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Site-directed mutagenesis of the foot-and-mouth diseasevirus RNA-polymerase gene disease virus RNA-polymerase gene

Rodrigo de M. Brindeiro; Marcelo A. Soares; Anna Luiza M.A. Vianna; Octavio H.A. de Pontes; Ana Beatriz F. Pacheco; Darcy F. de Almeida; Amilcar Tanuri

Laboratório de Fisiologia Celular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941 Rio de Janeiro, RI, Brasil. Send correspondence to A.T.

ABSTRACT

The foot-and-mouth disease virus RNA-polymerase gene was mutagenised in its active site. Pst I digestion of the polymerase gene (cDNA) generated a 790 bp fragment containing the critical sequence. This fragment was subcloned in M13mp8 for mutagenesis and four mutations were generated in vitro by the oligonucleotide site-directed mutagenesis method. The polymerase gene was then reconstructed and subcloned in pUC19. These mutants will be used to study the enzyme structure and activity and to develop "intracellular immunization" assays in eukaryotic cells.

Keywords: Mutagenesis; Foot-and-mouth disease; Virus RNA-polymerase.

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