Structured Review

Promega luciferase open reading frame
Luciferase Open Reading Frame, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/luciferase open reading frame/product/Promega
Average 89 stars, based on 17 article reviews
Price from $9.99 to $1999.99
luciferase open reading frame - by Bioz Stars, 2020-09
89/100 stars

Images

Related Articles

Clone Assay:

Article Title: MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor
Article Snippet: .. The PCR product was cloned into the EcoRΙ and HindΙΙΙ restriction sites downstream of the luciferase open reading frame in the pGL3-luciferase reporter plasmid (Promega, Madison, WI, USA). .. Luciferase reporter assays The cells (HTR-8/SVNEO and JEG-3) were grown to 70–80% confluence in 24-well plates and co-transfected with the luciferase reporter vector described above (200 ng) and the appropriate miRNA (50 nM) using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Article Title: Identifying targets of miR-143 using a SILAC-based proteomic approach
Article Snippet: .. 3’UTR fragments of selected genes were PCR amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega). .. Mutagenesis of 6-mer seed match in 3’UTR regions of each target was generated from corresponding wild type constructs using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions.

Article Title: Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease
Article Snippet: .. The SORL1 3′ UTR sequences (wild-type and mutated), containing the putative binding site of miR-1229-3p, were amplified and cloned into the pGL3 luciferase reporter vector downstream of the Luciferase open reading frame (Promega). .. Similarly, pre-miR-1229 sequence was amplified using a forward primer containing a XhoI restriction site and a reverse primer containing a EcoRI restriction site.

Article Title: Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology
Article Snippet: .. Luciferase assays The 3ʹ-UTR fragments of selected genes were amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega, USA). .. Twenty four hours prior to transfection, 5 × 104 cells were plated per well in a 48-well plate. pGL3 constructs (100 ng) plus 10 ng of the Renilla luciferase plasmid phRL-SV40 (Promega, USA) were co-transfected with miR-26a mimic or control oligo using Lipofectamine 2000 (Invitrogen, USA) in 293 T cells.

Amplification:

Article Title: Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs
Article Snippet: .. The luciferase open reading frame from sea pansy Renilla reniformis (Rluc) was amplified from the pGL4.70[ hRluc ] vector (Promega). .. The gene was integrated into the BglII restriction site of plasmid pIR1SAT ( ).

Article Title: Identifying targets of miR-143 using a SILAC-based proteomic approach
Article Snippet: .. 3’UTR fragments of selected genes were PCR amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega). .. Mutagenesis of 6-mer seed match in 3’UTR regions of each target was generated from corresponding wild type constructs using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions.

Article Title: Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease
Article Snippet: .. The SORL1 3′ UTR sequences (wild-type and mutated), containing the putative binding site of miR-1229-3p, were amplified and cloned into the pGL3 luciferase reporter vector downstream of the Luciferase open reading frame (Promega). .. Similarly, pre-miR-1229 sequence was amplified using a forward primer containing a XhoI restriction site and a reverse primer containing a EcoRI restriction site.

Article Title: Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology
Article Snippet: .. Luciferase assays The 3ʹ-UTR fragments of selected genes were amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega, USA). .. Twenty four hours prior to transfection, 5 × 104 cells were plated per well in a 48-well plate. pGL3 constructs (100 ng) plus 10 ng of the Renilla luciferase plasmid phRL-SV40 (Promega, USA) were co-transfected with miR-26a mimic or control oligo using Lipofectamine 2000 (Invitrogen, USA) in 293 T cells.

Construct:

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL
Article Snippet: .. Reporter plasmids were constructed using the pGL3 vector containing a firefly luciferase open reading frame (Promega). ..

Article Title: An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells
Article Snippet: .. The aforementioned 6.5-kb Kpn I- Mun I fragment was inserted into the Kpn I- Hind III site upstream of the luciferase open reading frame in the reporter gene vectors pGL3-basic and pGL2-basic (Promega, Madison, WI), resulting in constructs pTnNOS −6500/+81 pGL3 and pTnNOS −6500/+81 pGL2, respectively. .. A 2.2-kb Bgl II- Mun I fragment representing the 3′-portion of the putative promoter for TnNOS was also inserted into the Kpn I- Hind III site of the pGL3-basic and pGL2-basic, resulting in the constructs pTnNOS −2200/+81 pGL3 and pTnNOS −2200/+81 pGL2, respectively.

Sequencing:

Article Title: Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia
Article Snippet: .. Each of the reverse primers contained the splice acceptor site from the 3′ end of intron 1 and an NcoI site and were designed so that the FXN coding sequence in exon 1 would be translated in frame with the luciferase open reading frame from pGL3-Basic (Promega, Madison, WI, USA). .. After PCR amplification, the gel-purified fragment was digested with MluI and NcoI and cloned into pGL3-Basic.

Luciferase:

Article Title: Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia
Article Snippet: .. Each of the reverse primers contained the splice acceptor site from the 3′ end of intron 1 and an NcoI site and were designed so that the FXN coding sequence in exon 1 would be translated in frame with the luciferase open reading frame from pGL3-Basic (Promega, Madison, WI, USA). .. After PCR amplification, the gel-purified fragment was digested with MluI and NcoI and cloned into pGL3-Basic.

Article Title: Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs
Article Snippet: .. The luciferase open reading frame from sea pansy Renilla reniformis (Rluc) was amplified from the pGL4.70[ hRluc ] vector (Promega). .. The gene was integrated into the BglII restriction site of plasmid pIR1SAT ( ).

Article Title: MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor
Article Snippet: .. The PCR product was cloned into the EcoRΙ and HindΙΙΙ restriction sites downstream of the luciferase open reading frame in the pGL3-luciferase reporter plasmid (Promega, Madison, WI, USA). .. Luciferase reporter assays The cells (HTR-8/SVNEO and JEG-3) were grown to 70–80% confluence in 24-well plates and co-transfected with the luciferase reporter vector described above (200 ng) and the appropriate miRNA (50 nM) using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Article Title: Identifying targets of miR-143 using a SILAC-based proteomic approach
Article Snippet: .. 3’UTR fragments of selected genes were PCR amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega). .. Mutagenesis of 6-mer seed match in 3’UTR regions of each target was generated from corresponding wild type constructs using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions.

