RESUMEN
Two lines of investigation support a new hypothesis concerning the role of protein synthesis in the mitogenic pathway. The first is that a variety of mitogens and oncogene products increase phosphorylation and thereby activate eIF-4E, which is involved in the rate-limiting transfer of mRNA to ribosomes. The second is that overexpression or microinjection of eIF-4E induce rapid cell proliferation and oncogenic transformation.
Asunto(s)
División Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Biosíntesis de Proteínas , Animales , Factor 4E Eucariótico de Iniciación , Mitógenos , Proteínas Oncogénicas , Factores de Iniciación de Péptidos/metabolismo , FosforilaciónRESUMEN
Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Distrofina/metabolismo , Enterovirus Humano B/enzimología , Glicoproteínas de Membrana/metabolismo , Proteínas Virales , Animales , Cardiomiopatía Dilatada/patología , Células Cultivadas , Infecciones por Coxsackievirus/metabolismo , Citoesqueleto/patología , Distroglicanos , Enterovirus Humano B/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones SCID , Miocardio/citología , Ratas , Sarcolema/patologíaRESUMEN
HeLa cells were transformed to express antisense RNA against initiation factor eIF-4E mRNA from an inducible promoter. In the absence of inducer, these cells (AS cells) were morphologically similar to control cells but grew four- to sevenfold more slowly. Induction of antisense RNA production was lethal. Both eIF-4E mRNA and protein levels were reduced in proportion to the degree of antisense RNA expression, as were the rates of protein synthesis in vivo and in vitro. Polysomes were disaggregated with a concomitant increase in ribosomal subunits. Translation in vitro was restored by addition of the initiation factor complex eIF-4F but not by eIF-4E alone. Immunological analysis revealed that the p220 component of eIF-4F was decreased in extracts of AS cells and undetectable in AS cells treated with inducer, suggesting that p220 and eIF-4E levels are coordinately regulated. eIF-4A, another component of eIF-4F, was unaltered.
Asunto(s)
Factores de Iniciación de Péptidos/genética , ARN sin Sentido/genética , Actinas/genética , Secuencia de Bases , Northern Blotting , División Celular , Factor 4E Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Vectores Genéticos , Células HeLa , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Fenotipo , Reacción en Cadena de la Polimerasa , Polirribosomas/metabolismo , ARN Mensajero/genética , TransfecciónRESUMEN
The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.
Asunto(s)
Insulina/farmacología , Biosíntesis de Proteínas , Proteína Quinasa C/metabolismo , Actinas/biosíntesis , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática , Proteínas Sustrato del Receptor de Insulina , Ratones , Oligonucleótidos Antisentido/farmacología , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Quinasas S6 RibosómicasRESUMEN
Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Insulina/farmacología , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Factor 4E Eucariótico de Iniciación , Proteína Adaptadora GRB2 , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosfatidilinositol 3-Quinasas , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 RibosómicasRESUMEN
The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.
Asunto(s)
Oligorribonucleótidos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Globinas/genética , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Caperuzas de ARN/metabolismo , Conejos , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. METHODS AND RESULTS: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. CONCLUSIONS: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cisteína Endopeptidasas/metabolismo , Distrofina/metabolismo , Infecciones por Enterovirus/metabolismo , Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Péptido Hidrolasas/metabolismo , Proteínas Virales , Animales , Western Blotting , Células COS , Cardiomiopatía Dilatada/prevención & control , Dominio Catalítico/efectos de los fármacos , Cisteína/metabolismo , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/genética , Densitometría , Infecciones por Enterovirus/prevención & control , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Hidrazinas/farmacología , Ratones , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Espermina/análogos & derivados , Espermina/farmacología , Compuestos de Sulfhidrilo/metabolismo , Proteínas Virales de Fusión/genéticaRESUMEN
Topographic analyses were performed on the distribution of neuronal RNA loss in relation to local neuronal loss and neurofibrillary degeneration in the hippocampal region of brains of patients with Alzheimer's disease (AD). Compared to age-matched controls, pyramidal neuronal RNA was depressed (p less than 0.0001) in all areas of the hippocampus examined in AD, viz., the endplate (33%), Rose's H2 field (30%), Rose's H1 field (37%) and the subiculum (46%). Significant neuronal loss was observed in Rose's H1 field and the subiculum but not in the endplate or Rose's H2 field. The frequency of neurofibrillary tangle-bearing neurons was enhanced in all four regions of AD brains, the number of involved neurons being markedly greater in Rose's H1 field and the subiculum than in the endplate and Rose's H2 field. Overall, the data indicate the existence of a generalized disturbance in RNA metabolism within pyramidal neurons in the hippocampus in AD, occurring in regions of minimal (endplate, Rose's H2 field) as well as those of extensive (Rose's H1 field, subiculum) pathological alterations. Although there is focal accentuation of RNA depletion in the latter, the marked RNA depletion in regions of minimal pathologic change suggests that this effect is largely unrelated to local neuronal loss or neurofibrillary degeneration.
