RESUMEN
Lantibiotics are ribosomally synthesized oligopeptide antibiotics that contain lanthionine bridges derived by the posttranslational modification of amino acid residues. Here, we describe the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005, the first, to our knowledge, lantibiotic gene cluster from a high G+C bacterium to be cloned and sequenced. The cin cluster contains many genes not found in lantibiotic clusters from low G+C Gram-positive bacteria, including a Streptomyces antibiotic regulatory protein regulatory gene, and lacks others found in such clusters, such as a LanT-type transporter and a LanP-type protease. Transfer of the cin cluster to Streptomyces lividans resulted in heterologous production of cinnamycin. Furthermore, modification of the cinnamycin structural gene (cinA) led to production of two naturally occurring lantibiotics, duramycin and duramycin B, closely resembling cinnamycin, whereas attempts to make a more widely diverged derivative, duramycin C, failed to generate biologically active material. These results provide a basis for future attempts to construct extensive libraries of cinnamycin variants.
Asunto(s)
Antibacterianos/biosíntesis , Familia de Multigenes , Péptidos Cíclicos , Streptomyces/genética , Bacteriocinas , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
The estrogen receptor protein, in the nuclear receptor superfamily, carries two transactivator domains designated AF1 and AF2. The activity of AF2, localized in the carboxy-terminal region, is ligand-dependent, whereas AF1 (amino-terminal) seems to be activated via the MAPKkinase pathway. Uterine and mammary cells exhibiting large amounts of ERalpha were the first estrogen target organs demonstrated. The response intensity in these tissues is related to the affinity of the receptor and to the number of sites occupied by its ligand. Certain physiological and pharmacological phenomena of estrogen resistance associated with a truncated form of ERalpha (deleted in the AF1 domain) would seem however to challenge this assertion. The 45 kDa truncated form is unable to induce cell proliferation but can still increase the expression of certain genes. In this work we suggest that this 45 kDa ERalpha form may originate from differential regulation of translation of the mRNA encoding the ERalpha. In vitro translation studies and transient expression in COS-7 cells in vivo demonstrated a mechanism of translation regulation that produced from a given mRNA either the wild type ER 67 kDa form or the AF1 deleted ER 45 kDa isoform. Bicistronic vectors were used to demonstrate that the 45 kDa protein originates from translation initiation at AUG 174 induced by an internal ribosome entry.
Asunto(s)
Codón Iniciador/genética , Regulación de la Expresión Génica , Biosíntesis de Proteínas/genética , Receptores de Estrógenos/genética , Regiones no Traducidas 5'/química , Regiones no Traducidas 5'/genética , Animales , Western Blotting , Células COS , Codón de Terminación/genética , Receptor alfa de Estrógeno , Femenino , Genes/genética , Humanos , Peso Molecular , Mutación , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , Receptores de Estrógenos/biosíntesis , Receptores de Estrógenos/química , Ribosomas/metabolismo , Eliminación de Secuencia/genética , TransfecciónRESUMEN
Using ER 1D5 new antibody it is shown that the estrogen receptor has an extra-nuclear localisation in vascular cells. This work exhibits, moreover, that the polymorphism of the estrogen receptor is in part regulated at the translation step.
Asunto(s)
Endotelio Vascular/química , Estrógenos/fisiología , Polimorfismo Genético/genética , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Animales , Anticuerpos Monoclonales , Aorta/citología , Inmunohistoquímica , Músculo Liso Vascular/citología , Biosíntesis de Proteínas , Ratas , Ratas Wistar , Receptores de Estrógenos/efectos de los fármacosRESUMEN
Cell response to steroid stimulation is generally acknowledged to be mediated by an intracellular protein known as a receptor. Response intensity is related to the affinity of the receptor and to the number of sites occupied by its specific ligand. Although verified in the majority of experimental and clinical studies, certain phenomena of steroid hormone resistance would seem to challenge this assertion. Application of gene molecular biology to determine the action mechanisms of steroid hormones has partially explained cell resistance in terms of genetic modifications. The work presented here shows that in certain cases, estrogen resistance could be explained by regulation of translation of the single messenger RNA coding for the receptor.
Asunto(s)
Enfermedades del Sistema Endocrino/genética , Estradiol/farmacología , Biosíntesis de Proteínas , ARN Mensajero/genética , Receptores de Estrógenos/genética , Animales , Línea Celular , Resistencia a Medicamentos , HumanosRESUMEN
A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.
Asunto(s)
Cromosomas Bacterianos , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Mapeo Restrictivo , Streptococcus pneumoniae/genética , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Circular/genética , Marcadores Genéticos , Especificidad por SustratoRESUMEN
We are interested in the molecular mechanisms of the regulation of neurotransmitter related gene expression by neurotrophic factors and neuronal activity. Previous work has shown that conditioned medium of muscle cells induces choline acetyltransferase (CAT) activity and represses tyrosine hydroxylase, dopamine-beta-hydroxylase and aromatic L-amino acid decarboxylase (AADC) activities in cultured sympathetic neurons. Here, we show that a new muscle-derived cell line secretes two factors which induce CAT activity in spinal cord cultures; one of those is related to LIF, a CAT inducing factor for sympathetic neurons. Preliminary data are presented on the structure of the human AADC and CAT genes. Putative promoter regions have been coupled to reporter genes; transient transfection experiments will allow us to determine the promoter elements responsible for the regulation by neurotrophic factors. We also summarize the distribution of AADC-immunoreactive cells in rat and cat brain, which will be used as a reference for the study of the specificity of expression of AADC promoter in transgenic mice.
