RESUMEN
Mullerian Inhibiting Substance (MIS) functions to promote regression of the Mullerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas , Células de la Granulosa/fisiología , Inhibidores de Crecimiento/biosíntesis , Proteínas Nucleares , Células de Sertoli/metabolismo , Hormonas Testiculares/biosíntesis , Testículo/embriología , Factores de Transcripción/fisiología , Animales , Hormona Antimülleriana , Núcleo Celular/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Femenino , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Caracteres Sexuales , Proteína de la Región Y Determinante del Sexo , Factor Esteroidogénico 1 , Factores de Transcripción/biosíntesis , Activación TranscripcionalRESUMEN
The spatial and temporal distribution of soluble guanylyl cyclase and nitric oxide synthase mRNA was determined during embryonic and postnatal development of the mouse brain. This was achieved by in situ hybridization of specific probes for soluble beta 1 guanylyl cyclase subunit and nitric oxide synthase mRNA on mouse brain sections at late fetal development (19-day embryo) and different stages of postnatal development (3, 7, 15 days, and adult). In the embryo, soluble guanylyl cyclase transcripts are weakly expressed in the central nervous system. Following birth their expression increases in the striatum and neocortex, and they are widely distributed in the adult brain (layer II and V-VI of the cortex, olfactory bulb, striatum, Purkinje cell layer of the cerebellum). In contrast, nitric oxide synthase mRNA was expressed in several embryonic structures of the brain (different layers of the cortical neuroepithelium, colliculi neuroepithelium, pons), and markedly reduced at early postnatal stage, except in the accessory olfactory bulb and pediculopontine nuclei. Nitric oxide synthase transcripts progressively appear, within two weeks following birth, in the striatum and the cerebral cortex but they were specifically confined to isolated cells. During this period, this mRNA also increased in hippocampus, in discrete nuclei (hypothalamus, pontine) and in the molecular layer of the cerebellum. The situation in the adult was similar to the one observed at 15 days. These results show a general lack of regional colocalization of soluble guanylyl cyclase and NOS mRNA during ontogeny, thus suggesting an independent regulation of the related genes.
Asunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Encéfalo/enzimología , Guanilato Ciclasa/biosíntesis , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Tronco Encefálico/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa , Bulbo Olfatorio/metabolismo , Prosencéfalo/metabolismo , Solubilidad , Tálamo/metabolismoRESUMEN
We have determined the chromosomal location of the human genes coding for the alpha 3 and beta 3 subunits of soluble guanylyl cyclase (GC-S). The study was performed by in situ hybridization of human metaphase spreads with two human cDNA probes, containing the coding sequences of the GC-S alpha 3 and beta 3 subunits, respectively. Each probe gave a strong specific signal on chromosome 4 at the 4q31.3-4q33 region, with the maximal signal in the 4q32 band. The colocalization of both genes in 4q32 represents an interesting feature for the coordinated regulation of expression of both GC-S subunits.
Asunto(s)
Cromosomas Humanos Par 4 , Guanilato Ciclasa/genética , Mapeo Cromosómico , Guanilato Ciclasa/química , Humanos , Cariotipificación , SolubilidadRESUMEN
The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.
