Regulation of Gene Expression inNeurospora crassawith 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.

TheNeurosporaTranscription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

“…P tcu-1 -driven expression is activated by BCS (bathocuproinedisulfonic acid), a copper chelator, and repressed by high copper concentrations (Lamb et al 2013). Expression of adv-1 or control gene ef-1α was not dependent on the BCS/Cu concentrations in a WT cell as expected (Figure 2D).…”

“…To generate this strain, primers Pvu I adv-1 F and Pvu I/ Spe I adv-1 R (Supplemental Material, Table S1) were used to amplify the full-length adv-1 gene from WT N. crassa genomic DNA. This DNA fragment was digested with Pvu I and then cloned into the corresponding sites of pCR blunt bar::P tcu-1 (Lamb et al 2013), to generate pDBP506. Digestion of pDBP506 with Spe I yielded a 4.9 kb bar-P tcu-1 -adv-1 fragment that was ligated into Spe I cut pBM61 ( his-3 targeting vector) to generate pDBP508.…”

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

“…Because no enzymatic function of SO is known, we repressed the expression of the so gene by putting it under control of the copper repressible promoter Ptcu-1 (17). Full repression resulted in Δso-like phenotypes, whereas partial repression again produced Δerg-2-like deficiencies (Fig.…”

Neurospora crassa: Looking back and looking forward at a model microbe

“…As DNA manipulation and sequencing technology improved, the Neurospora community kept pace with identifying key insights from the genome, including identification of the 5S RNA genes (Free et al, 1979), characterization of telomeric sequences (Schechtman, 1987), and identification of coding sequences of many genes via expressed sequence tag (EST) libraries (Nelson et al, 1997), and followed by whole genome sequencing of N. crassa (Galagan et al, 2003), a first among filamentous fungal species. In recent years, molecular tools have been developed for targeted gene disruption and integration of recombinant DNA (Ninomiya et al, 2004), inducible and constitutive promoters (McNally and Free, 1988; Kupper et al, 1990; Campbell et al, 1994; Colot et al, 2006; Hurley et al, 2012; Lamb et al, 2013), and selectable markers (Orbach et al, 1986; Avalos et al, 1989; Staben et al, 1989; Austin et al, 1990; Yamashiro et al, 1992; Colot et al, 2006). These tools enabled production of the whole genome deletion library, which is an invaluable genetic resource to the Neurospora community (Colot et al, 2006; Dunlap et al, 2007), and has facilitated protein expression and thus protein biochemical studies (Honda and Selker, 2009).…”