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TaKaRa polymerase chain reaction pcr fragments
Ectopically expressed intronless <t>TSG101</t> gene (cDNA) is spliced and generates equivalent product as the endogenous aberrant mRNA in cancer cells. ( A ) The structure of the TSG101 mRNA. Red line and triangles indicate the postulated mRNA re-splicing via activated alternative 5′ and 3′ splice sites. Blue triangle indicates the PTC (1112–1114) generated by the postulated splicing. Black flags indicate the open reading frame (ORF; 127–1296). The positions of the <t>PCR</t> primers (P1–P4, P7–P10) are shown. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous TSG101 mRNAs were analyzed by RT-PCR (RT) and the genomic DNA was analyzed by PCR (Ge) with primer sets (P1–P4, P7–P10) annealed to the indicated exons. The black and red arrowheads denote the full-length and major aberrant mRNAs, respectively ( Figure 1 ). ( C ) The structures of three reporter TSG101-EGFP plasmids (see (A) for the red and blue triangles). The postulated splicing-dependent EGFP expression of these plasmids is indicated with (−) and (+). The positions of the PCR primer sets (P1–P5, P3–P6) are shown. ( D ) Cancer cells (MCF-7) were transfected with the indicated TSG101-EGFP reporter plasmids and the EGFP expression was analyzed by fluorescence microscopy. ( E ) These ectopically expressed TSG101-EGFP transcripts were analyzed by RT-PCR. The black and red arrowheads indicate the unspliced and spliced products via the alternative splice sites, respectively. Two minor spliced products (indicated with * and **) were generated from different alternative 3′ splice sites, i.e. Δ190–776 (587-nt deletion) and Δ190–1236 (1047-nt deletion), respectively.
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1) Product Images from "Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites"

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks520

Ectopically expressed intronless TSG101 gene (cDNA) is spliced and generates equivalent product as the endogenous aberrant mRNA in cancer cells. ( A ) The structure of the TSG101 mRNA. Red line and triangles indicate the postulated mRNA re-splicing via activated alternative 5′ and 3′ splice sites. Blue triangle indicates the PTC (1112–1114) generated by the postulated splicing. Black flags indicate the open reading frame (ORF; 127–1296). The positions of the PCR primers (P1–P4, P7–P10) are shown. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous TSG101 mRNAs were analyzed by RT-PCR (RT) and the genomic DNA was analyzed by PCR (Ge) with primer sets (P1–P4, P7–P10) annealed to the indicated exons. The black and red arrowheads denote the full-length and major aberrant mRNAs, respectively ( Figure 1 ). ( C ) The structures of three reporter TSG101-EGFP plasmids (see (A) for the red and blue triangles). The postulated splicing-dependent EGFP expression of these plasmids is indicated with (−) and (+). The positions of the PCR primer sets (P1–P5, P3–P6) are shown. ( D ) Cancer cells (MCF-7) were transfected with the indicated TSG101-EGFP reporter plasmids and the EGFP expression was analyzed by fluorescence microscopy. ( E ) These ectopically expressed TSG101-EGFP transcripts were analyzed by RT-PCR. The black and red arrowheads indicate the unspliced and spliced products via the alternative splice sites, respectively. Two minor spliced products (indicated with * and **) were generated from different alternative 3′ splice sites, i.e. Δ190–776 (587-nt deletion) and Δ190–1236 (1047-nt deletion), respectively.
Figure Legend Snippet: Ectopically expressed intronless TSG101 gene (cDNA) is spliced and generates equivalent product as the endogenous aberrant mRNA in cancer cells. ( A ) The structure of the TSG101 mRNA. Red line and triangles indicate the postulated mRNA re-splicing via activated alternative 5′ and 3′ splice sites. Blue triangle indicates the PTC (1112–1114) generated by the postulated splicing. Black flags indicate the open reading frame (ORF; 127–1296). The positions of the PCR primers (P1–P4, P7–P10) are shown. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous TSG101 mRNAs were analyzed by RT-PCR (RT) and the genomic DNA was analyzed by PCR (Ge) with primer sets (P1–P4, P7–P10) annealed to the indicated exons. The black and red arrowheads denote the full-length and major aberrant mRNAs, respectively ( Figure 1 ). ( C ) The structures of three reporter TSG101-EGFP plasmids (see (A) for the red and blue triangles). The postulated splicing-dependent EGFP expression of these plasmids is indicated with (−) and (+). The positions of the PCR primer sets (P1–P5, P3–P6) are shown. ( D ) Cancer cells (MCF-7) were transfected with the indicated TSG101-EGFP reporter plasmids and the EGFP expression was analyzed by fluorescence microscopy. ( E ) These ectopically expressed TSG101-EGFP transcripts were analyzed by RT-PCR. The black and red arrowheads indicate the unspliced and spliced products via the alternative splice sites, respectively. Two minor spliced products (indicated with * and **) were generated from different alternative 3′ splice sites, i.e. Δ190–776 (587-nt deletion) and Δ190–1236 (1047-nt deletion), respectively.

