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Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

Rodríguez-Lima O, García-Gutierrez P, Jiménez L, Zarain-Herzberg Á, Lazzarini R, Landa A - PLoS ONE (2015)

Bottom Line: The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s.These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium.Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F., México.

ABSTRACT
TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic mobility shift assay showing the interaction of wild type TsTBP1 pAT5 TATA-box probe.A) Lane 1: Labeled dsDNA-32P probe without nuclear extract; lane 2: TsTBP1-pAT5 TATA-box interaction with T. solium nuclear extract; lanes 3, 4, and 5: competence with pAT5 TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 6: super-shift interaction using anti-pTsTBP1-N; lane 7: consensus TATA-box probe interaction with T. solium nuclear extract (used as positive control); lane 8: consensus mutated TATA-box probe interaction with nuclear extract (used as negative control); lane 9, 10 and 11: cross-competence with Ts2-CysPrx TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 12: anti-TsTBP1-N antibody without T. solium nuclear extract (negative control). Shifted, super-shifted bands and the free-labeled dsDNA probe, are indicated by arrows. B) Densitometric analysis shows a decrease on the intensity of shifted bands in homologous and heterologous competition. Results are present as percentage mean ± SD of the shifted band in lane 2 (P < 0.005).
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pone.0141818.g004: Electrophoretic mobility shift assay showing the interaction of wild type TsTBP1 pAT5 TATA-box probe.A) Lane 1: Labeled dsDNA-32P probe without nuclear extract; lane 2: TsTBP1-pAT5 TATA-box interaction with T. solium nuclear extract; lanes 3, 4, and 5: competence with pAT5 TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 6: super-shift interaction using anti-pTsTBP1-N; lane 7: consensus TATA-box probe interaction with T. solium nuclear extract (used as positive control); lane 8: consensus mutated TATA-box probe interaction with nuclear extract (used as negative control); lane 9, 10 and 11: cross-competence with Ts2-CysPrx TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 12: anti-TsTBP1-N antibody without T. solium nuclear extract (negative control). Shifted, super-shifted bands and the free-labeled dsDNA probe, are indicated by arrows. B) Densitometric analysis shows a decrease on the intensity of shifted bands in homologous and heterologous competition. Results are present as percentage mean ± SD of the shifted band in lane 2 (P < 0.005).

Mentions: To establish the interaction of TsTBP1 with the TATA-box motif, we performed EMSA. As DNA targets, we used two putative TATA-box motifs located in the core promoter from two T. solium genes: actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx), both localized between -30 to -23 bp relative to the TSS. We also used as a control the consensus TATA-box of adenovirus major late promoter (TATA-box consensus, Table 1). The interaction of TsTBP1 with the putative TATA-box of pAT5 (Fig 4) and Ts2-CysPrx (Fig 5) labeled dsDNA probes showed a shifted band produced by TsTBP1 binding to the respective probe (see lanes 2, Figs 4 and 5). In lanes 3, 4, and 5 of Fig 4A a decrease in the intensity of the shifted band can be observed due to homologous competence with TATA-box of the pAT5. Densitometric analysis shows a decrease of 17%, 72%, and 89% for 25X, 50X and 100X with the non-labeled probe, respectively (Fig 4B). In lanes 3, 4, and 5 of Fig 5A also a decrease in the intensity of shifted band were observed with TATA-box of the Ts2-CysPrx. The densitometric analysis also shows a decrease of 61%, 73% and 87% when 25X, 50X and 100X with cold probe were used, respectively (Fig 5B). In lane 6 of Figs 4 and 5, a super-shifted band is observed due to the interaction with the anti-pTsTBP1-N antibody. In lane 7 of same figures, a shifted band is also observed when the consensus TATA-box labeled probe was added to the reaction. In contrast, in lanes 8, disappearance of the shifted band is observed, when the labeled mutated consensus TATA-box probe was added.


Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

Rodríguez-Lima O, García-Gutierrez P, Jiménez L, Zarain-Herzberg Á, Lazzarini R, Landa A - PLoS ONE (2015)

Electrophoretic mobility shift assay showing the interaction of wild type TsTBP1 pAT5 TATA-box probe.A) Lane 1: Labeled dsDNA-32P probe without nuclear extract; lane 2: TsTBP1-pAT5 TATA-box interaction with T. solium nuclear extract; lanes 3, 4, and 5: competence with pAT5 TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 6: super-shift interaction using anti-pTsTBP1-N; lane 7: consensus TATA-box probe interaction with T. solium nuclear extract (used as positive control); lane 8: consensus mutated TATA-box probe interaction with nuclear extract (used as negative control); lane 9, 10 and 11: cross-competence with Ts2-CysPrx TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 12: anti-TsTBP1-N antibody without T. solium nuclear extract (negative control). Shifted, super-shifted bands and the free-labeled dsDNA probe, are indicated by arrows. B) Densitometric analysis shows a decrease on the intensity of shifted bands in homologous and heterologous competition. Results are present as percentage mean ± SD of the shifted band in lane 2 (P < 0.005).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4631506&req=5

pone.0141818.g004: Electrophoretic mobility shift assay showing the interaction of wild type TsTBP1 pAT5 TATA-box probe.A) Lane 1: Labeled dsDNA-32P probe without nuclear extract; lane 2: TsTBP1-pAT5 TATA-box interaction with T. solium nuclear extract; lanes 3, 4, and 5: competence with pAT5 TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 6: super-shift interaction using anti-pTsTBP1-N; lane 7: consensus TATA-box probe interaction with T. solium nuclear extract (used as positive control); lane 8: consensus mutated TATA-box probe interaction with nuclear extract (used as negative control); lane 9, 10 and 11: cross-competence with Ts2-CysPrx TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 12: anti-TsTBP1-N antibody without T. solium nuclear extract (negative control). Shifted, super-shifted bands and the free-labeled dsDNA probe, are indicated by arrows. B) Densitometric analysis shows a decrease on the intensity of shifted bands in homologous and heterologous competition. Results are present as percentage mean ± SD of the shifted band in lane 2 (P < 0.005).
Mentions: To establish the interaction of TsTBP1 with the TATA-box motif, we performed EMSA. As DNA targets, we used two putative TATA-box motifs located in the core promoter from two T. solium genes: actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx), both localized between -30 to -23 bp relative to the TSS. We also used as a control the consensus TATA-box of adenovirus major late promoter (TATA-box consensus, Table 1). The interaction of TsTBP1 with the putative TATA-box of pAT5 (Fig 4) and Ts2-CysPrx (Fig 5) labeled dsDNA probes showed a shifted band produced by TsTBP1 binding to the respective probe (see lanes 2, Figs 4 and 5). In lanes 3, 4, and 5 of Fig 4A a decrease in the intensity of the shifted band can be observed due to homologous competence with TATA-box of the pAT5. Densitometric analysis shows a decrease of 17%, 72%, and 89% for 25X, 50X and 100X with the non-labeled probe, respectively (Fig 4B). In lanes 3, 4, and 5 of Fig 5A also a decrease in the intensity of shifted band were observed with TATA-box of the Ts2-CysPrx. The densitometric analysis also shows a decrease of 61%, 73% and 87% when 25X, 50X and 100X with cold probe were used, respectively (Fig 5B). In lane 6 of Figs 4 and 5, a super-shifted band is observed due to the interaction with the anti-pTsTBP1-N antibody. In lane 7 of same figures, a shifted band is also observed when the consensus TATA-box labeled probe was added to the reaction. In contrast, in lanes 8, disappearance of the shifted band is observed, when the labeled mutated consensus TATA-box probe was added.

Bottom Line: The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s.These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium.Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F., México.

ABSTRACT
TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

No MeSH data available.


Related in: MedlinePlus