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL
Article Snippet: .. Reporter plasmids were constructed using the pGL3 vector containing a firefly luciferase open reading frame (Promega). ..

Article Title: Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease
Article Snippet: .. The SORL1 3′ UTR sequences (wild-type and mutated), containing the putative binding site of miR-1229-3p, were amplified and cloned into the pGL3 luciferase reporter vector downstream of the Luciferase open reading frame (Promega). .. Similarly, pre-miR-1229 sequence was amplified using a forward primer containing a XhoI restriction site and a reverse primer containing a EcoRI restriction site.

Article Title: Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology
Article Snippet: .. Luciferase assays The 3ʹ-UTR fragments of selected genes were amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega, USA). .. Twenty four hours prior to transfection, 5 × 104 cells were plated per well in a 48-well plate. pGL3 constructs (100 ng) plus 10 ng of the Renilla luciferase plasmid phRL-SV40 (Promega, USA) were co-transfected with miR-26a mimic or control oligo using Lipofectamine 2000 (Invitrogen, USA) in 293 T cells.

Article Title: An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells
Article Snippet: .. The aforementioned 6.5-kb Kpn I- Mun I fragment was inserted into the Kpn I- Hind III site upstream of the luciferase open reading frame in the reporter gene vectors pGL3-basic and pGL2-basic (Promega, Madison, WI), resulting in constructs pTnNOS −6500/+81 pGL3 and pTnNOS −6500/+81 pGL2, respectively. .. A 2.2-kb Bgl II- Mun I fragment representing the 3′-portion of the putative promoter for TnNOS was also inserted into the Kpn I- Hind III site of the pGL3-basic and pGL2-basic, resulting in the constructs pTnNOS −2200/+81 pGL3 and pTnNOS −2200/+81 pGL2, respectively.

Polymerase Chain Reaction:

Article Title: MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor
Article Snippet: .. The PCR product was cloned into the EcoRΙ and HindΙΙΙ restriction sites downstream of the luciferase open reading frame in the pGL3-luciferase reporter plasmid (Promega, Madison, WI, USA). .. Luciferase reporter assays The cells (HTR-8/SVNEO and JEG-3) were grown to 70–80% confluence in 24-well plates and co-transfected with the luciferase reporter vector described above (200 ng) and the appropriate miRNA (50 nM) using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Article Title: Identifying targets of miR-143 using a SILAC-based proteomic approach
Article Snippet: .. 3’UTR fragments of selected genes were PCR amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega). .. Mutagenesis of 6-mer seed match in 3’UTR regions of each target was generated from corresponding wild type constructs using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions.

Binding Assay:

Article Title: Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease
Article Snippet: .. The SORL1 3′ UTR sequences (wild-type and mutated), containing the putative binding site of miR-1229-3p, were amplified and cloned into the pGL3 luciferase reporter vector downstream of the Luciferase open reading frame (Promega). .. Similarly, pre-miR-1229 sequence was amplified using a forward primer containing a XhoI restriction site and a reverse primer containing a EcoRI restriction site.

Plasmid Preparation:

Article Title: Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs
Article Snippet: .. The luciferase open reading frame from sea pansy Renilla reniformis (Rluc) was amplified from the pGL4.70[ hRluc ] vector (Promega). .. The gene was integrated into the BglII restriction site of plasmid pIR1SAT ( ).

Article Title: MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor
Article Snippet: .. The PCR product was cloned into the EcoRΙ and HindΙΙΙ restriction sites downstream of the luciferase open reading frame in the pGL3-luciferase reporter plasmid (Promega, Madison, WI, USA). .. Luciferase reporter assays The cells (HTR-8/SVNEO and JEG-3) were grown to 70–80% confluence in 24-well plates and co-transfected with the luciferase reporter vector described above (200 ng) and the appropriate miRNA (50 nM) using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Article Title: Identifying targets of miR-143 using a SILAC-based proteomic approach
Article Snippet: .. 3’UTR fragments of selected genes were PCR amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega). .. Mutagenesis of 6-mer seed match in 3’UTR regions of each target was generated from corresponding wild type constructs using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions.

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL
Article Snippet: .. Reporter plasmids were constructed using the pGL3 vector containing a firefly luciferase open reading frame (Promega). ..

Article Title: Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease
Article Snippet: .. The SORL1 3′ UTR sequences (wild-type and mutated), containing the putative binding site of miR-1229-3p, were amplified and cloned into the pGL3 luciferase reporter vector downstream of the Luciferase open reading frame (Promega). .. Similarly, pre-miR-1229 sequence was amplified using a forward primer containing a XhoI restriction site and a reverse primer containing a EcoRI restriction site.