Techniques Used: Generated, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Fluorescence, Microscopy

Ectopically expressed intronless FHIT gene (cDNA) is spliced, generating a product equivalent to the endogenous aberrant mRNA in cancer cells. ( A ) Schematic representation of the FHIT pre-mRNA and the observed aberrant splicing in cancer cells (red line). The aligned sequences of the normal (full-length) and aberrant FHIT mRNAs reveal the skipping of exon 3 to exon 6. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous FHIT mRNAs (schematically shown) were analyzed by RT-PCR with the indicated primer sets (P25–P26, P25′–P26′). The full-length mRNA (black arrowhead) and aberrantly spliced mRNA (red arrowhead) are indicated. Black flags indicate the ORF (372–815) and the red line indicates the postulated mRNA re-splicing that skips the whole exon 3 to exon 6 region. ( C ) Cancer cells (MCF-7) were transfected with the FHIT-EGFP reporter plasmid (schematically shown) and the spliced products were analyzed by RT-PCR with the indicated primer sets (P25–P5, P25′–P6). The black and red arrowheads indicate the unspliced and spliced products, respectively. Sequence analysis mapped the spliced sites (red triangles) to the 5′ end of exon 3 and the 3′ end of exon 6 (data not shown).
Figure Legend Snippet: Ectopically expressed intronless FHIT gene (cDNA) is spliced, generating a product equivalent to the endogenous aberrant mRNA in cancer cells. ( A ) Schematic representation of the FHIT pre-mRNA and the observed aberrant splicing in cancer cells (red line). The aligned sequences of the normal (full-length) and aberrant FHIT mRNAs reveal the skipping of exon 3 to exon 6. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous FHIT mRNAs (schematically shown) were analyzed by RT-PCR with the indicated primer sets (P25–P26, P25′–P26′). The full-length mRNA (black arrowhead) and aberrantly spliced mRNA (red arrowhead) are indicated. Black flags indicate the ORF (372–815) and the red line indicates the postulated mRNA re-splicing that skips the whole exon 3 to exon 6 region. ( C ) Cancer cells (MCF-7) were transfected with the FHIT-EGFP reporter plasmid (schematically shown) and the spliced products were analyzed by RT-PCR with the indicated primer sets (P25–P5, P25′–P6). The black and red arrowheads indicate the unspliced and spliced products, respectively. Sequence analysis mapped the spliced sites (red triangles) to the 5′ end of exon 3 and the 3′ end of exon 6 (data not shown).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Sequencing

The lariat RNA containing TSG101 exon 2 to exon 9 was identified by sequencing. ( A ) Detection of the lariat exons by lariat-specific RT-PCR amplification across the branch point with the indicated primer sets (P23–P24, P23′–P24′). The RT-PCR signal remained in the RNase R-digested (+) RNA sample, revealing a closed lariat structure. ( B ) Sequencing analysis of the lariat-specific RT-PCR product. Electropherogram of the sequence containing the branch point (arrow), followed by the alternative 5′ splice site (arrow) in the lariat exons. ( C ) A sequence alignment of the PCR fragment (blue) and the TSG101 gene (black) reveals a 2′–5′ branched connection between the end of the alternative 5′ splice site ( GT ) and the branch point ( A ), which is located upstream from the alternative 3′ splice site ( AG ).
Figure Legend Snippet: The lariat RNA containing TSG101 exon 2 to exon 9 was identified by sequencing. ( A ) Detection of the lariat exons by lariat-specific RT-PCR amplification across the branch point with the indicated primer sets (P23–P24, P23′–P24′). The RT-PCR signal remained in the RNase R-digested (+) RNA sample, revealing a closed lariat structure. ( B ) Sequencing analysis of the lariat-specific RT-PCR product. Electropherogram of the sequence containing the branch point (arrow), followed by the alternative 5′ splice site (arrow) in the lariat exons. ( C ) A sequence alignment of the PCR fragment (blue) and the TSG101 gene (black) reveals a 2′–5′ branched connection between the end of the alternative 5′ splice site ( GT ) and the branch point ( A ), which is located upstream from the alternative 3′ splice site ( AG ).

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

Detection of the lariat RNA consisting of only exons demonstrates the re-splicing of the endogenous TSG101 mRNA. ( A ) Schematic representation of the two postulated pathways leading to the generation of the aberrant mRNA, i.e. a conventional one-step direct aberrant splicing (right) and the proposed two-step process, including the re-splicing of the constitutively spliced mRNA (left). The pre-mRNA [1] and specific splicing products [2–5] from these two pathways were analyzed by RT-PCR with the indicated primers (arrows). ( B ) Detection of the specific splicing products [2–5] by RT-PCR using RNase R-digested (+) or RNase R-undigested (−) endogenous total RNA. The detected RT-PCR signals in the RNase R-digested sample (containing no linear RNAs) indicates an RNA species with either a 5′–2′ lariat or a 5′–3′ circular structure. However, the latter case was ruled out by the identification of a 5′–2′ branched structure ( Figure 4 A). Primers P19, P20, P21 and P22 anneal to introns 6, 7, 7 and 8, respectively. Primers P13/14 and P15/16 anneal to introns 7 and 8, respectively. Primers P11, P12, P17 and P18 anneal to exons 8, 10, 5 and 8, respectively. We used exactly the same PCR cycle numbers to amplify all these RNAs ( Supplementary Materials and Methods ).
Figure Legend Snippet: Detection of the lariat RNA consisting of only exons demonstrates the re-splicing of the endogenous TSG101 mRNA. ( A ) Schematic representation of the two postulated pathways leading to the generation of the aberrant mRNA, i.e. a conventional one-step direct aberrant splicing (right) and the proposed two-step process, including the re-splicing of the constitutively spliced mRNA (left). The pre-mRNA [1] and specific splicing products [2–5] from these two pathways were analyzed by RT-PCR with the indicated primers (arrows). ( B ) Detection of the specific splicing products [2–5] by RT-PCR using RNase R-digested (+) or RNase R-undigested (−) endogenous total RNA. The detected RT-PCR signals in the RNase R-digested sample (containing no linear RNAs) indicates an RNA species with either a 5′–2′ lariat or a 5′–3′ circular structure. However, the latter case was ruled out by the identification of a 5′–2′ branched structure ( Figure 4 A). Primers P19, P20, P21 and P22 anneal to introns 6, 7, 7 and 8, respectively. Primers P13/14 and P15/16 anneal to introns 7 and 8, respectively. Primers P11, P12, P17 and P18 anneal to exons 8, 10, 5 and 8, respectively. We used exactly the same PCR cycle numbers to amplify all these RNAs ( Supplementary Materials and Methods ).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

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Article Title: Functional Analysis of Cyclin-Dependent Kinase Inhibitors of Arabidopsis
Article Snippet: .. The resulting polymerase chain reaction (PCR) fragments were cut with EcoRI and BamHI and cloned into the EcoRI and BamHI sites of pGBT9 and pGAD424 (Clontech, Palo Alto, CA), resulting in the vectors pGADKRP4, pGADKRP5, pGADKRP6, pGADKRP7, pGBTKRP4, pGBTKRP5, pGBTKRP6, and pGBTKRP7. .. Binding specificity of the different Kip-related proteins (KRPs) toward CDKA;1, CDKB1;1, and CYCD4 was tested as described ( ).

