12 Rue des Professeurs Jeener et Brachet, Gosselies B-6041, Belgium. jantonio@dbm.ulb.ac.be
Gijón
Purificación
P
Amiguet-Vercher
Amelia
A
Pays
Etienne
E
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D015800:Protozoan Proteins / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014370:Tsetse Flies
D016054:DNA, Protozoan; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D018626:Retroelements; D015500:Chloramphenicol O-Acetyltransferase