RESUMEN

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis. In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.

Asunto(s)

Quilomicrones/farmacocinética , Vitamina A/análogos & derivados , Animales , Grasas de la Dieta/metabolismo , Diterpenos , Ayuno , Humanos , Lipoproteínas/farmacocinética , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Proteínas de Unión al Retinol/farmacocinética , Ésteres de Retinilo , Vitamina A/metabolismo

RESUMEN

The tissue-specific expression of lipoprotein lipase (LPL) in adipose tissue (AT), skeletal muscle (SM), and cardiac muscle (CM) is rate-limiting for the uptake of triglyceride (TG)-derived free fatty acids and decisive in the regulation of energy balance and lipoprotein metabolism. To investigate the tissue-specific metabolic effects of LPL, three independent transgenic mouse lines were established that expressed a human LPL (hLPL) minigene predominantly in CM. Through cross-breeding with heterozygous LPL knockout mice, animals were generated that produced hLPL mRNA and enzyme activity in CM but lacked the enzyme in SM and AT because of the absence of the endogenous mouse LPL gene (L0-hLPL). LPL activity in CM and postheparin plasma of L0-hLPL mice was reduced by 34% and 60%, respectively, compared with control mice. This reduced LPL expression was sufficient to rescue LPL knockout mice from neonatal death. L0-hLPL animals developed normally with regard to body weight and body-mass composition. Plasma TG levels in L0-hLPL animals were increased up to 10-fold during the suckling period but normalized after weaning and decreased in adult animals. L0-hLPL mice had normal plasma high-density lipoprotein (HDL)-cholesterol levels, indicating that LPL expression in CM alone was sufficient to allow for normal HDL production. The absence of LPL in SM and AT did not cause detectable morphological or histopathological changes in these tissues. However, the lipid composition in AT and SM exhibited a marked decrease in polyunsaturated fatty acids. From this genetic model of LPL deficiency in SM and AT, it can be concluded that CM-specific LPL expression is a major determinant in the regulation of plasma TG and HDL-cholesterol levels.

Asunto(s)

Tejido Adiposo/metabolismo , HDL-Colesterol/sangre , Lipoproteína Lipasa/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Triglicéridos/sangre , Animales , Peso Corporal , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Lipoproteína Lipasa/biosíntesis , Ratones , Ratones Mutantes , Ratones Transgénicos , Especificidad de Órganos , Transfección

RESUMEN

Lipoprotein lipase (LPL), the rate-limiting enzyme in triglyceride hydrolysis, is normally not expressed in the liver of adult humans and animals. However, liver LPL is found in the perinatal period, and in adults it can be induced by cytokines. To study the metabolic consequences of liver LPL expression, transgenic mice producing human LPL specifically in the liver were generated and crossed onto the LPL knockout (LPL0) background. LPL expression exclusively in liver rescued LPL0 mice from neonatal death. The mice developed a severe cachexia during high fat suckling, but caught up in weight after switching to a chow diet. At 18 h of age, compared with LPL0 mice, liver-only LPL-expressing mice had equally elevated triglycerides (10,700 vs. 14,800 mg/dl, P = NS), increased plasma ketones (4.3 vs. 1.7 mg/dl, P < 0.05) and glucose (28 vs. 15 mg/dl, P < 0.05), and excessive amounts of intracellular liver lipid droplets. Adult mice expressing LPL exclusively in liver had slower VLDL turnover than wild-type mice, but greater VLDL mass clearance, increased VLDL triglyceride production, and three- to fourfold more plasma ketones. In summary, it appears that liver LPL shunts circulating triglycerides to the liver, which results in a futile cycle of enhanced VLDL production and increased ketone production, and subsequently spares glucose. This may be important to sustain brain and muscle function at times of metabolic stress with limited glucose availability.