Asunto(s)
Enfermedad de Alzheimer/patología , Hipocampo/patología , Neurofibrillas/metabolismo , ARN/análisis , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Hipocampo/análisis , Histocitoquímica , Humanos , Masculino , Neurofibrillas/patología , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
eIF4G is a member of the class of translational initiation factors involved in mRNA recruitment to the 43S initiation complex. The proteins from yeast to mammals are present in multiple isoforms of 82-176 kDa. Mammalian eIF4G-1 is synthesized by internal initiation of translation and is specifically degraded by viral and host proteases activated by stress conditions. The role of eIF4G in protein synthesis is inferred from the presence of binding sites for other initiation factors that serve to co-localize the 5'- and 3'-termini of mRNA with RNA-helicase activity and the 40S ribosomal subunit. Growth-regulated mRNAs are preferentially translated under conditions of accentuated eIF4E-eIF4G interaction. Proteolysis of eIF4G or expression of competitor proteins interferes with its binding to either the 5'- or 3'-termini, changing the spectrum of mRNAs translated. Elevated eIF4G levels correlate with malignant cell transformation and diminished eIF4G levels, with nutritional deprivation and anoxia.
Asunto(s)
Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Animales , Factor 4G Eucariótico de Iniciación , Humanos , Factores de Iniciación de Péptidos/genética , Fosforilación , Biosíntesis de ProteínasRESUMEN
Comparative neuronal RNA analyses were conducted in disease-prone (frontal association, Brodmann's area 9) vs relatively disease-resistant (primary visual, occipital area 17) cortex of patients with autopsy-proved Pick's disease (PD) and Alzheimer's disease (AD). Azure B-RNA staining and scanning-integrating microdensitometry were used to determine total RNA contents of pyramidal neurons in layers 3 and 5. In both PD and AD (1) significant (15% to 47%) RNA loss was detected in neurons of both cortical areas and layers relative to those of aged, nondemented controls, and (2) RNA loss was not markedly enhanced in the damaged frontal cortex relative to that in the preserved occipital cortex. Neuronal RNA depletion was generally more marked in PD than AD. However, this impairment does not appear to be related to the formation of classic neuropathological abnormalities in either disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Corteza Cerebral/metabolismo , Demencia/metabolismo , Neuronas/metabolismo , ARN/metabolismo , Anciano , Enfermedad de Alzheimer/patología , Corteza Cerebral/patología , Demencia/patología , Susceptibilidad a Enfermedades , Femenino , Humanos , Masculino , Distribución TisularRESUMEN
Scanning-integrating microdensitometry of azure B-RNA- and Feulgen-Schiff-stained tissue sections was used to measure neostriatal neuronal RNA levels and susceptibility of neuronal and oligodendrocyte chromatin to acid hydrolysis in Alzheimer's disease (AD) patients and controls. AD was associated with neuronal RNA depletion (17 to 23%) in both caudate nucleus and putamen. While neuronal chromatin was found to be more acid-labile than that of the oligodendrocytes, there were no differences in either cell type between AD and controls. These data support the existence of a macromolecular disturbance (RNA loss) occurring within neostriatal neurons, perhaps related to the extrapyramidal dysfunction of AD, but fail to demonstrate that an alteration in chromatin is responsible for this effect.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Núcleo Caudado/análisis , Cromatina/análisis , Putamen/análisis , ARN/análisis , Anciano , Humanos , Persona de Mediana Edad , Neuronas/análisisRESUMEN
