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Regulation of gene expression in Neurospora crassa with a copper responsive promoter.

Lamb TM, Vickery J, Bell-Pedersen D - G3 (Bethesda) (2013)

Bottom Line: The kinetics of induction and repression of tcu-1 are rapid, and the effects are long lived.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, Texas 77843.

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.

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Effects of Ptcu-1-controlled wc-1 on circadian rhythms. (A) Pwc-1wc-1 and Ptcu-1wc-1 strains (denoted here, Pwc-1 and Ptcu-1) were inoculated on race tubes containing the indicated concentrations of CuSO4 (Cu in µM) and BCS (BCS in µM), grown in LL for 24 hr at 30°, and then shifted to DD at 25°. The solid black line on the left marks the growth front at the time of the shift. Every 24 hr the growth front was marked under safe red lights (smaller tick marks). Two independent tubes are shown for each strain and condition. (B) The period of the conidiation rhythm vs. the Cu/BCS levels is plotted for Pwc-1wc-1 and Ptcu-1wc-1 strains. Each data point is the average period calculated from at least three race tubes (±SEM). (C) Percent of growth that is linear hyphae (and not aerial hyphae/conidia) is plotted vs. the Cu/BCS levels for Pwc-1wc-1 and Ptcu-1wc-1 strains.
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fig5: Effects of Ptcu-1-controlled wc-1 on circadian rhythms. (A) Pwc-1wc-1 and Ptcu-1wc-1 strains (denoted here, Pwc-1 and Ptcu-1) were inoculated on race tubes containing the indicated concentrations of CuSO4 (Cu in µM) and BCS (BCS in µM), grown in LL for 24 hr at 30°, and then shifted to DD at 25°. The solid black line on the left marks the growth front at the time of the shift. Every 24 hr the growth front was marked under safe red lights (smaller tick marks). Two independent tubes are shown for each strain and condition. (B) The period of the conidiation rhythm vs. the Cu/BCS levels is plotted for Pwc-1wc-1 and Ptcu-1wc-1 strains. Each data point is the average period calculated from at least three race tubes (±SEM). (C) Percent of growth that is linear hyphae (and not aerial hyphae/conidia) is plotted vs. the Cu/BCS levels for Pwc-1wc-1 and Ptcu-1wc-1 strains.

Mentions: Finally, to assess growth and rhythmicity, Pwc-1wc-1 and Ptcu-1wc-1 strains were inoculated on race tubes containing 200, 100, and 50 µM final CuSO4, or 50 µM CuSO4 with 10, 25, 50, 100, and 200 µM BCS (Figure 5A). Growth rates of both strains were only very mildly affected by changes in the availability of CuSO4 (Figure S5), suggesting that copper toxicity or starvation is not achieved under these conditions. The Pwc-1wc-1 strain grew ~50% of the time as linear hyphae compared with conidiating aerial hyphae, and developed bands of conidia with a period of ~22.3 hr independent of copper availability (Figure 5, B and C). In high copper, the Ptcu-1wc-1 strain was largely arrhythmic with conidiation predominating (only ~10% of the growth was linear hyphae), and had a long period, when a period could be measured (~24.5 hr), as expected for strains with reduced or no expression of WC-1 (Lee et al. 2003). As copper was reduced and BCS increased, normal rhythmicity was restored at 50 µM CuSO4 with or without 10 µM BCS. Above this level of BCS, rhythmicity became unreliable, and the oscillations accelerated (shorter period), as expected for strains overexpressing WC-1 (Cheng et al. 2001).


Regulation of gene expression in Neurospora crassa with a copper responsive promoter.

Lamb TM, Vickery J, Bell-Pedersen D - G3 (Bethesda) (2013)

Effects of Ptcu-1-controlled wc-1 on circadian rhythms. (A) Pwc-1wc-1 and Ptcu-1wc-1 strains (denoted here, Pwc-1 and Ptcu-1) were inoculated on race tubes containing the indicated concentrations of CuSO4 (Cu in µM) and BCS (BCS in µM), grown in LL for 24 hr at 30°, and then shifted to DD at 25°. The solid black line on the left marks the growth front at the time of the shift. Every 24 hr the growth front was marked under safe red lights (smaller tick marks). Two independent tubes are shown for each strain and condition. (B) The period of the conidiation rhythm vs. the Cu/BCS levels is plotted for Pwc-1wc-1 and Ptcu-1wc-1 strains. Each data point is the average period calculated from at least three race tubes (±SEM). (C) Percent of growth that is linear hyphae (and not aerial hyphae/conidia) is plotted vs. the Cu/BCS levels for Pwc-1wc-1 and Ptcu-1wc-1 strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852388&req=5

fig5: Effects of Ptcu-1-controlled wc-1 on circadian rhythms. (A) Pwc-1wc-1 and Ptcu-1wc-1 strains (denoted here, Pwc-1 and Ptcu-1) were inoculated on race tubes containing the indicated concentrations of CuSO4 (Cu in µM) and BCS (BCS in µM), grown in LL for 24 hr at 30°, and then shifted to DD at 25°. The solid black line on the left marks the growth front at the time of the shift. Every 24 hr the growth front was marked under safe red lights (smaller tick marks). Two independent tubes are shown for each strain and condition. (B) The period of the conidiation rhythm vs. the Cu/BCS levels is plotted for Pwc-1wc-1 and Ptcu-1wc-1 strains. Each data point is the average period calculated from at least three race tubes (±SEM). (C) Percent of growth that is linear hyphae (and not aerial hyphae/conidia) is plotted vs. the Cu/BCS levels for Pwc-1wc-1 and Ptcu-1wc-1 strains.
Mentions: Finally, to assess growth and rhythmicity, Pwc-1wc-1 and Ptcu-1wc-1 strains were inoculated on race tubes containing 200, 100, and 50 µM final CuSO4, or 50 µM CuSO4 with 10, 25, 50, 100, and 200 µM BCS (Figure 5A). Growth rates of both strains were only very mildly affected by changes in the availability of CuSO4 (Figure S5), suggesting that copper toxicity or starvation is not achieved under these conditions. The Pwc-1wc-1 strain grew ~50% of the time as linear hyphae compared with conidiating aerial hyphae, and developed bands of conidia with a period of ~22.3 hr independent of copper availability (Figure 5, B and C). In high copper, the Ptcu-1wc-1 strain was largely arrhythmic with conidiation predominating (only ~10% of the growth was linear hyphae), and had a long period, when a period could be measured (~24.5 hr), as expected for strains with reduced or no expression of WC-1 (Lee et al. 2003). As copper was reduced and BCS increased, normal rhythmicity was restored at 50 µM CuSO4 with or without 10 µM BCS. Above this level of BCS, rhythmicity became unreliable, and the oscillations accelerated (shorter period), as expected for strains overexpressing WC-1 (Cheng et al. 2001).

Bottom Line: The kinetics of induction and repression of tcu-1 are rapid, and the effects are long lived.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, Texas 77843.

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.

Show MeSH
Related in: MedlinePlus