Amplification:

Article Title: Adjusting the attB Site in Donor Plasmid Improves the Efficiency of ?C31 Integrase System
Article Snippet: .. The attB site was amplified using the primers attBPF and attBPR , and the resulting polymerase chain reaction (PCR) fragments were inserted into the Pci I sites of pEGFP-N1 (Clontech, Mountain View, CA). .. The plasmids of pBtta-EGFP-N1 (reverse direction for attB ), pattB-EGFP-N1 (forward direction of attB ), and p2attB-EGFP-N1 (tandem with two attB s) were then constructed ( ).

Subcloning:

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites
Article Snippet: .. Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ). .. The FHIT-EGFP fusion plasmid (FHIT-EGFP; C) was constructed with the same procedure.

Construct:

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites
Article Snippet: .. Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ). .. The FHIT-EGFP fusion plasmid (FHIT-EGFP; C) was constructed with the same procedure.

Generated:

Article Title: Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor
Article Snippet: .. Polymerase chain reaction (PCR) fragments were generated using PrimeSTAR Max DNA Polymerase (TaKaRa). .. The method for plasmid construction is described below.

Expressing:

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites
Article Snippet: .. Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ). .. The FHIT-EGFP fusion plasmid (FHIT-EGFP; C) was constructed with the same procedure.

Polymerase Chain Reaction:

Article Title: Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor
Article Snippet: .. Polymerase chain reaction (PCR) fragments were generated using PrimeSTAR Max DNA Polymerase (TaKaRa). .. The method for plasmid construction is described below.

Article Title: The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA
Article Snippet: .. After transformation with polymerase chain reaction (PCR) fragments, yeast strains harboring mycoplasma genomes were grown in minimal SD Base medium (Clontech) with the supplements −HIS DO (SD minus HIS medium, Clontech), or with −HIS −URA DO (SD minus HIS minus URA medium, Clontech). ..

Article Title: Adjusting the attB Site in Donor Plasmid Improves the Efficiency of ?C31 Integrase System
Article Snippet: .. The attB site was amplified using the primers attBPF and attBPR , and the resulting polymerase chain reaction (PCR) fragments were inserted into the Pci I sites of pEGFP-N1 (Clontech, Mountain View, CA). .. The plasmids of pBtta-EGFP-N1 (reverse direction for attB ), pattB-EGFP-N1 (forward direction of attB ), and p2attB-EGFP-N1 (tandem with two attB s) were then constructed ( ).

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites
Article Snippet: .. Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ). .. The FHIT-EGFP fusion plasmid (FHIT-EGFP; C) was constructed with the same procedure.

Article Title: Functional Analysis of Cyclin-Dependent Kinase Inhibitors of Arabidopsis
Article Snippet: .. The resulting polymerase chain reaction (PCR) fragments were cut with EcoRI and BamHI and cloned into the EcoRI and BamHI sites of pGBT9 and pGAD424 (Clontech, Palo Alto, CA), resulting in the vectors pGADKRP4, pGADKRP5, pGADKRP6, pGADKRP7, pGBTKRP4, pGBTKRP5, pGBTKRP6, and pGBTKRP7. .. Binding specificity of the different Kip-related proteins (KRPs) toward CDKA;1, CDKB1;1, and CYCD4 was tested as described ( ).

Article Title: Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7
Article Snippet: .. Polymerase chain reaction (PCR) fragments were then digested via BglII/XhoI and ligated in pEYFP-C1 or mCherry-C1 (Clontech, Mountain View, CA). .. Cloning into pEGFP-N1 (Clontech)—for the expression of the LBR constructs without any tag—was performed using a stop codon for termination and the XhoI/BamHI restriction sites.

Transformation Assay:

Article Title: The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA
Article Snippet: .. After transformation with polymerase chain reaction (PCR) fragments, yeast strains harboring mycoplasma genomes were grown in minimal SD Base medium (Clontech) with the supplements −HIS DO (SD minus HIS medium, Clontech), or with −HIS −URA DO (SD minus HIS minus URA medium, Clontech). ..

Plasmid Preparation:

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites
Article Snippet: .. Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ). .. The FHIT-EGFP fusion plasmid (FHIT-EGFP; C) was constructed with the same procedure.

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    TaKaRa oct4 promoter
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    TaKaRa gbx2 fragment
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    Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

    Journal: Protein & Cell

    Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

    doi: 10.1007/s13238-019-0608-1

    Figure Lengend Snippet: Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

    Article Snippet: The modified genomic fragment of OCT4 promoter was amplified using LA Taq Hot StartVersion (TAKARA) as previously described (Duan et al., ).

