Limits...
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

Vectors and promoter fragments. (A) Targeting/tagging backbone vectors pRS426PVG/pnit-6 and pRS426PVG/ptcu-1. Yeast/E. coli shuttle vector pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data) is the backbone for both constructs. Both vectors confer uracil prototrophy to ura3 yeast mutants (URA3) and ampicillin resistance (ApR) in E. coli. pRS426PVG/ptcu-1 vectors contain fragments upstream of the tcu-1 ORF, whereas the pRS426PVG/pnit-6 group of vectors contain nit-6 upstream fragments. Both sets of vectors have the 5′ and 3′ flanking regions for the N. crassa pan-2 ORF (black arrows) surrounding the tcu-1 or nit-6 promoter fragment (violet arrow), a multiple cloning sequence (MCS; black bar), a 5-glycine linker (5XGly; gray bar), a V5 peptide tag (V5-Tag; black bar), the GFP gene (GFP; green arrow) and the bar gene (blue shading), conferring resistance to phosphinothricin in N. crassa (bar). The 5′ pan-2 flank extends from 1 kb upstream to the sequence just before the ATG, whereas the 3′ flank begins with the sequence just beyond the stop codon and extends 1 kb downstream. Other abbreviations: P(LAC), lac promoter; 2 MICRON, yeast 2 micrometer origin of replication; F1 ORI, origin of replication in E. coli. (B) Schematic representation of promoter fragments from nit-6 and tcu-1 cloned in the vectors. The regions 0.5, 1.0, and 1.5 kb upstream of the tcu-1 or nit-5 ORF were amplified using PCR and inserted into pRS426PVG using yeast recombinational cloning. The orientation of the promoter fragments relative to the MCS, 5XGly linker, V5 peptide tag, and GFP is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4555226&req=5

fig1: Vectors and promoter fragments. (A) Targeting/tagging backbone vectors pRS426PVG/pnit-6 and pRS426PVG/ptcu-1. Yeast/E. coli shuttle vector pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data) is the backbone for both constructs. Both vectors confer uracil prototrophy to ura3 yeast mutants (URA3) and ampicillin resistance (ApR) in E. coli. pRS426PVG/ptcu-1 vectors contain fragments upstream of the tcu-1 ORF, whereas the pRS426PVG/pnit-6 group of vectors contain nit-6 upstream fragments. Both sets of vectors have the 5′ and 3′ flanking regions for the N. crassa pan-2 ORF (black arrows) surrounding the tcu-1 or nit-6 promoter fragment (violet arrow), a multiple cloning sequence (MCS; black bar), a 5-glycine linker (5XGly; gray bar), a V5 peptide tag (V5-Tag; black bar), the GFP gene (GFP; green arrow) and the bar gene (blue shading), conferring resistance to phosphinothricin in N. crassa (bar). The 5′ pan-2 flank extends from 1 kb upstream to the sequence just before the ATG, whereas the 3′ flank begins with the sequence just beyond the stop codon and extends 1 kb downstream. Other abbreviations: P(LAC), lac promoter; 2 MICRON, yeast 2 micrometer origin of replication; F1 ORI, origin of replication in E. coli. (B) Schematic representation of promoter fragments from nit-6 and tcu-1 cloned in the vectors. The regions 0.5, 1.0, and 1.5 kb upstream of the tcu-1 or nit-5 ORF were amplified using PCR and inserted into pRS426PVG using yeast recombinational cloning. The orientation of the promoter fragments relative to the MCS, 5XGly linker, V5 peptide tag, and GFP is shown.

Mentions: We developed vectors for expression of proteins under control of regions corresponding to the tcu-1 and nit-6 promoters. The vectors were based on pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data), developed to insert genes at the pan-2 locus (Case and Giles 1958) in N. crassa. Mutation of pan-2 leads to a requirement for pantothenic acid. The ccg-1 promoter in pRS426PVG was replaced by fragments corresponding to upstream regions for the nit-6 and tcu-1 genes (Figure 1A). We amplified fragments corresponding to regions 0.5, 1.0, and 1.5 kb upstream of the start codon (Figure 1B). The tcu-1 promoter group of vectors includes pRS426PVG/tcu-1_0.5 kb, pRS426PVG/tcu-1_1.0 kb, and pRS426PVG/tcu-1_1.5 kb, whereas the three vectors with the nit-6 promoter fragments are pRS426PVG/pnit-6_0.5 kb, pRS426PVG/pnit-6_1.0 kb, and pRS426PVG/pnit-6_1.5 kb. All vectors contained a multiple cloning site, a 5-glycine linker, and V5 and GFP tags 3′ to the promoter fragment (Figure 1A). A bar cassette, conferring resistance to the antibiotic phosphinothricin (Pall 1993), was inserted 3′ to the GFP gene. The vectors were then transformed into the N. crassa recipient strain Δmus51-IV-8 (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data). A total of six strains were generated during this study (Table 1), each containing a different fragment of tcu-1 or nit-6 driving expression of GFP from the pan-2 locus in N. crassa.


