RESUMEN
The vanilla cream1 (vac1) albino mutant is defective in a gene encoding a chloroplast-localized pentatricopeptide repeat protein of the DYW subgroup. However, the carboxyl-terminal DYW motif is truncated in VAC1. To identify vac1-specific phenotypes, we compared 34 chloroplast RNA editing sites and approximately 90 chloroplast gene expression patterns among wild type, vac1 and another albino mutant ispH, which is defective in the plastid isoprenoid biosynthesis pathway. We found that the editing of accD and ndhF transcripts is partially affected in vac1. In addition, steady-state levels of chloroplast rRNAs are significantly decreased in vac1. The expression of plastid-encoded RNA polymerase transcribed genes is down-regulated, whereas the expression of nucleus-encoded RNA polymerase transcribed genes is up-regulated in vac1. Although the development and function of mutant chloroplasts are severely impaired, steady-state mRNA levels of nucleus-encoded photosynthetic genes are not affected or are only slightly decreased in vac1. The ZAT10 gene encodes a transcription factor and its expression is down-regulated by norflurazon treatment in wild type. This norflurazon effect was not observed in vac1. These results suggest that the VAC1 protein may be involved in plastid-to-nucleus retrograde signaling in addition to its role in chloroplast RNA editing and gene expression. A defect in a key biosynthetic pathway can have many indirect effects on chloroplast gene expression as is seen in the ispH mutant. Similarly, the vac1 mutant has pleiotropic molecular phenotypes and most of which may be indirect effects.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mutación , Edición de ARN , ARN del Cloroplasto/genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Clorofila/química , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Fluorescencia , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.