RESUMEN
The kappa immunoglobulin (Igk) light chain locus is transcriptionally silent in the mouse B-cell lymphoma 70Z/3. However, exposure to lipopolysaccharide (LPS) or interferon-gamma (IFN) causes a marked increase in Igk transcription. By immunoselection, we isolated two variants that are nonresponsive to IFN. One variant, AT7.2, has retained its response to LPS (IFN-LPS+), whereas the other, AT3.3, is also nonresponsive to LPS (IFN-LPS-). Stable transfection of an intact Igk gene does not rescue the phenotype of either variant. Both variants have intact Igk genes and neither is deficient in the binding or uptake of IFN. Nuclear extracts from LPS-treated wild-type 70Z/3 cells show strong increases in three transcription factors: OTF-2, NF-kappa B, and kBF-A. Remarkably, when the IFN-LPS- variant is treated with LPS, all three transcription factors are still observed in the nuclear extracts. Treatment of wild-type cells with either LPS or IFN also causes a decrease in nuclear complexes that bind to two other regions of the Igk intron enhancer, the octenh and the E kappa MHCIC regions. Both of these changes are also observed after LPS or IFN treatment of the IFN-LPS- variant. Thus, this variant transduces the IFN and LPS signals at least into the nuclear compartment, but still fails to activate Igk transcription. In contrast, the IFN-LPS+ variant decreases neither the octenh nor the E kappa MHCIC binding complexes in response to IFN. This variant may be defective in transducing the IFN signal to the nucleus. These variants will be useful in studying the activation of Igk transcription and the IFN signaling pathway in B cells.
Asunto(s)
Linfocitos B/inmunología , Proteínas de Unión al ADN , Interferón gamma/inmunología , Animales , Linfocitos B/metabolismo , Secuencia de Bases , ADN , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Variación Genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Interferón gamma/metabolismo , Intrones , Lipopolisacáridos/inmunología , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Factor 2 de Transcripción de Unión a Octámeros , Unión Proteica , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difficulties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 microliters of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA-2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory.
