Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii.
We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) interferes with transcription initiation while the chimeric transactivator, composed of the tetR fused to the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency. Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled transactivator, tTA2(s), the TetR(s )modulates parasite transcription over a range of approximately 15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level.
Meissner
M
M
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69102 Heidelberg, Germany.
Brecht
S
S
Bujard
H
H
Soldati
D
D
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D024507:Nonmuscle Myosin Type IIA / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013754:Tetracyclines / Q000494:pharmacology*; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C083139:Tet O resistance protein, Bacteria; D013754:Tetracyclines; D015534:Trans-Activators; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; C016229:4-epianhydrotetracycline; D015500:Chloramphenicol O-Acetyltransferase; D024507:Nonmuscle Myosin Type IIA; D004318:Doxycycline