Transient expression mediated by the Trypanosoma cruzi rRNA promoter.
Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3' trans-splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3' splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3' splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond 23298, USA.
R E
RE
Tyler-Cross
S L
SL
Short
L M
LM
Floeter-Winter
G A
GA
Buck
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016830:Kinetoplastida / Q000235:genetics; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000378:metabolism; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000235:genetics*
D015800:Protozoan Proteins; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases