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Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.

Ouyang S, Beecher CN, Wang K, Larive CK, Borkovich KA - G3 (Bethesda) (2015)

Bottom Line: However, relatively few highly tunable promoters have been developed for this species.We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source.Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, 900 University Avenue, Riverside, California 92521 College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China.

No MeSH data available.


Related in: MedlinePlus

Representative 1H NMR spectra with key resonances labeled. Although the crowded region between 3.59 and 3.92 ppm contains the resonances of several metabolites, the predominant peaks in this region are those of trehalose. (A) Spectra of extracts of the pnit-6_1.5 strain cultured on VM-Gln (top) or VM-nitrate (bottom). The resonances of Gln and Glu are increased when Gln is used as a nitrogen source, whereas the levels of Ala, mannitol, and trehalose are more intense when the strain is grown on nitrate-containing media (see also Figure S3 and Figure S4). (B) Spectra of extracts of the ptcu-1_1.5 strain cultured on media containing in BCS (top) and CuSO4 (bottom). These spectra are more similar than those shown in (A), with the most pronounced differences in the regions containing the resonances of BCS (see also Figure S5).
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fig4: Representative 1H NMR spectra with key resonances labeled. Although the crowded region between 3.59 and 3.92 ppm contains the resonances of several metabolites, the predominant peaks in this region are those of trehalose. (A) Spectra of extracts of the pnit-6_1.5 strain cultured on VM-Gln (top) or VM-nitrate (bottom). The resonances of Gln and Glu are increased when Gln is used as a nitrogen source, whereas the levels of Ala, mannitol, and trehalose are more intense when the strain is grown on nitrate-containing media (see also Figure S3 and Figure S4). (B) Spectra of extracts of the ptcu-1_1.5 strain cultured on media containing in BCS (top) and CuSO4 (bottom). These spectra are more similar than those shown in (A), with the most pronounced differences in the regions containing the resonances of BCS (see also Figure S5).

Mentions: 1H NMR provides an untargeted view of metabolic shifts resulting from expression of the inserted gene. Figure 4 presents representative 1H NMR spectra for each sample type with key resonances labeled. We were not surprised that use of Gln as a nitrogen source (Figure 4A) significantly increases the levels of Gln and glutamate (Glu) compared with the expression on media containing sodium nitrate. In contrast, the resonances of trehalose and mannitol are lower in the extracts of pnit-6_1.5 grown on Gln. The levels of alanine (Ala) also decrease in the pnit-6_1.5(Gln) samples, which can be more readily observed in the expansion of the spectra presented in Figure S3 at a lower vertical scale. Taken together, the lower levels of trehalose, Ala, and mannitol suggest a shift away from glycolytic metabolism compared to growth of the pnit-6_1.5 promoter strain on VM-nitrate (Dijkema et al. 1985). Expansions of NMR spectra measured for biological replicates (Figure S4A [pnit-6_1.5(Gln)] and Figure S4B [pnit-6_1.5(nitrate)]) demonstrate that the cell culture and extraction methods are highly reproducible and allow for a more detailed evaluation of the impact of culture conditions on the metabolite profile. It is important to note that the differences in the metabolic profiles for pnit-6_1.5(Gln) and pnit-6_1.5(nitrate) largely reflect the different growth conditions required to repress or induce the nit-6 promoter, and not the products of the expressed gene. This is confirmed by comparison of the 1H NMR spectra for the wild-type N. crassa strain 74-OR23-IVA (Table 1) grown on Gln and nitrate (Figure S5), with the results presented in Figure S3 for pnit-6_1.5.


Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.

Ouyang S, Beecher CN, Wang K, Larive CK, Borkovich KA - G3 (Bethesda) (2015)

Representative 1H NMR spectra with key resonances labeled. Although the crowded region between 3.59 and 3.92 ppm contains the resonances of several metabolites, the predominant peaks in this region are those of trehalose. (A) Spectra of extracts of the pnit-6_1.5 strain cultured on VM-Gln (top) or VM-nitrate (bottom). The resonances of Gln and Glu are increased when Gln is used as a nitrogen source, whereas the levels of Ala, mannitol, and trehalose are more intense when the strain is grown on nitrate-containing media (see also Figure S3 and Figure S4). (B) Spectra of extracts of the ptcu-1_1.5 strain cultured on media containing in BCS (top) and CuSO4 (bottom). These spectra are more similar than those shown in (A), with the most pronounced differences in the regions containing the resonances of BCS (see also Figure S5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555226&req=5

fig4: Representative 1H NMR spectra with key resonances labeled. Although the crowded region between 3.59 and 3.92 ppm contains the resonances of several metabolites, the predominant peaks in this region are those of trehalose. (A) Spectra of extracts of the pnit-6_1.5 strain cultured on VM-Gln (top) or VM-nitrate (bottom). The resonances of Gln and Glu are increased when Gln is used as a nitrogen source, whereas the levels of Ala, mannitol, and trehalose are more intense when the strain is grown on nitrate-containing media (see also Figure S3 and Figure S4). (B) Spectra of extracts of the ptcu-1_1.5 strain cultured on media containing in BCS (top) and CuSO4 (bottom). These spectra are more similar than those shown in (A), with the most pronounced differences in the regions containing the resonances of BCS (see also Figure S5).
Mentions: 1H NMR provides an untargeted view of metabolic shifts resulting from expression of the inserted gene. Figure 4 presents representative 1H NMR spectra for each sample type with key resonances labeled. We were not surprised that use of Gln as a nitrogen source (Figure 4A) significantly increases the levels of Gln and glutamate (Glu) compared with the expression on media containing sodium nitrate. In contrast, the resonances of trehalose and mannitol are lower in the extracts of pnit-6_1.5 grown on Gln. The levels of alanine (Ala) also decrease in the pnit-6_1.5(Gln) samples, which can be more readily observed in the expansion of the spectra presented in Figure S3 at a lower vertical scale. Taken together, the lower levels of trehalose, Ala, and mannitol suggest a shift away from glycolytic metabolism compared to growth of the pnit-6_1.5 promoter strain on VM-nitrate (Dijkema et al. 1985). Expansions of NMR spectra measured for biological replicates (Figure S4A [pnit-6_1.5(Gln)] and Figure S4B [pnit-6_1.5(nitrate)]) demonstrate that the cell culture and extraction methods are highly reproducible and allow for a more detailed evaluation of the impact of culture conditions on the metabolite profile. It is important to note that the differences in the metabolic profiles for pnit-6_1.5(Gln) and pnit-6_1.5(nitrate) largely reflect the different growth conditions required to repress or induce the nit-6 promoter, and not the products of the expressed gene. This is confirmed by comparison of the 1H NMR spectra for the wild-type N. crassa strain 74-OR23-IVA (Table 1) grown on Gln and nitrate (Figure S5), with the results presented in Figure S3 for pnit-6_1.5.

Bottom Line: However, relatively few highly tunable promoters have been developed for this species.We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source.Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, 900 University Avenue, Riverside, California 92521 College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China.

No MeSH data available.


Related in: MedlinePlus