Article Title: Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology
Article Snippet: .. Luciferase assays The 3ʹ-UTR fragments of selected genes were amplified from human cDNA and cloned downstream of the luciferase open reading frame in pGL3-control vector (Promega, USA). .. Twenty four hours prior to transfection, 5 × 104 cells were plated per well in a 48-well plate. pGL3 constructs (100 ng) plus 10 ng of the Renilla luciferase plasmid phRL-SV40 (Promega, USA) were co-transfected with miR-26a mimic or control oligo using Lipofectamine 2000 (Invitrogen, USA) in 293 T cells.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Promega tata box
    Activation of Gal4-driven reporter gene by Gal4(DBD)-p30 II fusion protein. (A) Schematic illustration of reporter and effector plasmids used in the Gal4 transcription assay. (B) HeLa-tat cells were transiently cotransfected with 0.3 μg of <t>p5XGT-TATA-luciferase</t> plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid as indicated. (C) HeLa-tat cells transfected with 0.3 μg of p5XGT-TATA-Luc or <t>pGL2-TATA-luciferase</t> reporter plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11 plasmid. Results are expressed as mean fold increase ± SD in luciferase activity (normalized to β-galactosidase activity) for four independent trials. (D) 293 cells were transiently cotransfected with 0.3 μg of p5XGT-TATA-luciferase plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid to confirm the results using HeLa-Tat cells.
    Tata Box, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tata box/product/Promega
    Average 90 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    tata box - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    89
    Promega western blot analysis transfected cells
    Translation initiation from the C0 initiation codon compared with that from C and P initiation codons. ( A ) Schematic representation of the DNA constructs used in this study (diagram not to scale). It contains the SV40 and T7 promoter at the 5′ end followed by DNA sequences corresponding to the HBV pregenomic RNA leader fused to the luciferase gene. The nucleotide sequence for the hepatitis B virus pregenomic RNA leader has been deposited in the DNA Data Bank of Japan under DDBJ accession no. AB037684. Immediately downstream of the luciferase gene is the 117 bases of repeated sequence to mimic the terminally redundant pgRNA which also contains the epsilon structure denoted by ε. At the 3′ end, A n denotes the poly(A) tract while the SV40 poly(A) signal is denoted by an enclosed A. Vertical bars with dots represent the initiation codons within the pgRNA labeled C0, C in a light and dark font. The darker font represents the initiation codon that is in-frame to the luciferase reporter gene. ORFs encoded by each initiation codon are represented by filled boxes. Any mutation introduced into the leader sequence is denoted with an asterisk (*). Other labels are as in Figure 1 . ( B ) Normalized expression level from the C0, C and P initiation codons after transfection into HepG2 cells. Expression from each initiation codon is expressed as a percentage of normalized CLUC expression. These results are averages of three replicates from two independent experiments. ( C ) Luciferase expression was confirmed via western blot using anti-luciferase antibody on HepG2 <t>transfected</t> lysates separated on 5–20% SDS–PAGE. ( D ) Fluorograph of the luciferase fusion proteins translated off capped transcript in rabbit reticulocyte. Proteins were separated on 5–20% SDS–PAGE. Lane 1, CLUC RNA; lane 2, C0LUC RNA; lane 3, PLUC RNA; M, protein or DNA marker. The integrity and quantitation of the capped RNA used for cell-free translation is shown on the RNA gel below. ( E ) Detection of the C0LUC fusion protein in HepG2 transfected lysates via immunoblot analysis using anti-C0 antibody. Lane 4 contains 50 ng of synthetic C0.
    Western Blot Analysis Transfected Cells, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/western blot analysis transfected cells/product/Promega
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    western blot analysis transfected cells - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    85
    Promega asamp p88 orf
    The 5′ SS and 3′ SS are required for the exo-endo trans-splicing. (A , B) Analysis of the products from the exo-endo trans-splicing event by western blot and RT-PCR assays. <t>Anti-asAmp-P88,</t> anti-ACAT1 and anti-Rluc antibodies were employed in the western blot analyses. Specific primer sets asAmp-F/A1-R1 (A) and asAmp-F/RL-R (B) were used in the RT-PCR assays. (C) Constructs (numbers 30-41) containing asAmp with mutated nucleotides (underlined) at the 5′ SS of GTAAGT were co-transfected with plasmid containing ACAT1 1-1786 (number 27 in Figure 4A ) into AC29 cells. Western blot analysis was performed with an anti-Flag antibody. (D , E) Constructs (numbers 42-59, containing ACAT1 1243-1786 and Amp r ) with mutated nucleotides (underlined) at the 3′ SS of CAG were transfected into AC29 cells. Western blot was performed with anti-ACAT1 (D) or anti-Rluc (E) antibodies.
    Asamp P88 Orf, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asamp p88 orf/product/Promega
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    asamp p88 orf - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    89
    Promega rluc coding sequence
    The peptides encoded by the five sequence-dependent CPuORFs act in cis. ( A ) Schematic representation of the <t>35S::UTR:FLUC</t> and <t>35S::UTR:RLUC</t> reporter constructs. ( B – F ) Transient expression studies of the co-transfected 35S::UTR:RLUC and 35S::UTR:FLUC reporter plasmids. MM2d protoplasts were co-transfected with three plasmids, the 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids and the 35S::GUS internal control plasmid, by PEG treatment. The 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids contained the WT or fs version of the ANAC082 (B), CIPK6 (C), At3g15430 (D), At5g27920 (E) or OTLD1 (F) CPuORFs. Co-transfection was carried out for all four combinations for each CPuORF, as indicated. After 48 h of incubation, the transfected cells were harvested and disrupted for luciferase and GUS assays. FLUC and RLUC activities were normalized to GUS activity, and the FLUC and RLUC activities relative to those in the experiment where both reporter plasmids had the WT CPuORF were calculated. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. In each graph, bars with the same colors are not significantly different, whereas bars with different colors differ significantly ( P
    Rluc Coding Sequence, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rluc coding sequence/product/Promega
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    rluc coding sequence - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    Image Search Results


    Activation of Gal4-driven reporter gene by Gal4(DBD)-p30 II fusion protein. (A) Schematic illustration of reporter and effector plasmids used in the Gal4 transcription assay. (B) HeLa-tat cells were transiently cotransfected with 0.3 μg of p5XGT-TATA-luciferase plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid as indicated. (C) HeLa-tat cells transfected with 0.3 μg of p5XGT-TATA-Luc or pGL2-TATA-luciferase reporter plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11 plasmid. Results are expressed as mean fold increase ± SD in luciferase activity (normalized to β-galactosidase activity) for four independent trials. (D) 293 cells were transiently cotransfected with 0.3 μg of p5XGT-TATA-luciferase plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid to confirm the results using HeLa-Tat cells.

    Journal: Journal of Virology

    Article Title: Human T-Lymphotropic Virus Type 1 p30II Functions as a Transcription Factor and Differentially Modulates CREB-Responsive Promoters

    doi:

    Figure Lengend Snippet: Activation of Gal4-driven reporter gene by Gal4(DBD)-p30 II fusion protein. (A) Schematic illustration of reporter and effector plasmids used in the Gal4 transcription assay. (B) HeLa-tat cells were transiently cotransfected with 0.3 μg of p5XGT-TATA-luciferase plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid as indicated. (C) HeLa-tat cells transfected with 0.3 μg of p5XGT-TATA-Luc or pGL2-TATA-luciferase reporter plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11 plasmid. Results are expressed as mean fold increase ± SD in luciferase activity (normalized to β-galactosidase activity) for four independent trials. (D) 293 cells were transiently cotransfected with 0.3 μg of p5XGT-TATA-luciferase plasmid and 0 to 1.5 μg of p30 II -Gal4-pCRG4-11, pCMV-p30 II -HA, or Gal4-pCRG4-11 plasmid to confirm the results using HeLa-Tat cells.