    Techniques: Mutagenesis, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, DNA Methylation Assay, Methylation, Cell Cycle Assay

    Transcriptional activity of the 56 Tktl1 in one- and two-cell-stage embryos. Plasmids (200 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos and the nuclei of early and late two-cell-stage embryos. Microinjection was performed at 9, 18 and 27 h after insemination in one-cell and early and late two-cell-stage embryos, respectively. Experiments were performed four times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: Transcriptional activity of the 56 Tktl1 in one- and two-cell-stage embryos. Plasmids (200 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos and the nuclei of early and late two-cell-stage embryos. Microinjection was performed at 9, 18 and 27 h after insemination in one-cell and early and late two-cell-stage embryos, respectively. Experiments were performed four times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: Activity Assay

    Effect of DNA synthesis inhibition on Tktl1 transcriptional activity in two-cell-stage embryos. Transcription of p Tktl1-2467+25 was measured in embryos in which DNA synthesis was inhibited by treatment with aphidicolin. Experiments were performed three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: Effect of DNA synthesis inhibition on Tktl1 transcriptional activity in two-cell-stage embryos. Transcription of p Tktl1-2467+25 was measured in embryos in which DNA synthesis was inhibited by treatment with aphidicolin. Experiments were performed three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: DNA Synthesis, Inhibition, Activity Assay

    The elements required for transcriptional activity of the core promoter region of Tktl1 . (A) Transcription-factor-binding sites in the 56 bp upstream of the TSS of Tktl1 . (B) Effects of mutation of the GC box and TATA box-like elements on the transcriptional activity of p Tktl1-56 . The sequences GGGCGG (GC box) and TTTTAA (TATA box like) were mutated to GGAAAG and TTGCGA , respectively. Plasmids (500 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos. Experiments were performed three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: The elements required for transcriptional activity of the core promoter region of Tktl1 . (A) Transcription-factor-binding sites in the 56 bp upstream of the TSS of Tktl1 . (B) Effects of mutation of the GC box and TATA box-like elements on the transcriptional activity of p Tktl1-56 . The sequences GGGCGG (GC box) and TTTTAA (TATA box like) were mutated to GGAAAG and TTGCGA , respectively. Plasmids (500 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos. Experiments were performed three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: Activity Assay, Binding Assay, Mutagenesis

    Transcriptional activity of the Tktl1 promoter in oocytes and preimplantation embryos. Plasmids containing the promoter regions of the Tktl1 (p Tktl1-2467+25 ) and Zp3 genes were microinjected into growing oocytes (A), one-cell-stage embryos (B), and early and late two-cell-stage embryos (C and D), and their transcriptional activities were then evaluated by luciferase reporter gene assay. The transcriptional activity of a plasmid without a promoter (None) was used as a control to determine the background level. Plasmids (200 ng/µl) were microinjected into the nucleus. Experiments were performed at least three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: Transcriptional activity of the Tktl1 promoter in oocytes and preimplantation embryos. Plasmids containing the promoter regions of the Tktl1 (p Tktl1-2467+25 ) and Zp3 genes were microinjected into growing oocytes (A), one-cell-stage embryos (B), and early and late two-cell-stage embryos (C and D), and their transcriptional activities were then evaluated by luciferase reporter gene assay. The transcriptional activity of a plasmid without a promoter (None) was used as a control to determine the background level. Plasmids (200 ng/µl) were microinjected into the nucleus. Experiments were performed at least three times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: Activity Assay, Luciferase, Reporter Gene Assay, Plasmid Preparation

    Transcriptional activity of Tktl1 -promoter deletion mutants in one-cell-stage embryos. (A) The upstream region of the Tktl1 promoter ( Tktl1-2467+25 ) was deleted to leave 227 bp ( Tktl1-227+25 ) and 56 bp ( Tktl1-56+25 ), and transcriptional activity was then evaluated. (B) The 25 bp downstream of the TSS was deleted from p Tktl1-56+25 and transcriptional activity was measured. Plasmids (500 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos. The experiments were performed at least 11 times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: Transcriptional activity of Tktl1 -promoter deletion mutants in one-cell-stage embryos. (A) The upstream region of the Tktl1 promoter ( Tktl1-2467+25 ) was deleted to leave 227 bp ( Tktl1-227+25 ) and 56 bp ( Tktl1-56+25 ), and transcriptional activity was then evaluated. (B) The 25 bp downstream of the TSS was deleted from p Tktl1-56+25 and transcriptional activity was measured. Plasmids (500 ng/µl) were microinjected into the male pronuclei of one-cell-stage embryos. The experiments were performed at least 11 times and the results are presented as means ± SEM. Asterisks indicate significant differences (Student's t -test; P

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: Activity Assay

    Expression of Tktl1 mRNA during preimplantation development. The expression of Tktl1 in MII stage oocytes and preimplantation embryos was assessed by semi-quantitative RT-PCR. Embryos were collected at the following time points after insemination: one-cell stage, 13 h; two-cell stage, 28 h; 4-cell stage, 45 h; morula, 60 h; and blastocyst, 96 h. The experiments were performed four times and the data are presented as means ± SEM. The value for two-cell-stage embryos was set to 1.0 and relative values were calculated for the other stages.

    Journal: PLoS ONE

    Article Title: Regulation of Transketolase Like 1 Gene Expression in the Murine One-Cell Stage Embryos

    doi: 10.1371/journal.pone.0082087

    Figure Lengend Snippet: Expression of Tktl1 mRNA during preimplantation development. The expression of Tktl1 in MII stage oocytes and preimplantation embryos was assessed by semi-quantitative RT-PCR. Embryos were collected at the following time points after insemination: one-cell stage, 13 h; two-cell stage, 28 h; 4-cell stage, 45 h; morula, 60 h; and blastocyst, 96 h. The experiments were performed four times and the data are presented as means ± SEM. The value for two-cell-stage embryos was set to 1.0 and relative values were calculated for the other stages.

    Article Snippet: A fragment of Tktl1 promoter was amplified by PCR using genome template from a C57BL6 mouse and a primer set shown in , and cloned into multi-cloning site in pEluc-test plasmid by In-fusion advantage cloning kit (Clontech, Shiga).

