Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence

This study continues the search for Plasmodium falciparum transcriptional machinery by isolating DNA binding pro­teins that could contribute towards discovering new therapeutic targets. The calmodulin gene was thus used as the model, using a 149 bp DNA fragment (PÆ’CAM149) specific for the 5' n...

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Autores:
Yara, Erika L
Rojas, María Orfa
Tipo de recurso:
Article of journal
Fecha de publicación:
2003
Institución:
Universidad Nacional de Colombia
Repositorio:
Universidad Nacional de Colombia
Idioma:
spa
OAI Identifier:
oai:repositorio.unal.edu.co:unal/22188
Acceso en línea:
https://repositorio.unal.edu.co/handle/unal/22188
http://bdigital.unal.edu.co/13222/
Palabra clave:
malaria
Plasmodium falciparum
maquinaria
transcripción
factores de transcripción
malaria
Plasmodium falciparum
machinery
transcription
transcription factors
Rights
closedAccess
License
Atribución-NoComercial 4.0 Internacional
id UNACIONAL2_d1c2a850561d24679f5a779d0fa3f2b1
oai_identifier_str oai:repositorio.unal.edu.co:unal/22188
network_acronym_str UNACIONAL2
network_name_str Universidad Nacional de Colombia
repository_id_str
dc.title.spa.fl_str_mv Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
title Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
spellingShingle Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
malaria
Plasmodium falciparum
maquinaria
transcripción
factores de transcripción
malaria
Plasmodium falciparum
machinery
transcription
transcription factors
title_short Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
title_full Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
title_fullStr Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
title_full_unstemmed Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
title_sort Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence
dc.creator.fl_str_mv Yara, Erika L
Rojas, María Orfa
dc.contributor.author.spa.fl_str_mv Yara, Erika L
Rojas, María Orfa
dc.subject.proposal.spa.fl_str_mv malaria
Plasmodium falciparum
maquinaria
transcripción
factores de transcripción
malaria
Plasmodium falciparum
machinery
transcription
transcription factors
topic malaria
Plasmodium falciparum
maquinaria
transcripción
factores de transcripción
malaria
Plasmodium falciparum
machinery
transcription
transcription factors
description This study continues the search for Plasmodium falciparum transcriptional machinery by isolating DNA binding pro­teins that could contribute towards discovering new therapeutic targets. The calmodulin gene was thus used as the model, using a 149 bp DNA fragment (PÆ’CAM149) specific for the 5' non-translated region. Partially purified proteins derived from total parasite nuclear extract were used to scale the PÆ’CAM149-biotina-streptavidin-magnesphere DNA affinity microsystem previously developed for capturing those proteins specifically binding to the DNA PÆ’CAM149 sequence. The scaling allowed the protein to be recycled three times through chromatographic matrix and determine which proteins remained bound to the system, indicating specific interaction with the calmodulin gene (PÆ’CAM149) promoter sequence. It also enabled obtaining the quantity of protein necessary for sequencing. Other assays allowed binding activity to be tracked by using eukaryotic transcription factor consensus sequences (CREB, OCT1, MIG, GATA), leading to selecting a 54 kDa protein whose possible identity corresponds to the E4BP4 transcription factor or related sequences. Problems regarding quantity were overcome in this work. Binding specificity was ascertained and it was determined that the 54 kDa protein had an intrinsic modification impeding its amino terminal being sequenced. These assays showed that CREB, or related transcription factors, form part of the parasite's transcriptional machinery. Key words: malaria; Plasmodium falciparum; machinery; transcription; transcription factors
publishDate 2003
dc.date.issued.spa.fl_str_mv 2003
dc.date.accessioned.spa.fl_str_mv 2019-06-25T20:33:52Z
dc.date.available.spa.fl_str_mv 2019-06-25T20:33:52Z
dc.type.spa.fl_str_mv Artículo de revista
dc.type.coar.fl_str_mv http://purl.org/coar/resource_type/c_2df8fbb1
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/article
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.coar.spa.fl_str_mv http://purl.org/coar/resource_type/c_6501
dc.type.coarversion.spa.fl_str_mv http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.content.spa.fl_str_mv Text
dc.type.redcol.spa.fl_str_mv http://purl.org/redcol/resource_type/ART
format http://purl.org/coar/resource_type/c_6501
status_str publishedVersion
dc.identifier.uri.none.fl_str_mv https://repositorio.unal.edu.co/handle/unal/22188
dc.identifier.eprints.spa.fl_str_mv http://bdigital.unal.edu.co/13222/
url https://repositorio.unal.edu.co/handle/unal/22188
http://bdigital.unal.edu.co/13222/
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.spa.fl_str_mv http://revistas.unal.edu.co/index.php/biotecnologia/article/view/595
dc.relation.ispartof.spa.fl_str_mv Universidad Nacional de Colombia Revistas electrónicas UN Revista Colombiana de Biotecnología
Revista Colombiana de Biotecnología
dc.relation.ispartofseries.none.fl_str_mv Revista Colombiana de Biotecnología; Vol. 5, núm. 1 (2003); 82-95 1909-8758 0123-3475
dc.relation.references.spa.fl_str_mv Yara, Erika L and Rojas, María Orfa (2003) Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence. Revista Colombiana de Biotecnología; Vol. 5, núm. 1 (2003); 82-95 1909-8758 0123-3475 .
dc.rights.spa.fl_str_mv Derechos reservados - Universidad Nacional de Colombia
dc.rights.coar.fl_str_mv http://purl.org/coar/access_right/c_14cb