    Article Snippet: Plasmid p5XGT-TATA-Luc, a kind gift of P. Quinn (The Pennsylvania State University, Hershey, Pa.), contains five tandem Gal4 DNA-binding sequences upstream of a TATA box, derived from positions −64 to +1 of the phosphoenolpyruvate carboxykinase (PEPCK) gene in a luciferase reporter gene plasmid ( ). pGL2-TATA-Luc was constructed by ligating the TATA box of the adenovirus E1b gene, derived from plasmid E1b-CAT , into the Xho I and Bgl II sites of pGL2-basic, a luciferase reporter gene vector (Promega), and then subcloning tandem copies of Gal4 DNA-binding sequence using Kpn I and Xho I sites.

    Techniques: Activation Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

    Effects of p30 II deletion mutants on Gal4-driven luciferase reporter gene activity. (A) Diagram of reporter plasmid p5XGT-TATA-Luc and structures of wild-type (WT) and deletion mutants of Gal4(DBD)-p30 II effector plasmids. (B) Expression of wild-type (WT) and deletion mutants of Gal4(DBD)-p30 II effector plasmids by immunoblot assay following transfection in HeLa-Tat cells. (C) Reporter gene activity from HeLa-tat cells cotransfected with 0.3 μg of p5XGT-TATA-luciferase reporter plasmids with 1 μg of each indicated Gal4(DBD)-p30 II expression plasmid (MT-1 to MT-6). The reporter gene activity by individual Gal4(DBD)-p30 II mutants is represented as a percentage of the reporter gene activity of wild-type (WT) Gal4(DBD)-p30 II normalized for β-galactosidase activity. Results are expressed as mean percent change in arbitrary light units (ALU) ± SD in luciferase activity (normalized to β-galactosidase activity) for four independent trials.

    Journal: Journal of Virology

    Article Title: Human T-Lymphotropic Virus Type 1 p30II Functions as a Transcription Factor and Differentially Modulates CREB-Responsive Promoters

    doi:

    Figure Lengend Snippet: Effects of p30 II deletion mutants on Gal4-driven luciferase reporter gene activity. (A) Diagram of reporter plasmid p5XGT-TATA-Luc and structures of wild-type (WT) and deletion mutants of Gal4(DBD)-p30 II effector plasmids. (B) Expression of wild-type (WT) and deletion mutants of Gal4(DBD)-p30 II effector plasmids by immunoblot assay following transfection in HeLa-Tat cells. (C) Reporter gene activity from HeLa-tat cells cotransfected with 0.3 μg of p5XGT-TATA-luciferase reporter plasmids with 1 μg of each indicated Gal4(DBD)-p30 II expression plasmid (MT-1 to MT-6). The reporter gene activity by individual Gal4(DBD)-p30 II mutants is represented as a percentage of the reporter gene activity of wild-type (WT) Gal4(DBD)-p30 II normalized for β-galactosidase activity. Results are expressed as mean percent change in arbitrary light units (ALU) ± SD in luciferase activity (normalized to β-galactosidase activity) for four independent trials.

    Article Snippet: Plasmid p5XGT-TATA-Luc, a kind gift of P. Quinn (The Pennsylvania State University, Hershey, Pa.), contains five tandem Gal4 DNA-binding sequences upstream of a TATA box, derived from positions −64 to +1 of the phosphoenolpyruvate carboxykinase (PEPCK) gene in a luciferase reporter gene plasmid ( ). pGL2-TATA-Luc was constructed by ligating the TATA box of the adenovirus E1b gene, derived from plasmid E1b-CAT , into the Xho I and Bgl II sites of pGL2-basic, a luciferase reporter gene vector (Promega), and then subcloning tandem copies of Gal4 DNA-binding sequence using Kpn I and Xho I sites.

    Techniques: Luciferase, Activity Assay, Plasmid Preparation, Expressing, Transfection

    Translation initiation from the C0 initiation codon compared with that from C and P initiation codons. ( A ) Schematic representation of the DNA constructs used in this study (diagram not to scale). It contains the SV40 and T7 promoter at the 5′ end followed by DNA sequences corresponding to the HBV pregenomic RNA leader fused to the luciferase gene. The nucleotide sequence for the hepatitis B virus pregenomic RNA leader has been deposited in the DNA Data Bank of Japan under DDBJ accession no. AB037684. Immediately downstream of the luciferase gene is the 117 bases of repeated sequence to mimic the terminally redundant pgRNA which also contains the epsilon structure denoted by ε. At the 3′ end, A n denotes the poly(A) tract while the SV40 poly(A) signal is denoted by an enclosed A. Vertical bars with dots represent the initiation codons within the pgRNA labeled C0, C in a light and dark font. The darker font represents the initiation codon that is in-frame to the luciferase reporter gene. ORFs encoded by each initiation codon are represented by filled boxes. Any mutation introduced into the leader sequence is denoted with an asterisk (*). Other labels are as in Figure 1 . ( B ) Normalized expression level from the C0, C and P initiation codons after transfection into HepG2 cells. Expression from each initiation codon is expressed as a percentage of normalized CLUC expression. These results are averages of three replicates from two independent experiments. ( C ) Luciferase expression was confirmed via western blot using anti-luciferase antibody on HepG2 transfected lysates separated on 5–20% SDS–PAGE. ( D ) Fluorograph of the luciferase fusion proteins translated off capped transcript in rabbit reticulocyte. Proteins were separated on 5–20% SDS–PAGE. Lane 1, CLUC RNA; lane 2, C0LUC RNA; lane 3, PLUC RNA; M, protein or DNA marker. The integrity and quantitation of the capped RNA used for cell-free translation is shown on the RNA gel below. ( E ) Detection of the C0LUC fusion protein in HepG2 transfected lysates via immunoblot analysis using anti-C0 antibody. Lane 4 contains 50 ng of synthetic C0.