    Techniques: Expressing, Quantitative RT-PCR

    Rmnd5 is part of an ubiquitin ligase complex. (A) Glycerol step gradient of Xenopus laevis NF stage 36 embryo lysates. Molecular mass (MW) standard: albumin (67 kDa), fraction 1, 2; LDH (140 kDa), fraction 4; catalase (232 kDa), fraction 6,7. Western blot analysis with α-RMND5A (Rmnd5; upper panel) (1:1000) and α-ARMC8 (lower panel) (1:1000). (B) In vitro polyubiquitination assay with recombinant Xenopus Rmnd5 and Rmnd5-C354S (lane 3, 4). Reactions are performed in the presence (+) or absence (-) of E1 (lane 1), E2 (lane 2) and purified Rmnd5 protein. HDM2 is used as a positive control (lane 5). Polyubiquitination (Poly-Ub) is detected with α-HA and α-RMND5A as control.

    Journal: PLoS ONE

    Article Title: RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    doi: 10.1371/journal.pone.0120342

    Figure Lengend Snippet: Rmnd5 is part of an ubiquitin ligase complex. (A) Glycerol step gradient of Xenopus laevis NF stage 36 embryo lysates. Molecular mass (MW) standard: albumin (67 kDa), fraction 1, 2; LDH (140 kDa), fraction 4; catalase (232 kDa), fraction 6,7. Western blot analysis with α-RMND5A (Rmnd5; upper panel) (1:1000) and α-ARMC8 (lower panel) (1:1000). (B) In vitro polyubiquitination assay with recombinant Xenopus Rmnd5 and Rmnd5-C354S (lane 3, 4). Reactions are performed in the presence (+) or absence (-) of E1 (lane 1), E2 (lane 2) and purified Rmnd5 protein. HDM2 is used as a positive control (lane 5). Polyubiquitination (Poly-Ub) is detected with α-HA and α-RMND5A as control.

    Article Snippet: Plasmid pTP250 contains the Xenopus laevis rmnd5 ORF amplified from pTP221 with primers TP250fwd and TP250rev, inserted via XhoI and HindIII into pEGFP-C1 (Clontech). pTP221 contains the Xenopus laevis rmnd5 ORF amplified from cDNA, which was inserted into EcoRI/XhoI linearized pTP213 by yeast homologous recombination and selection on CSD—URA medium. pTP230 is a pCA528 derivative containing Xenopus laevis rmnd5 that was cut from pTP221 and inserted into pCA528via XhoI and BamHI. pTP241 is a pTP230 derivative with a C354S mutation in the RMND5A gene SOMA-generated with primer TP226 [ ].

    Techniques: Western Blot, In Vitro, Recombinant, Purification, Positive Control

    rmnd5 is expressed during early embryonic development. (A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).

    Journal: PLoS ONE

    Article Title: RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    doi: 10.1371/journal.pone.0120342

    Figure Lengend Snippet: rmnd5 is expressed during early embryonic development. (A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).

    Article Snippet: Plasmid pTP250 contains the Xenopus laevis rmnd5 ORF amplified from pTP221 with primers TP250fwd and TP250rev, inserted via XhoI and HindIII into pEGFP-C1 (Clontech). pTP221 contains the Xenopus laevis rmnd5 ORF amplified from cDNA, which was inserted into EcoRI/XhoI linearized pTP213 by yeast homologous recombination and selection on CSD—URA medium. pTP230 is a pCA528 derivative containing Xenopus laevis rmnd5 that was cut from pTP221 and inserted into pCA528via XhoI and BamHI. pTP241 is a pTP230 derivative with a C354S mutation in the RMND5A gene SOMA-generated with primer TP226 [ ].

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, In Situ, Hybridization

    Xenopus laevis Rmnd5 protein is structurally and functionally related to human RMND5A. (A) Phylogenetic tree of Rmnd5 orthologs. The taxonomic tree of representative eukaryotic species rendered by Phylogeny.fr software [ 20 ]. Respective Gid2/Rmnd5 sequences obtained from NCBI with indicated accession numbers ( Saccharomyces cerevisiae [NP_010541.3], Candida albicans [XP_712238.1], Aspergillus niger [XP_001388791.2], Caenorhabditis elegans [NP_508444.1], Arabidopsis thaliana [NP_196525.1], Drosophila melanogaster [NP_611536.3], Xenopus laevis [NP_001086276.1], Falco peregrinus [XP_005229906.1], Gallus gallus [XP_004936301.1] Homo sapiens [NP_073617.1; NP_073599.2], Ornithorhynchus anatinus [XP_007670084.1; XP_001515875.2], Sarcophilus harrisii [XP_003758697.1; XP_003756956.1], Canis lupus familiaris [XP_852129.1; XP_531873.2], Mus musculus [NP_077250.2; NP_079622.1], Rattus norvegicus [XP_232051.4; NP_001017473.1]); homolog A (blue), homolog B (red). (B) Sequence alignment of Xenopus laevis Rmnd5 (top), Homo sapiens RMND5A (middle) and RMND5B (bottom). Identical residues (red), similar residues (blue), others (black). Identities (%): Xenopus laevis Rmnd5 to human RMND5A (94%), to human RMND5B (70%). (C) Localization of Homo sapiens RMND5A (RMND5a, middle panel), RMND5B (RMND5b, bottom panel) and Xenopus laevis Rmnd5 (Rmnd5, top panel) in HEK293 cells. GFP signal (left column), DAPI signal (middle column), merged signals (right column).

