Regulated expression of myosin II heavy chain and RacB using an inducible tRNA suppressor gene.
An inducible expression system that indirectly regulates gene expression through the use of an inducible suppressor tRNA has been used to express both endogenous and exogenous genes in Dictyostelium. The tetracycline repressor and tRNA suppressor (Glu) are expressed from a single G418 selectable vector, while a gene engineered to contain a stop codon is expressed from a separate hygromycin selectable vector. beta-Galactosidase could be induced over 300 fold with this system, and the extent of induction could be varied depending upon the amount of tetracycline added. It took 3 days to fully induce expression, and about 3 days for expression to decrease to baseline after removal of the tetracycline. Dictyostelium myosin II heavy chain could also be expressed in an inducible manner, although the induction ratio was not as high as beta-galactosidase and the maximum expression level was not as high as wild-type levels. A significant accumulation of the truncated peptide indicates that complete suppression of the stop codon was not achieved. Partial phenotypic reversion was observed in null mutants inducibly expressing myosin II. RacB could also be inducibly expressed, whereas the protein could not be expressed from a constitutive promoter, presumably because expression at high levels is lethal. Therefore, the inducible tRNA system can be used to control expression of endogenous Dictyostelium genes.
Pang
K M
KM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
Dingermann
T
T
Knecht
D A
DA
D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D016153:Genes, Suppressor; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012352:RNA, Transfer, Glu / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics; D013997:Time Factors; D001616:beta-Galactosidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D015800:Protozoan Proteins; D012352:RNA, Transfer, Glu; D001616:beta-Galactosidase; D018995:Myosin Heavy Chains; D013752:Tetracycline; D004318:Doxycycline