    Journal: Nucleic Acids Research

    Article Title: Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

    doi: 10.1093/nar/gki251

    Figure Lengend Snippet: Translation initiation from the C0 initiation codon compared with that from C and P initiation codons. ( A ) Schematic representation of the DNA constructs used in this study (diagram not to scale). It contains the SV40 and T7 promoter at the 5′ end followed by DNA sequences corresponding to the HBV pregenomic RNA leader fused to the luciferase gene. The nucleotide sequence for the hepatitis B virus pregenomic RNA leader has been deposited in the DNA Data Bank of Japan under DDBJ accession no. AB037684. Immediately downstream of the luciferase gene is the 117 bases of repeated sequence to mimic the terminally redundant pgRNA which also contains the epsilon structure denoted by ε. At the 3′ end, A n denotes the poly(A) tract while the SV40 poly(A) signal is denoted by an enclosed A. Vertical bars with dots represent the initiation codons within the pgRNA labeled C0, C in a light and dark font. The darker font represents the initiation codon that is in-frame to the luciferase reporter gene. ORFs encoded by each initiation codon are represented by filled boxes. Any mutation introduced into the leader sequence is denoted with an asterisk (*). Other labels are as in Figure 1 . ( B ) Normalized expression level from the C0, C and P initiation codons after transfection into HepG2 cells. Expression from each initiation codon is expressed as a percentage of normalized CLUC expression. These results are averages of three replicates from two independent experiments. ( C ) Luciferase expression was confirmed via western blot using anti-luciferase antibody on HepG2 transfected lysates separated on 5–20% SDS–PAGE. ( D ) Fluorograph of the luciferase fusion proteins translated off capped transcript in rabbit reticulocyte. Proteins were separated on 5–20% SDS–PAGE. Lane 1, CLUC RNA; lane 2, C0LUC RNA; lane 3, PLUC RNA; M, protein or DNA marker. The integrity and quantitation of the capped RNA used for cell-free translation is shown on the RNA gel below. ( E ) Detection of the C0LUC fusion protein in HepG2 transfected lysates via immunoblot analysis using anti-C0 antibody. Lane 4 contains 50 ng of synthetic C0.

    Article Snippet: Western blot analysis Transfected cells were harvested in 100 μl of 1× Passive Lysis Buffer (Promega) and centrifuged at 11 000 g for 1 min.

    Techniques: Construct, Luciferase, Sequencing, Labeling, Mutagenesis, Expressing, Transfection, Western Blot, SDS Page, Marker, Quantitation Assay

    The 5′ SS and 3′ SS are required for the exo-endo trans-splicing. (A , B) Analysis of the products from the exo-endo trans-splicing event by western blot and RT-PCR assays. Anti-asAmp-P88, anti-ACAT1 and anti-Rluc antibodies were employed in the western blot analyses. Specific primer sets asAmp-F/A1-R1 (A) and asAmp-F/RL-R (B) were used in the RT-PCR assays. (C) Constructs (numbers 30-41) containing asAmp with mutated nucleotides (underlined) at the 5′ SS of GTAAGT were co-transfected with plasmid containing ACAT1 1-1786 (number 27 in Figure 4A ) into AC29 cells. Western blot analysis was performed with an anti-Flag antibody. (D , E) Constructs (numbers 42-59, containing ACAT1 1243-1786 and Amp r ) with mutated nucleotides (underlined) at the 3′ SS of CAG were transfected into AC29 cells. Western blot was performed with anti-ACAT1 (D) or anti-Rluc (E) antibodies.

    Journal: Cell Research

    Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    doi: 10.1038/cr.2013.86

    Figure Lengend Snippet: The 5′ SS and 3′ SS are required for the exo-endo trans-splicing. (A , B) Analysis of the products from the exo-endo trans-splicing event by western blot and RT-PCR assays. Anti-asAmp-P88, anti-ACAT1 and anti-Rluc antibodies were employed in the western blot analyses. Specific primer sets asAmp-F/A1-R1 (A) and asAmp-F/RL-R (B) were used in the RT-PCR assays. (C) Constructs (numbers 30-41) containing asAmp with mutated nucleotides (underlined) at the 5′ SS of GTAAGT were co-transfected with plasmid containing ACAT1 1-1786 (number 27 in Figure 4A ) into AC29 cells. Western blot analysis was performed with an anti-Flag antibody. (D , E) Constructs (numbers 42-59, containing ACAT1 1243-1786 and Amp r ) with mutated nucleotides (underlined) at the 3′ SS of CAG were transfected into AC29 cells. Western blot was performed with anti-ACAT1 (D) or anti-Rluc (E) antibodies.

    Article Snippet: Identification of the recombined cryptic promoter by luciferase activity analysis Upstream regions of asAmp-P88 ORF were amplified from plasmid pcDNA3 by PCR with individual forward primers asAmp-u1000-F (5′-AAACTCGAGAAAGGCGGTAATACGGTTATCCACA-3′), asAmp-u850-F (5′-AAACTCGAGTCGACGCTCAAGTCAGAGGTGG-3′), asAmp-u500-F (5′-AAACTCGAGGCGGTGCTACAGAGTTCTTGAAGTG-3′), asAmp-u275-F (5′-AAACTCGAGGTCTGACGCTCAGTGGAACGAAAA-3′) and common reverse primer asAmp-ATG-R (5′-AAAAAGCTTTGCAGCACTGGGGCCAGATG-3′), and inserted into Xho I and Hind III sites of pGL3-B (Promega) to generate reporter plasmids pasAmp-u1000, pasAmp-u850, pasAmp-u500 and pasAmp-u275, respectively.

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Construct, Transfection, Plasmid Preparation

    Existence of asAmp transcripts in higher eukaryotic cells. (A) PCR analysis of genomic DNAs from various human cell lines. Primer sets asAmp-F/asAmp-R, RP-F/asAmp-R, A1C7-F/A1C7-R and Kan r -F/Kan r -R are the same as those in Figure 2E . Primer set gyrB-F/gyrB-R is complementary to the sequence of bacterial gyrB cDNA. NC, negative-control without DNA templates. (B) RT-PCR analysis of asAmp transcripts in human cell lines HEK293T and THP-1. Primer set asAmp-F/asAmp-R was used. NC, without RNA templates. (C) The PCR products from HEK293T cells (B) were subjected to DNA sequencing. The sequence matching the 267-bp asAmp ORF is shown. (D , E) Alignment analysis of the asAmp ORF in EST database. The 267-bp asAmp ORF sequence is entered as query sequence by using BLASTN. (D) Results are shown by taxonomy tree with hit numbers in each species indicated in brackets. (E) The species containing the 267-bp asAmp ORF (gray bar) with 100% identity. Branch lengths are not proportional to phylogeny time.