    Journal: PLoS ONE

    Article Title: RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    doi: 10.1371/journal.pone.0120342

    Figure Lengend Snippet: Xenopus laevis Rmnd5 protein is structurally and functionally related to human RMND5A. (A) Phylogenetic tree of Rmnd5 orthologs. The taxonomic tree of representative eukaryotic species rendered by Phylogeny.fr software [ 20 ]. Respective Gid2/Rmnd5 sequences obtained from NCBI with indicated accession numbers ( Saccharomyces cerevisiae [NP_010541.3], Candida albicans [XP_712238.1], Aspergillus niger [XP_001388791.2], Caenorhabditis elegans [NP_508444.1], Arabidopsis thaliana [NP_196525.1], Drosophila melanogaster [NP_611536.3], Xenopus laevis [NP_001086276.1], Falco peregrinus [XP_005229906.1], Gallus gallus [XP_004936301.1] Homo sapiens [NP_073617.1; NP_073599.2], Ornithorhynchus anatinus [XP_007670084.1; XP_001515875.2], Sarcophilus harrisii [XP_003758697.1; XP_003756956.1], Canis lupus familiaris [XP_852129.1; XP_531873.2], Mus musculus [NP_077250.2; NP_079622.1], Rattus norvegicus [XP_232051.4; NP_001017473.1]); homolog A (blue), homolog B (red). (B) Sequence alignment of Xenopus laevis Rmnd5 (top), Homo sapiens RMND5A (middle) and RMND5B (bottom). Identical residues (red), similar residues (blue), others (black). Identities (%): Xenopus laevis Rmnd5 to human RMND5A (94%), to human RMND5B (70%). (C) Localization of Homo sapiens RMND5A (RMND5a, middle panel), RMND5B (RMND5b, bottom panel) and Xenopus laevis Rmnd5 (Rmnd5, top panel) in HEK293 cells. GFP signal (left column), DAPI signal (middle column), merged signals (right column).

    Article Snippet: Plasmid pTP250 contains the Xenopus laevis rmnd5 ORF amplified from pTP221 with primers TP250fwd and TP250rev, inserted via XhoI and HindIII into pEGFP-C1 (Clontech). pTP221 contains the Xenopus laevis rmnd5 ORF amplified from cDNA, which was inserted into EcoRI/XhoI linearized pTP213 by yeast homologous recombination and selection on CSD—URA medium. pTP230 is a pCA528 derivative containing Xenopus laevis rmnd5 that was cut from pTP221 and inserted into pCA528via XhoI and BamHI. pTP241 is a pTP230 derivative with a C354S mutation in the RMND5A gene SOMA-generated with primer TP226 [ ].

    Techniques: Software, Sequencing

    Confirmation of murine GBX2 by Western blot and mass spectral analysis. (A) Western analysis of recombinant GBX2 proteins. The amino acid sequences for GBX2 (B) and GBX2Δ HD (B') recombinant proteins. Bold type indicates matched peptides identified by mass spectrometry. (C) MS/MS fragmentation data table includes: precursor mass (peptide chosen for MS/MS), approximate weight of the band analyzed by mass spectrometry, peptide sequence, location of the peptide in the protein sequence, the Mascot ion score, and the mass error for each peptide sequence. The low mass error score in parts per million (ppm = {[observed mass – theoretical mass]/theoretical mass}×10 6 ) suggests that the observed mass matches the theoretical mass.

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: Confirmation of murine GBX2 by Western blot and mass spectral analysis. (A) Western analysis of recombinant GBX2 proteins. The amino acid sequences for GBX2 (B) and GBX2Δ HD (B') recombinant proteins. Bold type indicates matched peptides identified by mass spectrometry. (C) MS/MS fragmentation data table includes: precursor mass (peptide chosen for MS/MS), approximate weight of the band analyzed by mass spectrometry, peptide sequence, location of the peptide in the protein sequence, the Mascot ion score, and the mass error for each peptide sequence. The low mass error score in parts per million (ppm = {[observed mass – theoretical mass]/theoretical mass}×10 6 ) suggests that the observed mass matches the theoretical mass.

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Western Blot, Recombinant, Mass Spectrometry, Sequencing

    GBX2 overexpression and ChIP in human PC-3 cells. (A) Schematic representation of the HA-GBX2 and HA-GBX2ΔHD recombinant proteins containing the proline-rich region (PR), DNA-binding homeodomain (HD), and the HA epitope tag located at the amino terminus. Immunoflouresence of transiently transfected human PC-3 cells with HA-GBX2 (C, D), and, HA-Gbx2 Δ HD (F, G). Blue channel identifies DAPI staining in the nucleus (B, E). Green channel identifies GFP-GBX2 fusion proteins. (D, G) Merge displays nuclear localization of GFP-GBX2 fusion proteins. Western blots of total lysates (H) and HA-immunoprecipitated samples (I) from mock, HA-Gbx2 , and HA-Gbx2 Δ HD transfected PC-3 cells.

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: GBX2 overexpression and ChIP in human PC-3 cells. (A) Schematic representation of the HA-GBX2 and HA-GBX2ΔHD recombinant proteins containing the proline-rich region (PR), DNA-binding homeodomain (HD), and the HA epitope tag located at the amino terminus. Immunoflouresence of transiently transfected human PC-3 cells with HA-GBX2 (C, D), and, HA-Gbx2 Δ HD (F, G). Blue channel identifies DAPI staining in the nucleus (B, E). Green channel identifies GFP-GBX2 fusion proteins. (D, G) Merge displays nuclear localization of GFP-GBX2 fusion proteins. Western blots of total lysates (H) and HA-immunoprecipitated samples (I) from mock, HA-Gbx2 , and HA-Gbx2 Δ HD transfected PC-3 cells.

