Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.
We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.
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Department of Biochemistry, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Tapping
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Capone
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D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D005786:Gene Expression Regulation / Q000187:drug effects; D016153:Genes, Suppressor; D006367:HeLa Cells; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012362:RNA, Transfer, Ser / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic
D012362:RNA, Transfer, Ser; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D004247:DNA