Asunto(s)

Regulación Enzimológica de la Expresión Génica/genética , Lipoproteína Lipasa/metabolismo , Hígado/enzimología , Factores de Edad , Animales , Animales Recién Nacidos , Peso Corporal/genética , Colesterol/sangre , Dieta , Genotipo , Histocitoquímica , Humanos , Cetonas/sangre , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Triglicéridos/sangre

RESUMEN

The erythroid-specific isoform of delta-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for beta-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

Asunto(s)

5-Aminolevulinato Sintetasa/deficiencia , Embrión de Mamíferos , Eritrocitos/enzimología , Globinas/biosíntesis , Hemo/biosíntesis , Células Madre/metabolismo , 5-Aminolevulinato Sintetasa/genética , Animales , Bencidinas , Diferenciación Celular , Colorantes , Expresión Génica , Marcación de Gen , Vectores Genéticos , Globinas/genética , Ratones , Mutación , ARN Mensajero/metabolismo

RESUMEN

To determine the physiological role of apolipoprotein (apo) A-IV, knockout mice were created by gene targeting in embryonic stem cells. In apoA-IV knockout mice, plasma cholesterol and triglyceride levels were reduced 25% and 44%, respectively, compared with controls. These changes were accounted for by decreased high density (HDL) and very low density lipoprotein (VLDL) levels, respectively, and metabolic studies indicated increased HDL-cholesteryl ester (CE) fractional catabolic rate (FCR) and reduced VLDL transport rate (TR), respectively. ApoA-IV knockout mice had greater than 70% reductions in both hepatic and intestinal apoC-III RNA levels and a similar reduction in the plasma apoC-III level. Complementation analysis, via crossbreeding of a mouse apoC-III transgene onto both the normal and apoA-IV knockout backgrounds, clearly demonstrated that the low triglyceride (VLDL) level in the apoA-IV knockout mice was due to alterations in apoC-III and not apoA-IV. ApoA-IV knockout mice had normal growth, feeding behavior, and lipid absorption, except male mice showed increased food intake in the 2 h after an 18-h fast, suggesting that under some circumstances apoA-IV might serve as a satiety factor. In summary, studies in apoA-IV-induced mutant mice have demonstrated a role for apoA-IV in increasing HDL cholesterol by inhibiting HDL cholesteryl ester FCR yet argue against the apolipoprotein as an overall important mediator of lipid absorption/metabolism.

Asunto(s)

Apolipoproteínas A/deficiencia , HDL-Colesterol/sangre , Metabolismo de los Lípidos , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/genética , Apolipoproteína C-III , Apolipoproteínas A/genética , Apolipoproteínas C/genética , Ésteres del Colesterol/sangre , HDL-Colesterol/metabolismo , Grasas de la Dieta/farmacocinética , Ingestión de Alimentos , Conducta Alimentaria , Femenino , Expresión Génica , Crecimiento , Absorción Intestinal , Lípidos/sangre , Lipoproteínas/sangre , Lipoproteínas HDL/sangre , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/metabolismo , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Aumento de Peso

RESUMEN

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the import of triglyceride-derived fatty acids by muscle, for utilization, and adipose tissue (AT), for storage. Relative ratios of LPL expression in these two tissues have therefore been suggested to determine body mass composition as well as play a role in the initiation and/or development of obesity. To test this, LPL knockout mice were mated to transgenics expressing LPL under the control of a muscle-specific promoter (MCK) to generate induced mutants with either relative (L2-MCK) or absolute AT LPL deficiency (L0-MCK). L0-MCK mice had normal weight gain and body mass composition. However, AT chemical composition indicated that LPL deficiency was compensated for by large increases in endogenous AT fatty acid synthesis. Histological analysis confirmed that such up-regulation of de novo fatty acid synthesis in L0-MCK mice could produce normal amounts of AT as early as 20 h after birth. To assess the role of AT LPL during times of profound weight gain, L0-MCK and L2-MCK genotypes were compared on the obese ob/ob background. ob/ob mice rendered deficient in AT LPL (L0-MCK-ob/ob) also demonstrated increased endogenous fatty acid synthesis but had diminished weight and fat mass. These findings reveal marked alterations in AT metabolism that occur during LPL deficiency and provide strong evidence for a role of AT LPL in one type of genetic obesity.