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Over Expression, Chromatin Immunoprecipitation, Recombinant, Binding Assay, Transfection, Staining, Western Blot, Immunoprecipitation

    GBX2 binds to and functionally interacts within the EEF1A1 core promoter. (A) EEF1A1 locus depicting non-coding exons (white boxes), coding exons (black boxes), and the ChIP-Seq identified location of the GBX2 DNA-binding sequence (red bar). Alignment of the human and mouse ChIP-Seq identified EEF1A1 promoter region using sequences obtained from Ensembl [61] , EEF1A1 TATA box (underlined sequence) and GBX2 DNA-binding sequence (red box). (B) Gel-shift analysis for identified GBX2 target EEF1A1 . A reduction in the mobility of [ÿ - 32 P] ATP labeled EEF1A1 100-mer probe is observed with the addition of GBX2 (black arrows). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical EEF1A1 100-mer unlabeled specific competitor probes at 100x, 300x, and 500x molar concentrations in lanes 4–6. Addition of EEF1A1 45-mer unlabeled non-specific competitor probes, omitting the GBX2 DNA-binding sequence in lanes 7–9. (C) EEF1A1 promoter luciferase reporter assay. HEK 293 cells were transiently transfected with either the empty pGL4.10[ luc2 ] vector (white bar), the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter sequence and the TATA box (TATATAA; black bars), the pGL4.10[ luc2 ] vector containing the mutated EEF1A1 promoter sequence with a mutated TATA box (TATATAA changed to GCGCGCC; striped bars), and the pGL4.70[ hRluc ] Renilla vector. Substantial luciferase activity was observed in cells transfected with the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter compared to the empty pGL4.10[ luc2 ] reporter construct (compare lane 3 to lane 1). Maximal luciferase activation is observed upon the addition of GBX2 in cells with the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter sequence (compare lane 4 to lane 3), and activation is reduced in GBX2Δ HD (lane 6) cells and cells expressing GBX2 and the pGL4.10[ luc2 ] mutated EEF1A1 reporter construct (compare lane 4 to lane 5). Luciferase activities were normalized to Renilla luciferase activities. * P = 0.0047 (two-tailed P value).

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: GBX2 binds to and functionally interacts within the EEF1A1 core promoter. (A) EEF1A1 locus depicting non-coding exons (white boxes), coding exons (black boxes), and the ChIP-Seq identified location of the GBX2 DNA-binding sequence (red bar). Alignment of the human and mouse ChIP-Seq identified EEF1A1 promoter region using sequences obtained from Ensembl [61] , EEF1A1 TATA box (underlined sequence) and GBX2 DNA-binding sequence (red box). (B) Gel-shift analysis for identified GBX2 target EEF1A1 . A reduction in the mobility of [ÿ - 32 P] ATP labeled EEF1A1 100-mer probe is observed with the addition of GBX2 (black arrows). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical EEF1A1 100-mer unlabeled specific competitor probes at 100x, 300x, and 500x molar concentrations in lanes 4–6. Addition of EEF1A1 45-mer unlabeled non-specific competitor probes, omitting the GBX2 DNA-binding sequence in lanes 7–9. (C) EEF1A1 promoter luciferase reporter assay. HEK 293 cells were transiently transfected with either the empty pGL4.10[ luc2 ] vector (white bar), the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter sequence and the TATA box (TATATAA; black bars), the pGL4.10[ luc2 ] vector containing the mutated EEF1A1 promoter sequence with a mutated TATA box (TATATAA changed to GCGCGCC; striped bars), and the pGL4.70[ hRluc ] Renilla vector. Substantial luciferase activity was observed in cells transfected with the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter compared to the empty pGL4.10[ luc2 ] reporter construct (compare lane 3 to lane 1). Maximal luciferase activation is observed upon the addition of GBX2 in cells with the pGL4.10[ luc2 ] vector containing the functional EEF1A1 promoter sequence (compare lane 4 to lane 3), and activation is reduced in GBX2Δ HD (lane 6) cells and cells expressing GBX2 and the pGL4.10[ luc2 ] mutated EEF1A1 reporter construct (compare lane 4 to lane 5). Luciferase activities were normalized to Renilla luciferase activities. * P = 0.0047 (two-tailed P value).

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Functional Assay, Activity Assay, Construct, Activation Assay, Expressing, Two Tailed Test

    Binding to ROBO1 and regulation of NC cell patterning by GBX2. (A) Gel-shift analysis for identified GBX2 target ROBO1 . A reduction in the mobility of [ÿ -32P] ATP labeled ROBO1 100-mer probe is observed with the addition of GBX2 (black arrows), whereas no shift is observed with the addition of GBX2ΔHD (compare lane 2 to lane 4). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical ROBO1 100-mer unlabeled specific competitor probe at 100x, 300x, and 500x molar concentrations in lanes 5–7. Addition of ROBO1 45-mer unlabeled non-specific competitor probe, omitting the GBX2 DNA-binding sequence in lanes 8–10. (B–I) Whole mount in situ hybridization for Robo1 (B–E) and the migrating neural crest marker, Sox10 (F–I) at gestational stage E9.5, demonstrates abnormal expression in Gbx2 −/− mutants. Image analysis of embryos in a right lateral view (B,C) and dorsal view (D,E) reveals a reduction in expression of Robo1 in rhombomere 4 (compare white arrows in B and D to C and E) and disorganized expression in the rhombomere 1 domain (compare black arrows in B and D to C and E) in Gbx2 –/– mutants compared to the WT control. Lateral and dorsal views reveal a reduction in Sox10 expression within the otic vesicle (compare F and H to G and I). Two distinct streams of NCCs into pharyngeal arch 1 and pharyngeal arch 2 are defined within control embryos (F, H) whereas in Gbx2 −/− mutants (G,I) the NC streams appear disrupted. In the mutant, expression of Sox10 within the NC stream into presumptive pharyngeal arch 1 is significantly downregulated (compare black arrows in F and H to G and I), and the NCCs within the pharyngeal arch 2 appear to be compacted more posteriorly, truncating the stream (compare white arrows in F and H to G and I) when compared to the WT control. ov = otic vesicle.

