RESUMEN
Tetracycline regulated gene expression in transgenic animals is potentially a very powerful technique (Furth et al., 1994; Gossen & Bujard 1992). We have utilized this system in an attempt to overcome the perinatal lethality resulting from constitutive transgenic expression in the heart (Valencik & McDonald, Am J Physiol Heart Circ Physiol 280: H361-H367). We found that compound hemizygous animals created by mating selected reverse tetracycline transactivator (rtTA) and transresponder (TR) lines display tightly regulated TR expression in the heart. However, we identified two fundamental problems. First, codon usage bias appeared to severely limit the expression of the rtTA driven by the cardiac alpha-myosin heavy chain promoter. Second, co-injection of rtTA and TR transgenes led to compound hemizygous animals that exhibited unregulated TR gene expression. Codon optimization of the rtTA construct leads to marked improvement (increasing the average induction from 20-fold to 832-fold) in cardiac myocyte expression. The resulting opt-rtTA lines can be bred to homozygosity, facilitating rapid screening of F0 TR animals for doxycycline regulated transgene expression.
Asunto(s)
Codón/genética , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Miocardio/metabolismo , Transgenes/genética , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Northern Blotting , Células Cultivadas , Genes Reporteros/genética , Immunoblotting , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Miocardio/citología , Cadenas Pesadas de Miosina/genética , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The molecular mechanisms that regulate the cardiac hypertrophic response and the progression from compensated hypertrophy to decompensated heart failure have not been thoroughly defined. Alteration in cardiac extracellular matrix is a distinguishing characteristic of these pathological processes. Integrins, cell surface receptors that mediate cellular adhesion to the extracellular matrix, are signaling molecules that possess mechanotransduction properties. Therefore, we hypothesized that integrins are likely candidates to play an important role in cardiac function. To test this hypothesis, transgenic mice were constructed in which normal integrin function was disrupted by expression of a chimeric molecule encoding the transmembrane and extracellular domains of the Tac subunit of the IL-2 receptor, fused to the cytoplasmic domain of beta(1A) integrin (Tacbeta(1A)). Using the alpha myosin heavy chain promoter to target expression of this chimera to the cardiac myocyte, transgenic mice were generated that had varied levels of transgene expression. Multiple transgenic founders that expressed the transgene at high levels, died perinatally and exhibited replacement fibrosis. Lines that survived showed 1) hypertrophic changes concordant with reduction in endogenous beta(1) integrin levels, or 2) reduced basal contractility and relaxation as well as alterations in components of integrin signaling pathways. These data support an important role for beta(1) integrin in normal cardiac function.
Asunto(s)
Cardiomegalia/etiología , Fibrosis/etiología , Corazón/fisiopatología , Integrina beta1/fisiología , Animales , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Adhesión Celular , Células Cultivadas , Matriz Extracelular/fisiología , Femenino , Muerte Fetal , Fibrosis/metabolismo , Fibrosis/patología , Fibrosis/fisiopatología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica , Hemodinámica , Integrina beta1/genética , Integrina beta1/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Miocardio/patología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-2/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. alpha(5)beta(1)-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of alpha(5)beta(1)-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human alpha(5)-integrin in cardiomyocytes: the full-length wild-type alpha(5)-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated alpha(5-1)-integrin. Overexpression of the wild-type alpha(5)-integrin has no detectable adverse effects in the mouse, whereas expression of alpha(5-1)-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.
Asunto(s)
Animales Recién Nacidos/fisiología , Genes Letales , Corazón/embriología , Corazón/fisiología , Miocardio/metabolismo , Receptores de Fibronectina/biosíntesis , Animales , Western Blotting , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Muerte Súbita/patología , Electrocardiografía , Fibrosis , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos , Miocardio/patología , Receptores de Fibronectina/genética , Receptores de Fibronectina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransgenesRESUMEN
The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.
Asunto(s)
Proteínas Fúngicas/fisiología , Levaduras/citología , División Celular/fisiología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Levaduras/química , Levaduras/metabolismoRESUMEN
Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor. This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation. While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure. The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression. Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression. Five of the ten mutants were respiration deficient. Two of these five mutants were defective in expression of COX3 but not in expression of COX1, while two other mutants had the opposite phenotype (primarily defective in expression of COX1). The fifth mutant was equally defective in expression of both genes. These results demonstrate that the two functions of PET54 are genetically separable and support the idea that the PET54 protein is multifunctional.
Asunto(s)
ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Mitocondrias/enzimología , Saccharomyces cerevisiae/genética , Transactivadores/genética , Deleción Cromosómica , Genes Supresores , Sustancias Macromoleculares , Mutagénesis Insercional , ARN Mensajero/genética , Mapeo Restrictivo , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.