Asunto(s)

Tejido Adiposo/metabolismo , Ácidos Grasos/metabolismo , Lipoproteína Lipasa/metabolismo , Tejido Adiposo/fisiología , Animales , Transporte Biológico , Ácidos Grasos/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Lipoproteína Lipasa/deficiencia , Lipoproteína Lipasa/genética , Ratones , Ratones Obesos , Ratones Transgénicos

RESUMEN

During periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and phospholipids provide energy for macrophages in areas that have limited amounts of ambient glucose, and during periods of intense metabolic activity.

Asunto(s)

Glucosa/deficiencia , Lipoproteína Lipasa/genética , Macrófagos/inmunología , Fagocitosis/inmunología , Receptores Fc/inmunología , Animales , Células Cultivadas , Medios de Cultivo , Regulación de la Expresión Génica , Humanos , Lipoproteína Lipasa/inmunología , Macrófagos/enzimología , Macrófagos/patología , Ratones , Ratones Transgénicos , Receptores Fc/genética

RESUMEN

To determine the contribution of muscle lipoprotein lipase (LPL) to lipoprotein metabolism, induced mutant mice were generated that express human LPL exclusively in muscle. By cross-breeding heterozygous LPL knockout mice with transgenic mice expressing human LPL only in muscle, animals were obtained that express human LPL primarily in skeletal muscle on either the null (L0-MCK) or normal (L2-MCK) LPL backgrounds, and these were compared with control littermates (L2). Fed and fasted post-heparin plasma (PHP) LPL activities were increased 1.4- and 2.3-fold, respectively, in L2-MCK mice and were normal in L0-MCK mice compared with controls. The specific enzyme activities of human LPL in mouse plasma was comparable to human LPL in human PHP. Skeletal muscle LPL activity was increased in both L2-MCK and L0-MCK mice in the fed (6.6-fold) and fasted (4.2-fold in L2-MCK; and 3.4-fold in L0-MCK) states. Adipose tissue LPL mRNA and activity were not detectable in L0-MCK mice. Growth and body mass composition were similar among all groups. In the fasted and fed state, L2-MCK mice had 31% and 53% reductions, respectively, in plasma triglycerides (TG), compatible with increased PHP LPL activity. Unexpectedly, both in the fasted and fed state the L0-MCK mice also had reduced TG (22%), despite normal PHP LPL activities. Very low density lipoprotein (VLDL) turnover studies revealed that the decreased TG were due to increased particle fractional catabolic rate in both L2-MCK and L0-MCK mice. Despite reduced TG, both L2-MCK and L0-MCK mice showed reduced high density lipoprotein (HDL) cholesterol levels (16% and 19%, respectively). HDL turnover studies indicated increased HDL cholesteryl ester fractional catabolic rate in the L2-MCK and L0-MCK compared with control mice. In summary, these studies suggest that muscle LPL is particularly potent with regard to VLDL metabolism and is sufficient to compensate for the lack of LPL in other tissues with regard to lipolyzing VLDL particles. With regard to HDL, muscle LPL expression does not result in normal levels due to enhanced breakdown either by mediating accelerated HDL clearance or by failing to establish normal HDL particles that are then cleared more quickly than normal. These studies provide new insights on the tissue-specific effects of LPL on lipoprotein metabolism.

Asunto(s)

HDL-Colesterol/sangre , Lipoproteína Lipasa/metabolismo , Músculos/enzimología , Triglicéridos/sangre , Animales , Metabolismo Energético , Femenino , Genotipo , Heparina , Humanos , Lípidos/sangre , Lipoproteína Lipasa/genética , Lipoproteínas/sangre , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos

