title: Regulation of gene expression in Neurospora crassa with a copper responsive promoter.
abstract: Precise control of gene expression is a powerful method to elucidate biological function, and protein overexpression is an important tool for industry and biochemistry. Expression of the Neurospora crassa tcu-1 gene (NCU00830), encoding a high-affinity copper transporter, is tightly controlled by copper availability. Excess copper represses, and copper depletion, via the use of a copper chelator, activates expression. The kinetics of induction and repression of tcu-1 are rapid, and the effects are long lived. We constructed a plasmid carrying the bar gene (for glufosinate selection) fused to the tcu-1 promoter. This plasmid permits the generation of DNA fragments that can direct integration of Ptcu-1 into any desired locus. We use this strategy to integrate Ptcu-1 in front of wc-1, a circadian oscillator and photoreceptor gene. The addition of excess copper to the Ptcu-1::wc-1 strain phenocopies a Δwc-1 strain, and the addition of the copper chelator, bathocuproinedisulfonic acid, phenocopies a wc-1 overexpression strain. To test whether copper repression can recapitulate the loss of viability that an essential gene knockout causes, we placed Ptcu-1 upstream of the essential gene, hpt-1. The addition of excess copper drastically reduced the growth rate as expected. Thus, this strategy will be useful to probe the biological function of any N. crassa gene through controlled expression.
authors.affiliation: Department of Biology, Texas A&M University, College Station, Texas 77843.
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Lamb
authors.forename: Justin
authors.initials: J
authors.lastname: Vickery
authors.forename: Deborah
authors.initials: D
authors.lastname: Bell-Pedersen
mesh_terms: D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D000079923:Copper Transporter 1; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D018390:Gene Targeting / Q000379:methods; D020043:Genes, Essential; D005800:Genes, Fungal; D007700:Kinetics; D009492:Neurospora crassa / Q000235:genetics*; D010618:Phenanthrolines / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000187:drug effects
chemical_list: D027682:Cation Transport Proteins; D002614:Chelating Agents; D000079923:Copper Transporter 1; D010618:Phenanthrolines; C028559:bathocuproine sulfonate; D003300:Copper
keywords: bathocuproinedisulfonic acid; copper sulfate (CuSO4); gene function; protein overexpression; regulation of essential genes