Tetracycline regulated gene expression in Leishmania donovani.
The prokaryotic tetracycline-responsive repressor/operator system has proven to be useful for studying the function of essential genes and the expression of toxic gene products in a number of organisms, including Trypanosoma brucei. We report here the adaptation of this system for use in Leishmania. The inducible promoter construct contains a bleomycin resistance-luciferase fusion (BLE-LUC) gene driven by an rRNA promoter with two copies of the TetO sequence inserted two nucleotides upstream of the transcriptional start site. This construct showed regulation of BLE-LUC expression by two orders of magnitude when targeted into the rDNA locus in the reverse orientation relative to transcription of the rRNA genes in a Leishmania donovani cell line expressing TETR. The luciferase expression level in the absence of tetracycline was approximately 50-fold lower than that in the tubulin locus (where it is transcribed by pol II), while the expression level in the presence of tetracycline was approximately five-fold higher than that from the tubulin locus. There was no linear relationship between the level of TETR expression and the regulation, and changing of positions of operator did not increase regulation.
Yan
S
S
Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109-1651, USA.
Myler
P J
PJ
Stuart
K
K
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D002352:Carrier Proteins; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D020459:Genes, rRNA; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D012335:RNA, Ribosomal; D012097:Repressor Proteins; C083139:Tet O resistance protein, Bacteria; C034811:tetracycline resistance-encoding transposon repressor protein; D001761:Bleomycin; D008156:Luciferases; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D013752:Tetracycline