Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.
Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems. Use of procedures (i) and (ii) in combination allowed a 50-fold induction. Three different reporter ORFs (rabbit ferritin L subunit, human chorionic gonadotropin alpha subunit, and human lutropin beta subunit), in three different chromosomal contexts, responded to these procedures. The maximum rate of expression achieved was estimated at over 10(9) molecules per cell per day, which is 20% of the transformed cell's protein synthetic capacity. At these extremely high levels some of the induced products were cytotoxic.
Walden
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WE
Department of Biology, Washington University, St. Louis, MO 63130.
Daniels-McQueen
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Smith
L L
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Thach
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D000818:Animals; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003513:Cycloheximide / Q000494:pharmacology; D004274:DNA, Recombinant; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D010455:Peptides / Q000032:analysis; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism
D004274:DNA, Recombinant; D010455:Peptides; D003609:Dactinomycin; D008668:Metallothionein; D003513:Cycloheximide; D015032:Zinc