RESUMO
To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; -812/-1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box. Comparison of the Rd-UG gene with that previously isolated from rabbits (Oryctolagus cuniculus) showed 93% in sequence identity as well as a number of conserved cis-acting elements, including the estrogen-response element (ERE; -265/-251), which differs from the consensus by two nucleotides. In MCF-7 cells, 17ß-estradiol (E(2)) induced transcription of a luciferase reporter driven by the Rd-UG promoter in a similar manner as in an equivalent rabbit UG reporter; the Rd-UG promoter was 30% more responsive to E(2) than the rabbit promoter. Mutagenesis studies on the Rd-ERE confirmed this cis-element as a target of E(2) as two luciferase mutant reporters of the Rd-promoter, one with the rabbit and the other with the consensus ERE, were more responsive to the hormone than the wild-type reporter. Gel shift and super-shift assays showed that estrogen receptor-α indeed binds to the imperfect palindromic sequence of the Rd-ERE.
Assuntos
Estrogênios/fisiologia , Lagomorpha/genética , Regiões Promotoras Genéticas , Elementos de Resposta , Uteroglobina/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Coelhos/genética , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , TATA Box/genética , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.
Assuntos
Arvicolinae/genética , Arvicolinae/metabolismo , Regiões Promotoras Genéticas , Receptores do FSH/química , Receptores do FSH/genética , Região 5'-Flanqueadora/genética , Sequência Rica em At , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Genes Reporter , Genes sry , Masculino , Camundongos , Camundongos da Linhagem 129 , Dados de Sequência Molecular , Receptores do FSH/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Células de Sertoli/metabolismoRESUMO
Analysis of the transcriptional regulation of the Clara cell secretory protein (CCSP) gene has resulted in the characterization of several trans-acting factors that regulate the activity of this gene. However, little is known about negative regulatory elements involved in CCSP gene transcription. Using transient transfections of luciferase reporter constructs driven by various fragments of the Neotomodon CCSP (nCCSP) promoter, we identified an inhibitory region that contains an inverted CCAAT box located -225 to -221 bp upstream of the transcriptional start site. Sequence analysis in a broad region of the nCCSP promoter (-744/+33) identified another potentially important CCAAT motif (-459/-455). Gel shift and supershift assays indicated that the transcription factor NF-Y binds to both CCAAT boxes. Mutation of the CCAAT motif prevented the in vitro binding of NF-Y and led to a significant increase of CCSP promoter activity in both pulmonary (H441) and non-pulmonary (HeLa and MCF-7) cells, suggesting that NF-Y is involved in a negative transcriptional regulation that may potentially contribute to the highly cell-specific expression of the anti-inflammatory CCSP gene.
Assuntos
Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Uteroglobina/metabolismo , Animais , Camundongos , Regiões Promotoras Genéticas/genética , Uteroglobina/genéticaRESUMO
To better understand the phylogenetic divergence and the species-specific characteristics of the Clara cell secretory protein (CCSP), we cloned the cDNA encoding the neotomodon CCSP (nCCSP) and analyzed its tissue-specific expression. The full-length cDNA is 451bp long and predicts an amino acid sequence of 93 residues. Northern blot analysis from different neotomodon tissues demonstrated that the mRNA of CCSP appears to be solely expressed in the lung. To study the transcriptional regulation of the CCSP gene, we cloned the 5'-flanking region of the nCCSP gene and compared its features with those previously reported for the hamster gene. The neotomodon and hamster genes share 89% sequence homology in their promoter region as well as a number of conserved cis-acting elements. However, in H441 cells the expression of a reporter gene driven by the nCCSP promoter was about 4-fold greater than its hamster counterpart. Functional analysis of progressive 5'-deletion mutants identified a region involved in the higher transcriptional activity of the neotomodon promoter.
Assuntos
Região 5'-Flanqueadora/genética , DNA Complementar/química , DNA Complementar/genética , Análise de Sequência de Proteína/métodos , Homologia de Sequência de Aminoácidos , Uteroglobina/química , Uteroglobina/genética , Sequência de Aminoácidos , Animais , Cricetinae , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA/métodos , Especificidade da EspécieRESUMO
El potencial eléctrico a través de la membrana (gama) se cuantificó mediante la acumulación del catión radiactivo trifenilmetilfosfonio (TPMP+) en la membrana espermática. Los espermatozoides previamente lavados se incubaron en presencia de TPMP+ en un medio denominado K+ - baja o K+ - elevada hasta que se logra el estado de equilibrio (20 min a 37 grados centigrados)., El valor obtenido por diferencia se inserta en la ecuación de Nernst y se obtiene el valor de -69 + - 2 mV. La presencia de cationes como el Zn ++ y Mg++ en el medio de incubación inducen una hiperpolarización de un 10 y 8.6% respectivamente. La adición de reactivos específicos como el p-cloromercuribenzoato-ácido sulofónico y etilendiaminotetra-acetato sódico, disminuye el gama en un 35 y 58% respectivamente. Los agentes que actúan sobre los componentes de la superficie de espermatozoide como el ditiotreitol y progesterona inducen una hiperpolarización y despolarización de la membrana en un 16 y 40%, respectivamente. La presencia de propanolol y L-alfa-lisofosfatidilcolina, los cuales afectan los gradientes iónicos presentes a través de la membrana inducen una despolarización en un 43 y 92%, respectivamente. Finalmente, la adición de tetraffenilboron (TPB -) en el medio de incubación aumentó el valor del gama 75%. La importancia de estos estudios es que mediante la utilización de agentes hiperpolarizantes y despolarizantes se obtienen cambios en el gama desde -80 + - 2.5 mV hasta -6 + - 0.6 mV es decir, cambios hasta de 74 +- 1.5 mV a través de la membrana espermática.(au)