RESUMEN
Aldehyde dehydrogenase (ALDH1) is highly expressed in the dorsal cells of the undifferentiated retina, where it has been proposed to play a role in the formation of a retinoic acid gradient along the ventrodorsal axis. In contrast to the retina, ALDH1 levels increase with differentiation in the liver and remain elevated in the adult tissue. To understand the molecular basis for differential expression of ALDH1 during development, we characterized the ALDH1 transcripts expressed in chick retina and liver. By sequencing, primer extension, and S1 nuclease analysis, we show that retina ALDH1 mRNA has an additional 300 nucleotides of 5'-untranslated sequence resulting from the transcription of two 5' noncoding exons. There is a 24-29-kilobase pair (kb) gap between exons 1 and 2 and a 290-base pair gap between exons 2 and 3. Exon 3, which contains the ALDH1 start codon, represents the first exon of the liver transcript. Using a reporter gene assay, we have identified tissue-specific regulatory elements that govern ALDH1 expression in primary retina and liver cultures. Constructs with >1.6 kb of DNA flanking the 5'-end of exon 1 showed elevated activity in retinal cultures but only basal activity in liver cultures. In contrast, constructs with <1 kb of 5'-flanking DNA were active in both retina and liver cultures. Our results suggest that an important mechanism for the control of ALDH1 transcriptional activity is through the presence of inhibitory elements located 0.7-1.6 kb upstream of the ALDH1 gene. DNase I footprint analysis reveal four sites of protein-DNA interaction within this region, one of which is specific to the liver and corresponds to a NF-kappaB/Rel binding site.
Asunto(s)
Aldehído Deshidrogenasa/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Hígado/enzimología , Retina/enzimología , Familia de Aldehído Deshidrogenasa 1 , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Secuencia de Consenso , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Exones , Humanos , Hígado/embriología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Retina/embriología , Retinal-Deshidrogenasa , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , TATA Box , TransfecciónRESUMEN
Brain fatty acid-binding protein (B-FABP) is expressed in the radial glial cells of the developing central nervous system as well as in a subset of human malignant glioma cell lines. Most of the malignant glioma lines that express B-FABP also express GFAP, an intermediate filament protein found in mature astrocytes. We are studying the regulation of the B-FABP gene to determine the basis for its differential expression in malignant glioma lines. By DNase I footprinting, we have identified five DNA-binding sites located within 400 base pairs (bp) of the B-FABP transcription start site, including two nuclear factor I (NFI)-binding sites at -35 to -58 bp (footprint 1, fp1) and -237 to -260 bp (fp3), respectively. Competition experiments, supershift experiments with anti-NFI antibody, and methylation interference experiments all indicate that the factor binding to fp1 and fp3 is NFI. By site-directed mutagenesis of both NFI-binding sites, we show that the most proximal NFI site is essential for B-FABP promoter activity in transiently transfected malignant glioma cells. Different band shift patterns are observed with nuclear extracts from B-FABP(+) and B-FABP(-) malignant glioma lines, with the latter generating complexes that migrate more slowly than those obtained with B-FABP(+) extracts. All bands are converted to a faster migrating form with potato acid phosphatase treatment, indicating that NFI is differentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results suggest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatase activity specific to B-FABP(+) lines.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Proteínas de Neoplasias , Regiones Promotoras Genéticas , Factores de Transcripción , Proteínas Supresoras de Tumor , Secuencia de Bases , Sitios de Unión/genética , Química Encefálica , Proteínas Portadoras/biosíntesis , Huella de ADN , Desoxirribonucleasa I/metabolismo , Proteína 3 de Unión a Ácidos Grasos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFI , Proteínas Nucleares , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Isoformas de Proteínas/metabolismo , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja YRESUMEN
The pathogenetic role of interferon-gamma (IFN-gamma) in acute graft-versus-host disease (GVHD) was examined in a murine model. IFN-gamma gene expression was evaluated by northern blotting and mRNA in situ hybridization. The temporal and tissue specific patterns of IFN-gamma gene expression were related to the patterns of major histocompatibility complex (MHC) antigen induction and of tissue injury. Markedly increased levels of IFN-gamma transcripts were seen in the spleen during the early lymphoproliferative phase and coincided with widespread MHC induction in non-lymphoid tissues. Increased IFN-gamma transcripts were also found in the non-lymphoid target tissues during the phase of subsequent tissue injury. These findings support a role for IFN-gamma in leading to widespread MHC induction during acute GVHD and suggest that IFN-gamma may also contribute to target tissue injury during acute GVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Antígenos de Histocompatibilidad Clase I/análisis , Interferón gamma/genética , Animales , Northern Blotting , Encéfalo/inmunología , Expresión Génica , Hibridación in Situ , Hígado/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Miocardio/inmunología , Bazo/inmunología , Lengua/inmunologíaRESUMEN
Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5' flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5' flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5' flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Retina/embriología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Embrión de Pollo , ADN/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión a Ácidos Grasos , Genes/genética , Datos de Secuencia Molecular , Mutación , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Retina/citología , Factor de Transcripción AP-2 , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
1. Extracellular fluid deficits of 33% were produced in male Long-Evans rats by peritoneal dialysis. The conscious, unrestrained animals were then infused I.V. for 30 min with atriopeptin III at doses of 0.01, 0.1, 0.5 and 1.0 micrograms/min. At 5 min into the infusion, the rats were offered water and subsequent intakes were monitored. Since atrial natriuretic peptide (ANP) causes hypotension, one group of control animals was given an injection of diazoxide sufficient to match this fall in blood pressure. 2. A similar group of rats was prepared for measurement of plasma ANP achieved by infusion. 3. Relative to the saline-infused controls, atriopeptin III did not reduce water intake. Indeed, intake was increased at the highest dose of 1.0 micrograms/min. 4. Relative to the diazoxide controls, water intake was influenced by atriopeptin III in a dose-dependent manner, the greatest attenuation being observed at infusions of 0.1 microgram/min. 5. Infusion of atriopeptin III at 0.1 microgram/min caused plasma ANP levels to rise from 252 +/- 21 to 532 +/- 136 pg/ml (n = 9, P less than 0.05) at 15 min. The lowest dose (0.01 microgram/min) caused no detectable increase in plasma levels. 6. It is concluded that, in groups of hypovolaemic rats matched for blood pressure, atriopeptin III caused a dose-related reduction in water intake.