RESUMEN

We previously showed that human apoC-III expression in transgenic mice causes hypertriglyceridemia due to the accumulation of enlarged very low density lipoprotein (VLDL)-like particles, with increased triglycerides and apoC-III and decreased apoE. In vivo turnover studies indicated the metabolic basis was decreased particle fractional catabolic rate. The presence of enlarged triglyceride-rich particles with prolonged residence time in plasma implied defective lipolysis, but in vitro these particles were good substrates for purified lipoprotein lipase (LPL). In the current study we further characterize the metabolic properties of these particles. We show that expression of a mouse apoC-III transgene can also cause hypertriglyceridemia with a similar accumulation of a VLDL-like particle with increased apoC-III and decreased apoE. A vitamin A fat tolerance test was used to show that MoCIIITg and HuCIIITg mice had similarly delayed clearance of triglyceride-rich postprandial particles. Thus, the previously observed hypertriglyceridemia caused by human apoC-III transgene expression was not due interspecies incompatibility but a property of apoC-III. In further experiments we showed VLDL from apoC-III transgenic mice interacted poorly with fibroblast lipoprotein receptors and this could be corrected by adding exogenous apoE. In addition, control VLDL interaction could be decreased by exogenous apoC-III. Moreover, the hypertriglyceridemia of HuCIIITg mice could be normalized by crossbreeding with HuETg mice. Thus, a functionally significant reciprocal relationship of apoC-III and apoE exists, presumably due to competition for space on the surface of triglyceride-rich lipoproteins. Finally, VLDL from HuCIITg and MoCIIITg mice showed decreased binding to heparin-Sepharose. This suggests and additional locus of the defect in these mice could potentially be in the binding of triglyceride-rich lipoproteins to heparan sulfate proteoglycan matrix on the surface of endothelial cells in which LPL is embedded. This could explain the predicted functional lipase deficiency in apoC-III transgenic mice based on the observation of a prolonged residence time of enlarged triglyceride-rich lipoproteins.

Asunto(s)

Apolipoproteínas C/genética , Regulación de la Expresión Génica/genética , Hipertrigliceridemia/etiología , Lipoproteínas VLDL/metabolismo , Triglicéridos/metabolismo , Animales , Apolipoproteína C-III , Grasas de la Dieta/metabolismo , Ayuno , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Lípidos/sangre , Lipoproteínas VLDL/análisis , Masculino , Ratones , Ratones Transgénicos , Periodo Posprandial , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Tiempo , Transgenes/genética , Triglicéridos/análisis , Vitamina A/metabolismo

RESUMEN

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.

Asunto(s)

Hipertrigliceridemia/fisiopatología , Lipoproteína Lipasa/deficiencia , Lipoproteína Lipasa/genética , Lipoproteínas HDL/sangre , Triglicéridos/sangre , Tejido Adiposo/patología , Animales , Animales Recién Nacidos , Southern Blotting , Composición Corporal , Colesterol/sangre , ADN/análisis , Muerte , Femenino , Genotipo , Heterocigoto , Hipertrigliceridemia/genética , Hipertrigliceridemia/patología , Absorción Intestinal , Lipoproteínas LDL/sangre , Hígado/patología , Hígado/ultraestructura , Ratones , Ratones Noqueados , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Embarazo , Células Madre , Vitamina A/metabolismo

RESUMEN

In extrahepatic tissues lipoprotein lipase (LPL) hydrolyzes triglycerides thereby generating FFA for tissue uptake and metabolism. To study the effects of increased FFA uptake in muscle tissue, transgenic mouse lines were generated with a human LPL minigene driven by the promoter of the muscle creatine kinase gene. In these mice human LPL was expressed in skeletal muscle and cardiac muscle, but not in other tissues. In proportion to the level of LPL overexpression, decreased plasma triglyceride levels, elevated FFA uptake by muscle tissue, weight loss, and premature death were observed in three independent transgenic mouse lines. The animals developed a severe myopathy characterized by muscle fiber degeneration, fiber atrophy, glycogen storage, and extensive proliferation of mitochondria and peroxisomes. This degree of proliferation suggests that FFA play an important role in the biogenesis of these organelles. Our experiments indicate that LPL is rate limiting for the supply of muscle tissue with triglyceride-derived FFA. Improper regulation of muscle LPL can lead to major pathological changes and may be important in the pathogenesis of some human myopathies. Muscle-specific LPL transgenic mouse lines will serve as a useful animal model for the investigation of myopathies and the biogenesis of mitochondria and peroxisomes.

