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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

Identification of promoter fragments from tcu-1 and nit-6 that drive highest expression of GFP. (A) Northern analysis of GFP mRNA in the strains with different promoter fragments. Total RNA was isolated from strains cultured overnight in VM-Gln and then transferred to VM-nitrate medium for the indicated times. Samples containing 20 μg of total RNA were subjected to Northern analysis using GFP as a probe. 18s rRNA bands from the ethidium bromide-stained gel served as the loading control. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, pnit-6_0.5, pnit-6_1.0, pnit-6_1.5, ptcu-1_0.5, ptcu-1_1.0, and ptcu-1_1.5. The blot shown is representative of at least three experiments. (B) Western analysis of protein extracts from strains with different promoter fragments. Whole cell extracts were isolated from the strains with different promoter fragments and samples containing 50 μg total protein subjected to Western analysis using GFP antiserum (see Materials and Methods for details). The asterisk indicates a higher-molecular-weight, nonspecific background band. Strains are the same as in (A). The blot shown is representative of at least three experiments. (C) Time course expression of GFP proteins driven by 1.5-kb promoter fragments for tcu-1 and nit-6. Whole cell extracts were isolated and subjected to Western analysis as described for (B). The asterisk indicates a nonspecific background band. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, ptcu-1_1.5, and pnit-6_1.5. Treatments: “-” refers to repressing conditions, which are VM-Gln for the pnit-6_1.5 strain and VM-Cu, for the ptcu-1_1.5 strain. “+” corresponds to inducing conditions, which are VM-nitrate for the pnit-6_1.5 strain and VM-BCS for the ptcu-1_1.5 strain. The blot shown is representative of at least three experiments.
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fig2: Identification of promoter fragments from tcu-1 and nit-6 that drive highest expression of GFP. (A) Northern analysis of GFP mRNA in the strains with different promoter fragments. Total RNA was isolated from strains cultured overnight in VM-Gln and then transferred to VM-nitrate medium for the indicated times. Samples containing 20 μg of total RNA were subjected to Northern analysis using GFP as a probe. 18s rRNA bands from the ethidium bromide-stained gel served as the loading control. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, pnit-6_0.5, pnit-6_1.0, pnit-6_1.5, ptcu-1_0.5, ptcu-1_1.0, and ptcu-1_1.5. The blot shown is representative of at least three experiments. (B) Western analysis of protein extracts from strains with different promoter fragments. Whole cell extracts were isolated from the strains with different promoter fragments and samples containing 50 μg total protein subjected to Western analysis using GFP antiserum (see Materials and Methods for details). The asterisk indicates a higher-molecular-weight, nonspecific background band. Strains are the same as in (A). The blot shown is representative of at least three experiments. (C) Time course expression of GFP proteins driven by 1.5-kb promoter fragments for tcu-1 and nit-6. Whole cell extracts were isolated and subjected to Western analysis as described for (B). The asterisk indicates a nonspecific background band. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, ptcu-1_1.5, and pnit-6_1.5. Treatments: “-” refers to repressing conditions, which are VM-Gln for the pnit-6_1.5 strain and VM-Cu, for the ptcu-1_1.5 strain. “+” corresponds to inducing conditions, which are VM-nitrate for the pnit-6_1.5 strain and VM-BCS for the ptcu-1_1.5 strain. The blot shown is representative of at least three experiments.

Mentions: We next investigated expression of GFP mRNA and protein in the six N. crassa strains carrying the different promoter fragments. For these studies, we used a strain in which the ccg-1 promoter controls expression of GFP as a positive control (Figure 2A). The results showed that the growth regimens used for the different promoters did not influence expression of GFP mRNA or protein from ccg-1 (Figure 2, A and B).


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)

Identification of promoter fragments from tcu-1 and nit-6 that drive highest expression of GFP. (A) Northern analysis of GFP mRNA in the strains with different promoter fragments. Total RNA was isolated from strains cultured overnight in VM-Gln and then transferred to VM-nitrate medium for the indicated times. Samples containing 20 μg of total RNA were subjected to Northern analysis using GFP as a probe. 18s rRNA bands from the ethidium bromide-stained gel served as the loading control. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, pnit-6_0.5, pnit-6_1.0, pnit-6_1.5, ptcu-1_0.5, ptcu-1_1.0, and ptcu-1_1.5. The blot shown is representative of at least three experiments. (B) Western analysis of protein extracts from strains with different promoter fragments. Whole cell extracts were isolated from the strains with different promoter fragments and samples containing 50 μg total protein subjected to Western analysis using GFP antiserum (see Materials and Methods for details). The asterisk indicates a higher-molecular-weight, nonspecific background band. Strains are the same as in (A). The blot shown is representative of at least three experiments. (C) Time course expression of GFP proteins driven by 1.5-kb promoter fragments for tcu-1 and nit-6. Whole cell extracts were isolated and subjected to Western analysis as described for (B). The asterisk indicates a nonspecific background band. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, ptcu-1_1.5, and pnit-6_1.5. Treatments: “-” refers to repressing conditions, which are VM-Gln for the pnit-6_1.5 strain and VM-Cu, for the ptcu-1_1.5 strain. “+” corresponds to inducing conditions, which are VM-nitrate for the pnit-6_1.5 strain and VM-BCS for the ptcu-1_1.5 strain. The blot shown is representative of at least three experiments.
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Related In: Results  -  Collection

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fig2: Identification of promoter fragments from tcu-1 and nit-6 that drive highest expression of GFP. (A) Northern analysis of GFP mRNA in the strains with different promoter fragments. Total RNA was isolated from strains cultured overnight in VM-Gln and then transferred to VM-nitrate medium for the indicated times. Samples containing 20 μg of total RNA were subjected to Northern analysis using GFP as a probe. 18s rRNA bands from the ethidium bromide-stained gel served as the loading control. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, pnit-6_0.5, pnit-6_1.0, pnit-6_1.5, ptcu-1_0.5, ptcu-1_1.0, and ptcu-1_1.5. The blot shown is representative of at least three experiments. (B) Western analysis of protein extracts from strains with different promoter fragments. Whole cell extracts were isolated from the strains with different promoter fragments and samples containing 50 μg total protein subjected to Western analysis using GFP antiserum (see Materials and Methods for details). The asterisk indicates a higher-molecular-weight, nonspecific background band. Strains are the same as in (A). The blot shown is representative of at least three experiments. (C) Time course expression of GFP proteins driven by 1.5-kb promoter fragments for tcu-1 and nit-6. Whole cell extracts were isolated and subjected to Western analysis as described for (B). The asterisk indicates a nonspecific background band. Strains are wild-type 74-OR23-IVA (WT), pccg-1_GFP, ptcu-1_1.5, and pnit-6_1.5. Treatments: “-” refers to repressing conditions, which are VM-Gln for the pnit-6_1.5 strain and VM-Cu, for the ptcu-1_1.5 strain. “+” corresponds to inducing conditions, which are VM-nitrate for the pnit-6_1.5 strain and VM-BCS for the ptcu-1_1.5 strain. The blot shown is representative of at least three experiments.
Mentions: We next investigated expression of GFP mRNA and protein in the six N. crassa strains carrying the different promoter fragments. For these studies, we used a strain in which the ccg-1 promoter controls expression of GFP as a positive control (Figure 2A). The results showed that the growth regimens used for the different promoters did not influence expression of GFP mRNA or protein from ccg-1 (Figure 2, A and B).

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