RESUMEN
We hypothesized that certain proteins encoded by temperature-responsive genes in brown adipose tissue (BAT) contribute to the remarkable metabolic shifts observed in this tissue, thus prompting a differential mRNA expression analysis to identify candidates involved in this process in mouse BAT. An mRNA species corresponding to a novel partial-length gene was found to be induced 2-3-fold above the control following cold exposure (4 degrees C), and repressed approximately 70% by warm acclimation (33 degrees C, 3 weeks) compared with controls (22 degrees C). The gene displayed robust BAT expression (i.e. approximately 7-100-fold higher than other tissues in controls). The full-length murine gene encodes a 594 amino acid ( approximately 67 kDa) open reading frame with significant homology to the human hypothetical acyl-CoA thioesterase KIAA0707. Based on cold-inducibility of the gene and the presence of two acyl-CoA thioesterase domains, we termed the protein brown-fat-inducible thioesterase (BFIT). Subsequent analyses and cloning efforts revealed the presence of a novel splice variant in humans (termed hBFIT2), encoding the orthologue to the murine BAT gene. BFIT was mapped to syntenic regions of chromosomes 1 (human) and 4 (mouse) associated with body fatness and diet-induced obesity, potentially linking a deficit of BFIT activity with exacerbation of these traits. Consistent with this notion, BFIT mRNA was significantly higher ( approximately 1.6-2-fold) in the BAT of obesity-resistant compared with obesity-prone mice fed a high-fat diet, and was 2.5-fold higher in controls compared with ob/ob mice. Its strong, cold-inducible BAT expression in mice suggests that BFIT supports the transition of this tissue towards increased metabolic activity, probably through alteration of intracellular fatty acyl-CoA concentration.
Asunto(s)
Tejido Adiposo/enzimología , Obesidad/genética , Palmitoil-CoA Hidrolasa/biosíntesis , Palmitoil-CoA Hidrolasa/química , Palmitoil-CoA Hidrolasa/genética , Empalme Alternativo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Clonación Molecular , Frío , ADN Complementario/metabolismo , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Mapeo de Híbrido por Radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Temperatura , Distribución TisularRESUMEN
Since their development in the early 1990s, differential gene expression (DGE) technologies have been applied to a multitude of biological challenges, both for the purpose of basic biological research and as a valuable tool for the discovery and development of pharmaceuticals. In this review we survey a class of DGE technologies collectively referred to as 'open' architecture systems. These technologies are distinct from the 'closed' DGE technologies (quantitative PCR, chip technologies), in that no pre-existing biological or sequence information is necessary and they are applicable to any species. Examples of open systems include GeneCalling, SAGE, TOGA, READS, and their progenitor DGE technologies, differential display and cDNA representational difference analysis. We review these technologies and summarize a specific application using GeneCalling for novel gene discovery. Additionally, the significance of data management and experimental design in this new age of expression analysis is discussed.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Animales , Línea Celular , Quimiocinas/genética , ADN Complementario/genética , HumanosRESUMEN
Using differential mRNA expression analysis, a previously uncharacterized gene was found to be up-regulated 2-fold in brown adipose tissue (BAT) of mice exposed to cold (4 degrees C) for 48 h. Contig and homology analysis revealed that the gene represents the murine orthologue to a sequence from a public database encoding a putative human protein (CGI-69). The presence of mitochondrial carrier domains in the human protein, its transmembrane topology and cold-induction of the mouse CGI-69 gene in BAT prompted an analysis of the idea that CGI-69 may represent a new uncoupling protein (UCP) functional homologue. However, transfection of human CGI-69 isoforms in HEK-293 cells yielded no change in mitochondrial membrane potential (Deltapsi(m)), despite localization of FLAG-tagged CGI-69 to mitochondria of MCF7 cells. Surprisingly, overexpression of the human 2-oxoglutarate carrier (OGC) protein (originally designed as a negative control) sparked a significant drop in Deltapsi(m), possibly signalling a previously unappreciated uncoupling activity for the OGC.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteínas Portadoras/biosíntesis , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas Mitocondriales , Proteínas Recombinantes de Fusión , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular , Frío , Masculino , Potenciales de la Membrana , Proteínas de Transporte de Membrana Mitocondrial , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de AminoácidoRESUMEN
In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.
Asunto(s)
Expresión Génica , Neovascularización Fisiológica/genética , Venas/fisiología , Células Cultivadas , Dermatoglifia del ADN , Fragmentación del ADN , ADN Complementario/genética , Endotelio Vascular/fisiología , Genoma , Humanos , Hibridación in Situ , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Absence of the hormone leptin leads to dramatic increases in appetite, food intake, and adiposity. The primary site of action, at least with respect to appetite, is the hypothalamus. Leptin also has significant effects on the function(s) of peripheral organs involved in maintaining body composition. Some of these effects are mediated through direct interaction of leptin with its receptor on the target tissue, and some effects are indirectly mediated through secondary hormonal and neural pathways. Few of the genes that are responsible for regulating body composition and the peripheral effects of leptin are known. We have used a new gene profiling technology to characterize gene expression changes that occur in the pituitary, hypothalamus, fat, muscle, and liver in response to both obesity and treatment with exogenous leptin. These differences were then overlaid to allow the identification of genes that are regulated by obesity and at least partially normalized by leptin treatment. By using this process we have identified five genes (POMC, PC2, prolactin, HSGP25L2G, and one novel) that are both abnormally expressed in the pituitaries of obese mice and are sensitive to the effects of leptin. We also show that adrenocorticotropic hormone appears to be involved in a regulatory loop involving leptin.
Asunto(s)
Hormona Adrenocorticotrópica/genética , Regulación de la Expresión Génica/fisiología , Leptina/fisiología , Obesidad/genética , Hipófisis/metabolismo , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/metabolismo , Leptina/farmacología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Biológicos , Músculo Esquelético/metabolismo , Obesidad/fisiopatología , Especificidad de ÓrganosRESUMEN
Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation.
Asunto(s)
Quimiocinas/fisiología , Hematopoyesis/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Quimiocinas/genética , Quimiocinas CXC , Quimiotaxis/fisiología , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We describe the cloning, expression, and localization of gamma2-adaptin, a novel isoform of gamma-adaptin. The predicted human and mouse gamma2-adaptin proteins are approximately 90 kDa and 64.4% and 61.7%) identical to gamma-adaptin, respectively. gamma2-Adaptin was localized to the Golgi, its localization distinct from gamma-adaptin. The membrane association of gamma- and gamma2-adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, gamma2-adaptin binding being insensitive to drug treatment. Together, these results suggest that gamma2-adaptin plays a role in membrane transport distinct from that played by gamma-adaptin.
Asunto(s)
Proteínas de la Membrana/genética , Subunidades gamma de Complejo de Proteína Adaptadora , Secuencia de Aminoácidos , Animales , Clonación Molecular , Expresión Génica , Células HeLa , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Análisis de SecuenciaRESUMEN
We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.