Asunto(s)

Ácidos Grasos no Esterificados/metabolismo , Lipoproteína Lipasa/genética , Microcuerpos/patología , Mitocondrias Musculares/patología , Miopatías Mitocondriales/etiología , Proteínas Musculares/genética , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Creatina Quinasa/genética , Inducción Enzimática , Genes Sintéticos , Glucosa/metabolismo , Humanos , Lipólisis , Lipoproteína Lipasa/biosíntesis , Longevidad , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Miopatías Mitocondriales/enzimología , Miopatías Mitocondriales/genética , Proteínas Musculares/biosíntesis , Músculos/metabolismo , Músculos/patología , Especificidad de Órganos , Regiones Promotoras Genéticas , Triglicéridos/sangre , Pérdida de Peso

RESUMEN

Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.

Asunto(s)

ADN de Cadena Simple/metabolismo , ADN/metabolismo , Técnicas Genéticas , Hibridación de Ácido Nucleico , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Estabilidad de Enzimas , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Temperatura

RESUMEN

In nerve cells, a specialized protein synthetic machinery is thought to operate in local compartments of dendrites, in particular beneath synaptic junctions, and thereby to facilitate swift adjustments of the postsynaptic protein repertoire in situ. This notion has been supported by the identification of polyribosomes and selected mRNAs in those compartments. In this study, we report the discovery of a specific RNA polymerase III transcript in dendrites. This RNA, a noncoding, 152-nucleotide-long, single-gene transcript known as BC1 RNA, is expressed almost exclusively in the nervous system. In adult rats as well as in immature rats in late developmental stages, BC1 RNA has been located in the dendrites and somata of a subset of neurons in the central and peripheral nervous system. The colocalization of BC1 RNA with dendritic mRNAs and polyribosomes may indicate a role--possibly within the functional unit of a high molecular mass ribonucleoprotein particle--in specific pre- or posttranslational processes in postsynaptic compartments of neurons.

Asunto(s)

Encéfalo/fisiología , Dendritas/fisiología , Neuronas/fisiología , ARN/análisis , Animales , Encéfalo/citología , Células Cultivadas , Dendritas/ultraestructura , Proteína GAP-43 , Glicoproteínas de Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Hibridación de Ácido Nucleico , Especificidad de Órganos , Fosfoproteínas/genética , ARN/genética , ARN Polimerasa III/metabolismo , ARN Mensajero/análisis , Ratas , Médula Espinal/fisiología , Transcripción Genética

RESUMEN

Branch capture reactions (BCR) contain two DNA species: (i) a recipient restriction fragment terminating in an overhang and (ii) a displacer-linker duplex terminating in a displacer tail complementary to the overhang as well as contiguous nucleotides within the recipient duplex. Branched complexes containing both species are captured by ligation of the linker to the recipient overhang. Specificity depends upon branch migration and is increased by substitution of bromodeoxycytidine for deoxycytidine in the displacer. BCR rates and specificities were determined for recipient overhangs that were (i) 5' and 3', (ii) 3 and 4 nucleotides long, and (iii) 0-100% G+C. Model systems permitted independent determination of G+C and branching effects on ligation rates and verification of rapid equilibrium between the branched complex and its component species. With all 4-base overhangs, recipient duplexes permitting extensive branch migration became saturated with displacer-linker duplexes. With increasing G+C, increasing ligation at competing sites led to decreased BCR specificity. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce a new overhang sequence compatible with a restriction endonuclease site in a cloning vector. A protocol was confirmed for mapping restriction sites in cloned DNA.

Asunto(s)

ADN/genética , Mapeo Restrictivo , Secuencia de Bases , Técnicas Genéticas , Cinética , Matemática , Datos de Secuencia Molecular