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: Binding to ROBO1 and regulation of NC cell patterning by GBX2. (A) Gel-shift analysis for identified GBX2 target ROBO1 . A reduction in the mobility of [ÿ -32P] ATP labeled ROBO1 100-mer probe is observed with the addition of GBX2 (black arrows), whereas no shift is observed with the addition of GBX2ΔHD (compare lane 2 to lane 4). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical ROBO1 100-mer unlabeled specific competitor probe at 100x, 300x, and 500x molar concentrations in lanes 5–7. Addition of ROBO1 45-mer unlabeled non-specific competitor probe, omitting the GBX2 DNA-binding sequence in lanes 8–10. (B–I) Whole mount in situ hybridization for Robo1 (B–E) and the migrating neural crest marker, Sox10 (F–I) at gestational stage E9.5, demonstrates abnormal expression in Gbx2 −/− mutants. Image analysis of embryos in a right lateral view (B,C) and dorsal view (D,E) reveals a reduction in expression of Robo1 in rhombomere 4 (compare white arrows in B and D to C and E) and disorganized expression in the rhombomere 1 domain (compare black arrows in B and D to C and E) in Gbx2 –/– mutants compared to the WT control. Lateral and dorsal views reveal a reduction in Sox10 expression within the otic vesicle (compare F and H to G and I). Two distinct streams of NCCs into pharyngeal arch 1 and pharyngeal arch 2 are defined within control embryos (F, H) whereas in Gbx2 −/− mutants (G,I) the NC streams appear disrupted. In the mutant, expression of Sox10 within the NC stream into presumptive pharyngeal arch 1 is significantly downregulated (compare black arrows in F and H to G and I), and the NCCs within the pharyngeal arch 2 appear to be compacted more posteriorly, truncating the stream (compare white arrows in F and H to G and I) when compared to the WT control. ov = otic vesicle.

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Sequencing, In Situ Hybridization, Marker, Expressing, Mutagenesis

    GBX2 directly targets multiple genes associated with Usher syndrome and inner ear development. (A) Reverse transcription (RT)-PCR analysis of Gbx2 and identified targets, Pcdh15 , Ush2a , and Notch2 , in E13.5 wild-type mouse cochlear or vestibular inner ear tissues. Myo15 positive control expression is observed in cochlear and vestibular tissues. (B,C,D) Gel-shift analysis for identified GBX2 targets USH2A , PCDH15 , and NOTCH2 . A reduction in the mobility of [ÿ - 32 P] ATP labeled USH2A , PCDH15 , and NOTCH2 100-mer probes is observed with the addition of GBX2 (black arrows), whereas no shift is observed with the addition of GBX2ΔHD (compare lane 2 to lane 4). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical USH2A , PCDH15 , and NOTCH2 100-mer unlabeled specific competitor probes at 100x, 300x, and 500x molar concentrations in lanes 5–7. Addition of USH2A , PCDH15 , and NOTCH2 45-mer unlabeled non-specific competitor probe, omitting the GBX2 DNA-binding sequence in lanes 8–10.

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: GBX2 directly targets multiple genes associated with Usher syndrome and inner ear development. (A) Reverse transcription (RT)-PCR analysis of Gbx2 and identified targets, Pcdh15 , Ush2a , and Notch2 , in E13.5 wild-type mouse cochlear or vestibular inner ear tissues. Myo15 positive control expression is observed in cochlear and vestibular tissues. (B,C,D) Gel-shift analysis for identified GBX2 targets USH2A , PCDH15 , and NOTCH2 . A reduction in the mobility of [ÿ - 32 P] ATP labeled USH2A , PCDH15 , and NOTCH2 100-mer probes is observed with the addition of GBX2 (black arrows), whereas no shift is observed with the addition of GBX2ΔHD (compare lane 2 to lane 4). A supershift is observed in lane 3 with the addition of anti-GBX2. Addition of identical USH2A , PCDH15 , and NOTCH2 100-mer unlabeled specific competitor probes at 100x, 300x, and 500x molar concentrations in lanes 5–7. Addition of USH2A , PCDH15 , and NOTCH2 45-mer unlabeled non-specific competitor probe, omitting the GBX2 DNA-binding sequence in lanes 8–10.

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Positive Control, Expressing, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Sequencing

    Bioinformatic analysis of the top 286 GBX2 target genes. (A) Tissue expression analysis for the top 286 genes targeted by GBX2 determined by DAVID. Of the top 286 genes targeted by GBX2, 51% are expressed in the nervous system: 38% brain, 1% brain stem, 2% brain cortex, 3% hippocampus, 7% fetal brain. (B) The top two GBX2 DNA-binding consensus motifs bioinformatically determined by Motif Sampler analysis of GBX2 ChIP-Seq target sequence fragments.

    Journal: PLoS ONE

    Article Title: Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2

    doi: 10.1371/journal.pone.0047366

    Figure Lengend Snippet: Bioinformatic analysis of the top 286 GBX2 target genes. (A) Tissue expression analysis for the top 286 genes targeted by GBX2 determined by DAVID. Of the top 286 genes targeted by GBX2, 51% are expressed in the nervous system: 38% brain, 1% brain stem, 2% brain cortex, 3% hippocampus, 7% fetal brain. (B) The top two GBX2 DNA-binding consensus motifs bioinformatically determined by Motif Sampler analysis of GBX2 ChIP-Seq target sequence fragments.

    Article Snippet: Plasmid Constructs and ChIP Transfection The full-length coding sequence for mouse Gbx2 and a truncated Gbx2 fragment omitting the homeobox, Gbx2- ΔHD , were subcloned into the pCMV-HA mammalian expression vector (Clontech).

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Sequencing