RESUMEN

Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Factores de Transcripción Paired Box , Hipófisis/citología , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.

Asunto(s)

Cardiomegalia/genética , Miocardio/patología , Receptores de Angiotensina/genética , Remodelación Ventricular/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Unión Competitiva/efectos de los fármacos , Northern Blotting , Cardiomegalia/patología , Regulación de la Expresión Génica , Atrios Cardíacos/química , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Ventrículos Cardíacos/química , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Losartán/farmacología , Ratones , Ratones Transgénicos , Miocardio/citología , Miocardio/metabolismo , Fenotipo , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Distribución Tisular , Transgenes/genética

RESUMEN

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

Asunto(s)

Vectores Genéticos , Glucuronidasa/metabolismo , Toxina del Pertussis , Streptomyces/genética , Factores de Virulencia de Bordetella/metabolismo , Xilosidasas/genética , Bordetella pertussis/genética , Clonación Molecular , Endo-1,4-beta Xilanasas , Escherichia coli/genética , Genes Bacterianos , Glucuronidasa/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Factores de Virulencia de Bordetella/genética , Xilosidasas/metabolismo

RESUMEN

The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles.

Asunto(s)

Glicósido Hidrolasas/metabolismo , Bacterias Anaerobias Gramnegativas/enzimología , Secuencia de Aminoácidos , Catálisis , Secuencia Conservada , Análisis Mutacional de ADN , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/genética , Bacterias Anaerobias Gramnegativas/genética , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Xilano Endo-1,3-beta-Xilosidasa , Xilanos/metabolismo

RESUMEN

The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.

Asunto(s)

Glicósido Hidrolasas/genética , Bacterias Anaerobias Gramnegativas/enzimología , Bacterias Anaerobias Gramnegativas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Catálisis , Clonación Molecular , Análisis Mutacional de ADN , Escherichia coli/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Xilano Endo-1,3-beta-Xilosidasa

RESUMEN

The gene coding for a beta-mannanase was cloned homologously from Streptomyces lividans and its DNA sequence was determined. The fully secreted enzyme was isolated and purified from culture filtrates of the hyperproducing clone S. lividans IAF36 grown in mineral salt media containing galactomannan as the main carbon source. It had a molecular mass of 36 kDa and a specific activity of 876 i.u./mg of protein. Under the assay conditions used, the optimal enzyme activity was obtained at 58 degrees C and a pH of 6.8. The pI was 3.5. The kinetic constants of this mannanase determined with galactomannan as substrate were a Vmax. of 205 i.u./mg of enzyme and a Km of 0.77 mg/ml. Data from SDS/PAGE and Western blotting show that the cloned enzyme was identical to that of the wild-type strain.

Asunto(s)

Clonación Molecular , ADN Bacteriano/química , Manosidasas/genética , Streptomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Manosidasas/química , Manosidasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Streptomyces/genética , Temperatura , beta-Manosidasa

RESUMEN

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.

Asunto(s)

Escherichia coli/genética , Vectores Genéticos , Streptomyces/genética , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos

RESUMEN

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.

Asunto(s)

Brevibacterium/genética , Celulasa/genética , Bacterias Grampositivas/genética , Clonación Molecular , Regulación de la Expresión Génica , Genes Bacterianos , Vectores Genéticos , Bacterias Grampositivas/enzimología , Transformación Genética