    Journal: Cell Research

    Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    doi: 10.1038/cr.2013.86

    Figure Lengend Snippet: Existence of asAmp transcripts in higher eukaryotic cells. (A) PCR analysis of genomic DNAs from various human cell lines. Primer sets asAmp-F/asAmp-R, RP-F/asAmp-R, A1C7-F/A1C7-R and Kan r -F/Kan r -R are the same as those in Figure 2E . Primer set gyrB-F/gyrB-R is complementary to the sequence of bacterial gyrB cDNA. NC, negative-control without DNA templates. (B) RT-PCR analysis of asAmp transcripts in human cell lines HEK293T and THP-1. Primer set asAmp-F/asAmp-R was used. NC, without RNA templates. (C) The PCR products from HEK293T cells (B) were subjected to DNA sequencing. The sequence matching the 267-bp asAmp ORF is shown. (D , E) Alignment analysis of the asAmp ORF in EST database. The 267-bp asAmp ORF sequence is entered as query sequence by using BLASTN. (D) Results are shown by taxonomy tree with hit numbers in each species indicated in brackets. (E) The species containing the 267-bp asAmp ORF (gray bar) with 100% identity. Branch lengths are not proportional to phylogeny time.

    Article Snippet: Identification of the recombined cryptic promoter by luciferase activity analysis Upstream regions of asAmp-P88 ORF were amplified from plasmid pcDNA3 by PCR with individual forward primers asAmp-u1000-F (5′-AAACTCGAGAAAGGCGGTAATACGGTTATCCACA-3′), asAmp-u850-F (5′-AAACTCGAGTCGACGCTCAAGTCAGAGGTGG-3′), asAmp-u500-F (5′-AAACTCGAGGCGGTGCTACAGAGTTCTTGAAGTG-3′), asAmp-u275-F (5′-AAACTCGAGGTCTGACGCTCAGTGGAACGAAAA-3′) and common reverse primer asAmp-ATG-R (5′-AAAAAGCTTTGCAGCACTGGGGCCAGATG-3′), and inserted into Xho I and Hind III sites of pGL3-B (Promega) to generate reporter plasmids pasAmp-u1000, pasAmp-u850, pasAmp-u500 and pasAmp-u275, respectively.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing

    An extra N-terminal region contained in human ACAT1 56-kDa isoform is encoded by a sequence present in recombinant Amp r -plasmids. (A - C) Western blot analysis of the expression of WT and mutant ACAT1 constructs. Constructs (numbers 1-23) containing the entire ACAT1 (A) , partial ACAT1 (B , C) or Rluc (C) were transfected separately into AC29 cells. Western blot analyses were performed with anti-ACAT1 or anti-Rluc antibodies. Filled circle, GGC 1274-1276 codon; hollow circle, ATG 1397-1399 codon; and underlined characters, mutated nucleotides. (D) Calculated molecular weights (MW) of proteins initiated from the GGC 1274-1276 , ATG 1274-1276 and ATG 1397-1399 codons. ACAT1-50NT, 130 amino acids encoded by the sequence from ATG 1397-1399 to GAT 1784-1786 . ACAT1-56uNT, 41 amino acids upstream of ACAT1-50NT. Filled circle, glycine; hollow circle, methionine; and aa, amino acids. (E) Purification and 2-D electrophoresis of the 26-kDa ACAT1 protein. Construct pNTF-number 10 (B) was expressed in AC29 cells to generate the 26-kDa ACAT1 protein. The proteins were purified with ANTI-FLAG M2 Affinity Gel and separated by 2-D electrophoresis. Arrows indicate spots of different 26-kDa proteins designated as P1, P2 and P3. P CMV , CMV promoter (black arrow); ori, ColE1 origin (gray arrow). (F) MALDI-TOF MS analysis of the purified 26-kDa proteins. Mass spectra are recorded in the positive mode and listed for the purified 26-kDa protein P2. (G) The N-terminal region of the purified P2 protein and its origin. The 15 amino acids (bold) were determined by the N-terminal sequencing of the purified P2 protein; they are encoded by the underlined sequence that matches the sequence of asAmp derived from recombinant plasmids (asAmp, tessellated box). The amino acid sequence of asAmp-encoding p88 protein (asAmp-P88) and the region of a recombined cryptic promoter are shown. (H) Schematic representation of human ACAT1-56NT region. Italics indicate the N-terminal 15 amino acids of the purified P2 protein. Asterisks indicate modifications by iodoacetamide on cysteines. Trypsin cleavage sites are indicated by bold characters and calculated MWs of representative peptides (underlined) after trypsin cleavage are shown. ACAT1-56eNT, 43 or 46 amino acids from the asAmp-P88. (I) Activity of a recombined cryptic promoter upstream of the asAmp ORF region. Reporter plasmids containing the luciferase gene under the control of various-sized upstream sequences of the asAmp ORF region were transfected into HEK293 cells, and their relative luciferase activities were determined. Means and SDs are shown ( n = 3). Value with the promoter-less plasmid is defined as 1.0.

    Journal: Cell Research

    Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    doi: 10.1038/cr.2013.86

    Figure Lengend Snippet: An extra N-terminal region contained in human ACAT1 56-kDa isoform is encoded by a sequence present in recombinant Amp r -plasmids. (A - C) Western blot analysis of the expression of WT and mutant ACAT1 constructs. Constructs (numbers 1-23) containing the entire ACAT1 (A) , partial ACAT1 (B , C) or Rluc (C) were transfected separately into AC29 cells. Western blot analyses were performed with anti-ACAT1 or anti-Rluc antibodies. Filled circle, GGC 1274-1276 codon; hollow circle, ATG 1397-1399 codon; and underlined characters, mutated nucleotides. (D) Calculated molecular weights (MW) of proteins initiated from the GGC 1274-1276 , ATG 1274-1276 and ATG 1397-1399 codons. ACAT1-50NT, 130 amino acids encoded by the sequence from ATG 1397-1399 to GAT 1784-1786 . ACAT1-56uNT, 41 amino acids upstream of ACAT1-50NT. Filled circle, glycine; hollow circle, methionine; and aa, amino acids. (E) Purification and 2-D electrophoresis of the 26-kDa ACAT1 protein. Construct pNTF-number 10 (B) was expressed in AC29 cells to generate the 26-kDa ACAT1 protein. The proteins were purified with ANTI-FLAG M2 Affinity Gel and separated by 2-D electrophoresis. Arrows indicate spots of different 26-kDa proteins designated as P1, P2 and P3. P CMV , CMV promoter (black arrow); ori, ColE1 origin (gray arrow). (F) MALDI-TOF MS analysis of the purified 26-kDa proteins. Mass spectra are recorded in the positive mode and listed for the purified 26-kDa protein P2. (G) The N-terminal region of the purified P2 protein and its origin. The 15 amino acids (bold) were determined by the N-terminal sequencing of the purified P2 protein; they are encoded by the underlined sequence that matches the sequence of asAmp derived from recombinant plasmids (asAmp, tessellated box). The amino acid sequence of asAmp-encoding p88 protein (asAmp-P88) and the region of a recombined cryptic promoter are shown. (H) Schematic representation of human ACAT1-56NT region. Italics indicate the N-terminal 15 amino acids of the purified P2 protein. Asterisks indicate modifications by iodoacetamide on cysteines. Trypsin cleavage sites are indicated by bold characters and calculated MWs of representative peptides (underlined) after trypsin cleavage are shown. ACAT1-56eNT, 43 or 46 amino acids from the asAmp-P88. (I) Activity of a recombined cryptic promoter upstream of the asAmp ORF region. Reporter plasmids containing the luciferase gene under the control of various-sized upstream sequences of the asAmp ORF region were transfected into HEK293 cells, and their relative luciferase activities were determined. Means and SDs are shown ( n = 3). Value with the promoter-less plasmid is defined as 1.0.