Asunto(s)
Azacitidina/farmacología , gamma-Glutamiltransferasa/metabolismo , Animales , Separación Celular , Células Clonales , ADN/metabolismo , Expresión Génica/efectos de los fármacos , Genoma , Metilación/efectos de los fármacos , Ratas , Células Tumorales Cultivadas/metabolismo , gamma-Glutamiltransferasa/genéticaRESUMEN
We have investigated, using DNA methylation patterning, the site-specific methylation of promoters I and II of the rat gamma-glutamyl transpeptidase gene. This analysis was done in fetal, newborn and adult rat kidney, in which promoters I and II are progressively active during development, as well as in rat liver, which never expresses mRNAs from these two promoters. During kidney development, a progressive demethylation occurs in the promoter I and II region, specially at the level of the most proximal MspI site of promoter II. A progressive reorganization of the methylated sites within the 5' end of the gene also occurs during liver development.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Riñón/enzimología , Regiones Promotoras Genéticas , gamma-Glutamiltransferasa/genética , Animales , Southern Blotting , ADN/metabolismo , Riñón/crecimiento & desarrollo , Metilación , Especificidad de Órganos/genética , Ratas , gamma-Glutamiltransferasa/metabolismoRESUMEN
Cyclic nucleotides are major intracellular mediators in the signal transduction events in synaptic neurotransmission of the CNS. Intracellular Ca2+ is known to regulate adenylyl cyclase (AC) in a calmodulin (CaM)-dependent manner, and guanylyl cyclase (GC), in an indirect manner through CaM-sensitive nitric oxide synthase. To ascertain the physiological significance of cyclic nucleotide second messenger systems, we have localized the mRNAs encoding AC, GC, and CaM in the rat brain by in situ hybridization using 35S-labeled RNA probes. The AC mRNA is widely distributed throughout the brain; strong hybridization signal was observed in the granular layers of the cerebellum, in the pyramidal and granule cells of the hippocampus, and in the olfactory system. These AC mRNA localizations are compatible with the distribution of Ca2+/CaM-sensitive AC activities. In contrast to AC mRNA distribution, GC mRNA has a more limited distribution. Significant signals were observed in the striatum, in the pyramidal and granule cells of the hippocampus, in the olfactory system, in the inferior and superior colliculus, in the Purkinje cells of the cerebellum, in the locus coeruleus, and in many pyramidal cells in the layers II-III and V of the cerebral cortex, and mainly, in the occipital cortex. In some discrete brain regions, a close correlation was found between enzyme activity and mRNA hybridization signal of GC. The distinct distribution of AC and GC mRNAs suggests that different cyclic nucleotide second messenger systems have specialized functions. On the other hand, CaM mRNA was colocalized with the AC and GC mRNA, but its distribution was more abundant and specific for neuronal cells, since there was little hybridization signal with CaM probe in neuronal fiber regions such as the corpus callosum and the anterior commissure. The high expression of CaM mRNA in neuronal cells is in agreement with its biochemical role in the regulation of various enzymes. Results of the present study should help in analyzing the role of cyclic nucleotides and CaM in physiological and pathological situations in the CNS.
Asunto(s)
Adenilil Ciclasas/metabolismo , Encéfalo/enzimología , Calmodulina/genética , Guanilato Ciclasa/metabolismo , Hibridación de Ácido Nucleico , ARN Mensajero/metabolismo , Animales , Northern Blotting , Femenino , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
Complementary DNA clones corresponding to the 70 and 82 kDa subunits of soluble guanylyl cyclase from human adult brain have been isolated and sequenced. Their respective open reading frames correspond to 619 amino acids (M(r) 70,469) and 717 amino acids (M(r) 81,324). Southern blots of human genomic DNA using these clones as probes give patterns which might be compatible with the presence of more than one copy per gene, or pseudogenes, for each subunit in the human genome. Comparison of the protein sequence of the large subunit from adult brain with the subunit cloned from human fetal brain (Harteneck, C., Wedel, B., Koesling, D., Malekewitz, J., Böhme, E., and Schultz, G. (1991) FEBS Lett. 292, 217-222) revealed only 34% homology. This result demonstrates the existence of a novel large subunit isoform for soluble guanylyl cyclase in human tissues.
Asunto(s)
Encéfalo/enzimología , Guanilato Ciclasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN , Guanilato Ciclasa/metabolismo , Humanos , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , SolubilidadRESUMEN
The methylation status of the rat gamma-glutamyl transpeptidase (GGT) gene was investigated during liver development and hepatocarcinogenesis. The analysis with the restriction enzymes MspI/HpaII revealed that, during ontogeny, there is a progressive methylation of the GGT gene that coincides with a progressive decrease in GGT activity. Thus, there is an inverse correlation between methylation and expression of the GGT gene, suggesting a role for DNA methylation in the regulation of the gene during normal differentiation. The methylation patterns of the GGT gene in liver tumours induced by aflatoxin B1 exhibit heterogeneity. Nevertheless, a band of 5.7 kb was observed in all the DNA samples from aflatoxin B1-induced tumours which was not present in control liver DNA. The specificity of the DNA methylation changes was assessed using nafenopin, which induces hepatic tumours without elevation of GGT activity. We conclude that, during hepatocarcinogenesis, there is a modification of the DNA methylation pattern of the GGT gene, but there is no simple correlation with GGT activity. In no case was the GGT gene methylation in hepatocarcinogenesis found to be equivalent to the pattern observed in fetal liver. Thus if methylation is involved in the regulation of GGT gene transcription, the mechanisms must be different in fetal liver and hepatocarcinoma.