Chimeric gene able to confer heat-stable resistance to kanamycin in thermophilic bacteria of the Thermus genus

The chimeric gene able to confer heat-stable resistance to kanamycin in thermophilic bacteria of the Thermus genus is characterized by conferring resistance to the antibiotic kanamycin in Thermus thermophilus and in Escherichia coli, within a range of temperatures between 30 and 80degreeC. The chimeric gene was constructed by means of a transcriptional fusion between the promoter of a gene of high expression level from Thermus thermophilus HB8, called slpA, and a moderately stable kanamycin resistance gene (kat) from Bacillus stearothermophilus. The construction was carried out by means of the polymerase chain reaction (PCR), introducing by directed mutagenesis the recognition sequence for the restriction endonuclease Ndel on the first coding triplet of the kanamycin resistance gene. A powerful transcription terminator was subsequently introduced at the end of the coding region which stabilizes the construction. Said chimeric gene was used as a selection marker for obtaining insertion mutants of previously cloned Thermus genes and for the cloning of replication origins of cryptic plasmids from Thermus and the subsequent development of bifunctional Escherichia coli-Thermus plasmids. EL GEN QUIMERICO CAPAZ DE CONFERIR RESISTENCIA TERMOESTABLE A KANAMICINA EN BACTERIAS TERMOFILAS DEL GENERO THERMUS ESTA CARACTERIZADO PARA CONFERIR RESISTENCIA AL ANTIBIOTICO KANAMICINA EN THERMUS THERMOPHILUS Y EN ESCHERICHIA COLI, DENTRO DE UN RANGO DE TEMPERATURAS COMPRENDIDO ENTRE 30 Y 80GC. EL GEN QUIMERICO HA SIDO CONSTRUIDO MEDIANTE UNA FUSION TRANSCRIPCIONAL ENTRE EL PROMOTOR DE UN GEN DE ALTO NIVEL DE EXPRESION PROCEDENTE DE THERMUS THERMOPHILUS HB8 DENOMINADO SLPA Y UN GEN DE RESISTENCIA A KANAMICINA MODERADAMENTE ESTABLE (KAT) PROCEDENTE DE BACILLUS STEAROTHERMOPHILUS. LA CONSTRUCCION HA SIDO REALIZADA MEDIANTE LA REACCION DE LA POLIMERASA EN CADENA (PCR), INTRODUCIENDO POR MUTAGENESIS DIRIGIDA LA SECUENCIA DE RECONOCIMIENTO PARA LA ENDONUCLEASA DE RESTRICCION NDEI SOBRE EL PRIMER TRIPLETE CODIFICANTE DEL GEN DE RESISTENCIA A KANAMICINA. POSTERIORMENTE, HA SIDO INTRODUCIDO UN POTENTE TERMINADOR DE TRANSCRIPCION AL EXTREMO DE LA REGION CODIFICANTE QUE ESTABILIZA LA CONSTRUCCION.DICHO GEN QUIMERICO HA SIDO UTILIZADO COMO MARCADOR DE SELECCION PARA LA OBTENCION DE MUTANTES INSERCIONALES DE GENES DE THERMUS PREVIAMENTE CLONADOS Y PARA EL CLONAJE DE ORIGENES DE REPLICACION DE PLASMIDOS CRIPTICOS PROCEDENTES DE THERMUS Y EL SUBSIGUIENTE DESARROLLO DE PLASMIDOS BIFUNCIONALE