    Article Snippet: Identification of the recombined cryptic promoter by luciferase activity analysis Upstream regions of asAmp-P88 ORF were amplified from plasmid pcDNA3 by PCR with individual forward primers asAmp-u1000-F (5′-AAACTCGAGAAAGGCGGTAATACGGTTATCCACA-3′), asAmp-u850-F (5′-AAACTCGAGTCGACGCTCAAGTCAGAGGTGG-3′), asAmp-u500-F (5′-AAACTCGAGGCGGTGCTACAGAGTTCTTGAAGTG-3′), asAmp-u275-F (5′-AAACTCGAGGTCTGACGCTCAGTGGAACGAAAA-3′) and common reverse primer asAmp-ATG-R (5′-AAAAAGCTTTGCAGCACTGGGGCCAGATG-3′), and inserted into Xho I and Hind III sites of pGL3-B (Promega) to generate reporter plasmids pasAmp-u1000, pasAmp-u850, pasAmp-u500 and pasAmp-u275, respectively.

    Techniques: Sequencing, Recombinant, Western Blot, Expressing, Mutagenesis, Construct, Transfection, Purification, Electrophoresis, Mass Spectrometry, Derivative Assay, Activity Assay, Luciferase, Plasmid Preparation

    The peptides encoded by the five sequence-dependent CPuORFs act in cis. ( A ) Schematic representation of the 35S::UTR:FLUC and 35S::UTR:RLUC reporter constructs. ( B – F ) Transient expression studies of the co-transfected 35S::UTR:RLUC and 35S::UTR:FLUC reporter plasmids. MM2d protoplasts were co-transfected with three plasmids, the 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids and the 35S::GUS internal control plasmid, by PEG treatment. The 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids contained the WT or fs version of the ANAC082 (B), CIPK6 (C), At3g15430 (D), At5g27920 (E) or OTLD1 (F) CPuORFs. Co-transfection was carried out for all four combinations for each CPuORF, as indicated. After 48 h of incubation, the transfected cells were harvested and disrupted for luciferase and GUS assays. FLUC and RLUC activities were normalized to GUS activity, and the FLUC and RLUC activities relative to those in the experiment where both reporter plasmids had the WT CPuORF were calculated. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. In each graph, bars with the same colors are not significantly different, whereas bars with different colors differ significantly ( P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: The peptides encoded by the five sequence-dependent CPuORFs act in cis. ( A ) Schematic representation of the 35S::UTR:FLUC and 35S::UTR:RLUC reporter constructs. ( B – F ) Transient expression studies of the co-transfected 35S::UTR:RLUC and 35S::UTR:FLUC reporter plasmids. MM2d protoplasts were co-transfected with three plasmids, the 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids and the 35S::GUS internal control plasmid, by PEG treatment. The 35S::UTR:FLUC and 35S::UTR:RLUC reporter plasmids contained the WT or fs version of the ANAC082 (B), CIPK6 (C), At3g15430 (D), At5g27920 (E) or OTLD1 (F) CPuORFs. Co-transfection was carried out for all four combinations for each CPuORF, as indicated. After 48 h of incubation, the transfected cells were harvested and disrupted for luciferase and GUS assays. FLUC and RLUC activities were normalized to GUS activity, and the FLUC and RLUC activities relative to those in the experiment where both reporter plasmids had the WT CPuORF were calculated. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. In each graph, bars with the same colors are not significantly different, whereas bars with different colors differ significantly ( P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Sequencing, Activated Clotting Time Assay, Construct, Expressing, Transfection, Plasmid Preparation, Cotransfection, Incubation, Luciferase, Activity Assay

    Alanine scanning of the five sequence-dependent CPuORFs. ( A – E ) Effects of Ala substitutions and conservation scores of amino acid residues in the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E) CPuORFs. The 35S::UTR:RLUC reporter plasmid harboring a WT CPuORF or its mutant with an Ala substitution was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of at least two separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences from the corresponding WT at P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: Alanine scanning of the five sequence-dependent CPuORFs. ( A – E ) Effects of Ala substitutions and conservation scores of amino acid residues in the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E) CPuORFs. The 35S::UTR:RLUC reporter plasmid harboring a WT CPuORF or its mutant with an Ala substitution was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of at least two separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences from the corresponding WT at P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Sequencing, Plasmid Preparation, Mutagenesis, Transfection, Electroporation

    Effect of the fs mutation in the presence and absence of the uORF start codon. ( A – E ) Transient expression studies of the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E) CPuORFs. The 35S::UTR:RLUC reporter plasmids carrying the WT CPuORF, the mutant CPuORF lacking the start codon (ΔAUG), the fs mutant CPuORF or fs ΔAUG double mutant CPuORF of each gene was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Double asterisks indicate a significant difference between two constructs ( P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: Effect of the fs mutation in the presence and absence of the uORF start codon. ( A – E ) Transient expression studies of the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E) CPuORFs. The 35S::UTR:RLUC reporter plasmids carrying the WT CPuORF, the mutant CPuORF lacking the start codon (ΔAUG), the fs mutant CPuORF or fs ΔAUG double mutant CPuORF of each gene was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Double asterisks indicate a significant difference between two constructs ( P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Electroporation, Construct

