RESUMEN
In the present study, we investigated the function of the heterotrimeric G protein ß-subunit (Gß) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gßγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Oryza/fisiología , Semillas/fisiología , Proliferación Celular , ADN de Plantas/genética , Flores/anatomía & histología , Flores/genética , Flores/fisiología , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Oryza/anatomía & histología , Oryza/genética , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Tallos de la Planta/anatomía & histología , Tallos de la Planta/genética , Tallos de la Planta/fisiología , Plantas Modificadas Genéticamente/anatomía & histología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Mensajero/genética , ARN de Planta/genética , Plantones/anatomía & histología , Plantones/genética , Plantones/fisiología , Semillas/anatomía & histología , Semillas/genética , Eliminación de Secuencia , Transducción de Señal/fisiologíaRESUMEN
Clinical studies overseas using the therapeutic anti-EGFR monoclonal antibodies, cetuximab or panitumumab against metastatic colorectal cancer(mCRC), have revealed KRAS mutations as a negative predictive marker of response. Accordingly, the Ministry of Health, Labour and Welfare in Japan approved medical reimbursement of the KRAS mutation test in April 2010. Anti-EGFR monoclonal antibody therapies are now used as first-line treatment for patients with mCRC. To advance the simple high-throughput KRAS mutation test, we established a high-throughput screening system for detecting KRAS mutations utilizing Luminex(xMAP)technology(the fluorescent bead-based multiplex analyte profiling method), in combination with the polymerase chain reaction-reverse sequence-specific oligonucleotide method. Here we evaluated the basic performance of our system and confirmed its high specificity and reproducibility in detecting KRAS mutations at codons 12 and 13 in both plasmid DNAs carrying mutant KRAS genes and formalin-fixed paraffin-embedded tissues from mCRC patients. We demonstrated the KRAS mutation status in paraffin-embedded tissues of mCRC and confirmed that the results were comparable to those of the direct sequencing method. Our high-throughput method has an advantage in simultaneous analysis of multiple mutations in one well of 96-well PCR plates, and will advance the KRAS mutation test in clinical laboratories.
Asunto(s)
Codón/genética , Neoplasias del Colon/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Mutación , Adhesión en Parafina/métodos , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Animales , Línea Celular Tumoral , Neoplasias del Colon/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein ß subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein ß subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.