dc.rights.license.spa.fl_str_mv Atribución-NoComercial 4.0 Internacional
dc.rights.uri.spa.fl_str_mv http://creativecommons.org/licenses/by-nc/4.0/
dc.rights.accessrights.spa.fl_str_mv info:eu-repo/semantics/closedAccess
rights_invalid_str_mv Atribución-NoComercial 4.0 Internacional
Derechos reservados - Universidad Nacional de Colombia
http://creativecommons.org/licenses/by-nc/4.0/
http://purl.org/coar/access_right/c_14cb
eu_rights_str_mv closedAccess
dc.publisher.spa.fl_str_mv Universidad Nacional de Colombia
institution Universidad Nacional de Colombia
repository.name.fl_str_mv Repositorio Institucional Universidad Nacional de Colombia
repository.mail.fl_str_mv repositorio_nal@unal.edu.co
_version_ 1806885949134602240
spelling Atribución-NoComercial 4.0 InternacionalDerechos reservados - Universidad Nacional de Colombiahttp://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/closedAccesshttp://purl.org/coar/access_right/c_14cbYara, Erika Lf65ac70f-f39f-43c2-a643-5e5a35fc8b25300Rojas, María Orfa3dd3433e-ec23-4cbb-bd0c-628800e0aff33002019-06-25T20:33:52Z2019-06-25T20:33:52Z2003https://repositorio.unal.edu.co/handle/unal/22188http://bdigital.unal.edu.co/13222/This study continues the search for Plasmodium falciparum transcriptional machinery by isolating DNA binding pro­teins that could contribute towards discovering new therapeutic targets. The calmodulin gene was thus used as the model, using a 149 bp DNA fragment (PÆ’CAM149) specific for the 5' non-translated region. Partially purified proteins derived from total parasite nuclear extract were used to scale the PÆ’CAM149-biotina-streptavidin-magnesphere DNA affinity microsystem previously developed for capturing those proteins specifically binding to the DNA PÆ’CAM149 sequence. The scaling allowed the protein to be recycled three times through chromatographic matrix and determine which proteins remained bound to the system, indicating specific interaction with the calmodulin gene (PÆ’CAM149) promoter sequence. It also enabled obtaining the quantity of protein necessary for sequencing. Other assays allowed binding activity to be tracked by using eukaryotic transcription factor consensus sequences (CREB, OCT1, MIG, GATA), leading to selecting a 54 kDa protein whose possible identity corresponds to the E4BP4 transcription factor or related sequences. Problems regarding quantity were overcome in this work. Binding specificity was ascertained and it was determined that the 54 kDa protein had an intrinsic modification impeding its amino terminal being sequenced. These assays showed that CREB, or related transcription factors, form part of the parasite's transcriptional machinery. Key words: malaria; Plasmodium falciparum; machinery; transcription; transcription factorsEn este trabajo se sigue la búsqueda de la maquinaria transcripcional de Plasmodium falciparum, aislando proteínas de unión a DNA, que podrían contribuir en un futuro a encontrar nuevos blancos terapéuticos. Con este fin se tomó como modelo el gen de calmodulina, utilizando específicamente un fragmento de DNA de 149 pb (PfCAM149) de la región 5' no traducida. Proteínas parcialmente purificadas provenientes del extracto nuclear total del parásito, se usaron para escalar el microsistema de afinidad DNA PfCAM149-biotina- estreptavidina- magnesferas, previamente desarrollado, para capturar aquellas proteínas de unión específica a la secuencia de DNA PfCAM149. El escalamiento permitió reci-clar tres veces la proteína a través de la matriz cromatográfica y determinar cuáles proteínas permanecían unidas al sistema, indicando interacción específica a la secuencia del promotor del gen de calmodulina (PfCAM149); además, condujo a obtener la cantidad de proteína necesaria para secuenciación. Por otra parte, se realizaron ensayos para rastrear la actividad de unión con secuencias consenso para factores de transcripción eucariotes (CREB, OCT1, MIG y GATA), que permitieron elegir una proteína de 54 kDa, cuya posible identidad corresponde al factor de transcripción E4BP4 o secuencias relacionadas. Con este trabajo se superaron problemas de cantidad, se comprobó la especificidad de la unión y se determinó que la proteína de 54 kDa tenía una modificación intrínseca que impidió la secuenciación de su extremo aminoterminal. Los ensayos realizados evidencian que el factor de trascripción CREB, o factores de trans­cripción relacionados forman parte de la maquinaria transcripcional del parásito. Palabras claves: malaria; Plasmodium falciparum; maquinaria; transcripción; factores de transcripción.spaUniversidad Nacional de Colombiahttp://revistas.unal.edu.co/index.php/biotecnologia/article/view/595Universidad Nacional de Colombia Revistas electrónicas UN Revista Colombiana de BiotecnologíaRevista Colombiana de BiotecnologíaRevista Colombiana de Biotecnología; Vol. 5, núm. 1 (2003); 82-95 1909-8758 0123-3475Yara, Erika L and Rojas, María Orfa (2003) Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequence. Revista Colombiana de Biotecnología; Vol. 5, núm. 1 (2003); 82-95 1909-8758 0123-3475 .Isolating and identifying proteins binding to a specific plasmodium falciparum calmodulin gene promotor sequenceArtículo de revistainfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1http://purl.org/coar/version/c_970fb48d4fbd8a85Texthttp://purl.org/redcol/resource_type/ARTmalariaPlasmodium falciparummaquinariatranscripciónfactores de transcripciónmalariaPlasmodium falciparummachinerytranscriptiontranscription factorsunal/22188oai:repositorio.unal.edu.co:unal/221882021-04-23 10:13:27.353Repositorio Institucional Universidad Nacional de Colombiarepositorio_nal@unal.edu.co

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