    Search for sequence-dependent regulatory uORFs. ( A ) Schematic representation of the WT ( 35S::UTR(WT):RLUC ) and fs mutant ( 35S::UTR(fs):RLUC ) reporter constructs. The hatched box in the fs mutant CPuORF shows the frame-shifted region. Although only a single uORF is depicted in each construct, the actual 5′-UTRs of some genes have multiple uORFs. See Figure 1 and Supplementary Figure S1 for the exact 5′-UTR structure of each gene and the exact positions of the fs mutations in each CPuORF. The polyadenylation signal of the Agrobacterium tumefaciens NOS gene is designated as ‘ter’. ( B and C ) Transient expression studies of class I (B) and class II (C) CPuORFs. The reporter plasmids containing the WT or fs mutant CPuORF of each gene were co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation. After 48 h of incubation, the transfected cells were harvested and disrupted for luciferase assay. RLUC activity was normalized to FLUC activity, and the relative activity to that of the corresponding WT construct was calculated. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences between the WT and fs constructs at P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: Search for sequence-dependent regulatory uORFs. ( A ) Schematic representation of the WT ( 35S::UTR(WT):RLUC ) and fs mutant ( 35S::UTR(fs):RLUC ) reporter constructs. The hatched box in the fs mutant CPuORF shows the frame-shifted region. Although only a single uORF is depicted in each construct, the actual 5′-UTRs of some genes have multiple uORFs. See Figure 1 and Supplementary Figure S1 for the exact 5′-UTR structure of each gene and the exact positions of the fs mutations in each CPuORF. The polyadenylation signal of the Agrobacterium tumefaciens NOS gene is designated as ‘ter’. ( B and C ) Transient expression studies of class I (B) and class II (C) CPuORFs. The reporter plasmids containing the WT or fs mutant CPuORF of each gene were co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation. After 48 h of incubation, the transfected cells were harvested and disrupted for luciferase assay. RLUC activity was normalized to FLUC activity, and the relative activity to that of the corresponding WT construct was calculated. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences between the WT and fs constructs at P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Sequencing, Mutagenesis, Construct, Expressing, Transfection, Plasmid Preparation, Electroporation, Incubation, Luciferase, Activity Assay

    The 3′-non-conserved region and the stop codon of the OTLD1 CPuORF are not important for the peptide sequence-dependent repressive effect. ( A ) Amino acid sequences of the WT OTLD1 CPuORF and its mutants with a deletion (ΔPWDI, ΔWDIL or ΔPWDIL) and/or a fs (fs2) mutation. The frameshifted region in the fs2 mutant is underlined. Hyphens indicate the deleted amino acid residues. ( B ) Transient expression assay to test the effect of the C-terminal deletion series. ( C ) Amino acid sequences of the WT OTLD1 CPuORF and its mutants with the Ala substitution of the stop codon (Stop39A) and/or a fs mutation. Asterisks represent stop codons. The frameshifted region in the fs mutant is underlined. ( D ) Transient expression assay to examine the effect of uORF stop codon elimination. In (B) and (D), the 35S::UTR:RLUC reporter plasmids containing the WT or mutant OTLD1 CPuORF whose sequence is presented in (A) and (C), respectively, was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of four and three biological replicates are shown in (B) and (D), respectively. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Double asterisks indicate a significant difference between two constructs ( P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: The 3′-non-conserved region and the stop codon of the OTLD1 CPuORF are not important for the peptide sequence-dependent repressive effect. ( A ) Amino acid sequences of the WT OTLD1 CPuORF and its mutants with a deletion (ΔPWDI, ΔWDIL or ΔPWDIL) and/or a fs (fs2) mutation. The frameshifted region in the fs2 mutant is underlined. Hyphens indicate the deleted amino acid residues. ( B ) Transient expression assay to test the effect of the C-terminal deletion series. ( C ) Amino acid sequences of the WT OTLD1 CPuORF and its mutants with the Ala substitution of the stop codon (Stop39A) and/or a fs mutation. Asterisks represent stop codons. The frameshifted region in the fs mutant is underlined. ( D ) Transient expression assay to examine the effect of uORF stop codon elimination. In (B) and (D), the 35S::UTR:RLUC reporter plasmids containing the WT or mutant OTLD1 CPuORF whose sequence is presented in (A) and (C), respectively, was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of four and three biological replicates are shown in (B) and (D), respectively. Each graph is representative of two or more separate experiments using independently prepared protoplasts. Double asterisks indicate a significant difference between two constructs ( P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Sequencing, Mutagenesis, Expressing, Transfection, Plasmid Preparation, Electroporation, Construct

    Effects of synonymous codon changes in the five CPuORFs. ( A – F ) Transient expression studies to compare the effects of Ala substitutions and synonymous codon changes in the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E and F) CPuORFs. The 35S::UTR:RLUC reporter plasmid harboring a WT CPuORF or its mutant with an Ala substitution or a synonymous codon change was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of three separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences between two constructs at P

    Journal: Nucleic Acids Research

    Article Title: Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    doi: 10.1093/nar/gkv018

    Figure Lengend Snippet: Effects of synonymous codon changes in the five CPuORFs. ( A – F ) Transient expression studies to compare the effects of Ala substitutions and synonymous codon changes in the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E and F) CPuORFs. The 35S::UTR:RLUC reporter plasmid harboring a WT CPuORF or its mutant with an Ala substitution or a synonymous codon change was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2 . Means ± S.D. of at least three biological replicates are shown. Each graph is representative of three separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences between two constructs at P

    Article Snippet: To construct this vector, we first digested plasmid pSY209 , which contains the AdoMetDC1 5′-UTR and the RLUC coding sequence in the pSP64 Poly(A) vector (Promega), with XbaI and SmaI at sites downstream of RLUC , treated the digested DNA with a T4 DNA polymerase to fill in the XbaI end, and then ligated the blunt-ended XbaI site to the SmaI site to remove the XbaI and SmaI sites.

    Techniques: Expressing, Plasmid Preparation, Mutagenesis, Transfection, Electroporation, Construct