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)

Vectors and promoter fragments. (A) Targeting/tagging backbone vectors pRS426PVG/pnit-6 and pRS426PVG/ptcu-1. Yeast/E. coli shuttle vector pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data) is the backbone for both constructs. Both vectors confer uracil prototrophy to ura3 yeast mutants (URA3) and ampicillin resistance (ApR) in E. coli. pRS426PVG/ptcu-1 vectors contain fragments upstream of the tcu-1 ORF, whereas the pRS426PVG/pnit-6 group of vectors contain nit-6 upstream fragments. Both sets of vectors have the 5′ and 3′ flanking regions for the N. crassa pan-2 ORF (black arrows) surrounding the tcu-1 or nit-6 promoter fragment (violet arrow), a multiple cloning sequence (MCS; black bar), a 5-glycine linker (5XGly; gray bar), a V5 peptide tag (V5-Tag; black bar), the GFP gene (GFP; green arrow) and the bar gene (blue shading), conferring resistance to phosphinothricin in N. crassa (bar). The 5′ pan-2 flank extends from 1 kb upstream to the sequence just before the ATG, whereas the 3′ flank begins with the sequence just beyond the stop codon and extends 1 kb downstream. Other abbreviations: P(LAC), lac promoter; 2 MICRON, yeast 2 micrometer origin of replication; F1 ORI, origin of replication in E. coli. (B) Schematic representation of promoter fragments from nit-6 and tcu-1 cloned in the vectors. The regions 0.5, 1.0, and 1.5 kb upstream of the tcu-1 or nit-5 ORF were amplified using PCR and inserted into pRS426PVG using yeast recombinational cloning. The orientation of the promoter fragments relative to the MCS, 5XGly linker, V5 peptide tag, and GFP is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Vectors and promoter fragments. (A) Targeting/tagging backbone vectors pRS426PVG/pnit-6 and pRS426PVG/ptcu-1. Yeast/E. coli shuttle vector pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data) is the backbone for both constructs. Both vectors confer uracil prototrophy to ura3 yeast mutants (URA3) and ampicillin resistance (ApR) in E. coli. pRS426PVG/ptcu-1 vectors contain fragments upstream of the tcu-1 ORF, whereas the pRS426PVG/pnit-6 group of vectors contain nit-6 upstream fragments. Both sets of vectors have the 5′ and 3′ flanking regions for the N. crassa pan-2 ORF (black arrows) surrounding the tcu-1 or nit-6 promoter fragment (violet arrow), a multiple cloning sequence (MCS; black bar), a 5-glycine linker (5XGly; gray bar), a V5 peptide tag (V5-Tag; black bar), the GFP gene (GFP; green arrow) and the bar gene (blue shading), conferring resistance to phosphinothricin in N. crassa (bar). The 5′ pan-2 flank extends from 1 kb upstream to the sequence just before the ATG, whereas the 3′ flank begins with the sequence just beyond the stop codon and extends 1 kb downstream. Other abbreviations: P(LAC), lac promoter; 2 MICRON, yeast 2 micrometer origin of replication; F1 ORI, origin of replication in E. coli. (B) Schematic representation of promoter fragments from nit-6 and tcu-1 cloned in the vectors. The regions 0.5, 1.0, and 1.5 kb upstream of the tcu-1 or nit-5 ORF were amplified using PCR and inserted into pRS426PVG using yeast recombinational cloning. The orientation of the promoter fragments relative to the MCS, 5XGly linker, V5 peptide tag, and GFP is shown.
Mentions: We developed vectors for expression of proteins under control of regions corresponding to the tcu-1 and nit-6 promoters. The vectors were based on pRS426PVG (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data), developed to insert genes at the pan-2 locus (Case and Giles 1958) in N. crassa. Mutation of pan-2 leads to a requirement for pantothenic acid. The ccg-1 promoter in pRS426PVG was replaced by fragments corresponding to upstream regions for the nit-6 and tcu-1 genes (Figure 1A). We amplified fragments corresponding to regions 0.5, 1.0, and 1.5 kb upstream of the start codon (Figure 1B). The tcu-1 promoter group of vectors includes pRS426PVG/tcu-1_0.5 kb, pRS426PVG/tcu-1_1.0 kb, and pRS426PVG/tcu-1_1.5 kb, whereas the three vectors with the nit-6 promoter fragments are pRS426PVG/pnit-6_0.5 kb, pRS426PVG/pnit-6_1.0 kb, and pRS426PVG/pnit-6_1.5 kb. All vectors contained a multiple cloning site, a 5-glycine linker, and V5 and GFP tags 3′ to the promoter fragment (Figure 1A). A bar cassette, conferring resistance to the antibiotic phosphinothricin (Pall 1993), was inserted 3′ to the GFP gene. The vectors were then transformed into the N. crassa recipient strain Δmus51-IV-8 (S. Ouyang, I. E. Cabrera, A. J. Campbell, K. A. Borkovich, unpublished data). A total of six strains were generated during this study (Table 1), each containing a different fragment of tcu-1 or nit-6 driving expression of GFP from the pan-2 locus in N. crassa.

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