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Biotecnologia Aplicada
Elfos Scientiae
ISSN: 0684-4551
Vol. 13, No. 4, 1996
Bioline Code: ba96103
Full paper language: Spanish
Document type: Research Article
Document available free of charge

Biotecnologia Aplicada, Vol. 13, No. 4, 1996

 es Modificacion del Gen de la Esporamina de Boniato con un Fragmento de ADN Sintetico. Secuencia Nucleotidica y Expresion en Escherichia coli check for this species in other resources
Lopez, Alina; Zaldua, Zurima; Pimentel, Eulogio; Garcia, Melba; Garcia, Rolando; Mena, Jesus; Moran, Rolando & Selman, Guillermo

Resumen

La esporamina, principal proteina del tuberculo de boniato ( Ipomoea batatas check for this species in other resources ), presenta un contenido de aminoacidos relativamente balanceado pero limitado a su vez en lisina y metionina. Se diseno y sintetizo un fragmento de ADN de 240 pares de bases con el objetivo de mejorar la calidad aminoacidica de la esporamina. A partir de una preparacion de ADN total de boniato y por reaccion en cadena de la polimerasa, se amplifico la region que contiene el gen que codifica la esporamina y las secuencias reguladoras adyacentes. El ADN amplificado se clono y secuencio. Tambien por PCR, se amplifico un fragmento que contiene solo el gen estructural de la esporamina y las secuencias que codifican los peptidos senal y de transito a vacuolas. Esta region se clono, secuencio y expreso en Escherichia coli. Con el uso de endonucleasas especificas de restriccion se realizaron dos estrategias en las que el fragmento de ADN sintetico fue insertado en posiciones diferentes de la secuencia del gen estructural para la esporamina. Las predicciones de los posibles atributos de las esporaminas modificadas fueron realizadas a traves de programas de computo. Las variantes geneticas obtenidas fueron chequeadas por restriccion enzimatica, hibridacion y secuencia. Su expresion en E. coli fue detectada por Western blotting.

Palabras-clave
aminoacidos esenciales, calidad proteica

 
 en
Lopez, Alina; Zaldua, Zurima; Pimentel, Eulogio; Garcia, Melba; Garcia, Rolando; Mena, Jesus; Moran, Rolando & Selman, Guillermo

Abstract

Sporamin, the major soluble protein of sweet potato tuberous roots ( Ipomoea batatas check for this species in other resources ), has a relatively balanced aminoacid content, but it is limited in lysine and methionine. A 240 base pair DNA fragment was designed and synthesized in order to improve the aminoacidic quality of the sporamin. From a total DNA preparation and using polimerase chain reaction, the region containing the sporamin gene and its regulatory regions was amplified. The DNA obtained was cloned and sequenced. Besides, we amplified by PCR a fragment containing the sporamin gene, the sequences coding for transit to vacuoles and the signal peptides. This region was cloned, sequenced and expressed in Escherichia coli. With the use of specific restriction endonucleases, two strategies were developed to insert the DNA synthetic fragment in two different positions of the structural gene for sporamin. Predictions of the possible features of the modified sporamins were performed using appropriate softwares. The genetic variants obtained were checked by restriction, hibridization and sequencing. Their expression in E. coli was detected by Western blotting.

Keywords
essential aminoacids, protein quality

 
© Copyright 1996 Elfos Scientiae
Alternative site location: http://elfosscientiae.cigb.edu.cu/Archivo.asp?Id=6

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