The step of protein synthesis which is normally rate limiting, formation of the 48S initiation complex, is catalyzed by the group 4 initiation factors. Collectively they recognize the 7-methylguanosine-containing cap of mRNA, unwind mRNA secondary structure, and allow scanning for the initiation codon by the small ribosomal subunit. The activities of the eIF-4 polypeptides are modulated by phosphorylation. Recent studies shed new light on the mechanism of assembly of the 48S initiation complex and the effect of phosphorylation of one of the eIF-4 polypeptides, the cap-binding protein eIF-4E.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Animales , Humanos , Factores de Iniciación de Péptidos/genética , ARN Mensajero/genética , Proteínas Ribosómicas/biosíntesisAsunto(s)
Factores de Iniciación de Péptidos/fisiología , ARN Mensajero/metabolismo , Células 3T3/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Células Eucariotas/metabolismo , Factor 4E Eucariótico de Iniciación , Células HeLa/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Fosforilación , Caperuzas de ARN/metabolismo , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , Conejos , Ratas , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Insulina/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , División Celular , Línea Celular , Activación Enzimática , Factor 2B Eucariótico de Iniciación/metabolismo , Glucógeno Sintasa Quinasa 3 , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TORAsunto(s)
Ovalbúmina/biosíntesis , Biosíntesis de Proteínas , ARN Mensajero/análisis , Reticulocitos/análisis , Anemia/sangre , Animales , Centrifugación por Gradiente de Densidad , Pollos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Femenino , Cabras/inmunología , Concentración de Iones de Hidrógeno , Cinética , Leucina , Métodos , Oviductos/metabolismo , Fenilhidrazinas , Pruebas de Precipitina , ARN Mensajero/sangre , ARN Mensajero/metabolismo , Conejos , Reticulocitos/metabolismo , Espectrofotometría Ultravioleta , Factores de Tiempo , TritioRESUMEN
The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.
Asunto(s)
Antivirales/farmacología , Células Dendríticas/virología , Interferón-alfa/farmacología , Interferón beta/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Virus Sindbis/patogenicidad , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células de la Médula Ósea , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Células Dendríticas/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacología , Proteínas de Unión al ARN , Virus Sindbis/genética , Virus Sindbis/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Ovalbumin messenger RNA was purified from hen oviduct by immunoprecipitation of polysomes and oligo(dT)-cellulose chromatography. Two steps were introduced to improve the separation of mRNA and rRNA by oligo(dT)-cellulose. First, aggregates of mRNA and rRNA were dissociated by heating at 65degrees for 10 min before chromatography. Second, elution of the mRNA was achieved by stepwise increases in temperature rather than by lowering the ionic strength. Ovalbumin mRNA activity was eluted primarily in RNA fractions eluting between 45degrees and 55degrees. Polyacrylamide gel electrophoresis indicated that the ovalbumin mRNA thus obtained was essentiallyyy free of rRNA. The poly(A) content of various thermally eluted fractions was assayed by two methods. In the first (Favre, A., Bertazzoni, V., Berns, A.J.M., and Bloemendal, H. (1974) Biochem. Biophys. Res. Commun. 56, 273-280), the increase in fluorescence intensity of bound ethidium bromide was used to follow the formation of double-stranded RNA during titration of the mRNA with poly(U). In the second (Bishop, J. O., Rosbash, M., and Evans, D. (1974) J. Mol. Biol. 85, 75-86), poly(A) content was derived from the radioactivity remaining acid-insoluble after annealing mRNA fractions with [3H]poly(U) and treating with ribonuclease A. Both methods indicated that ovalbumin mRNA fractions eluting at higher temperatures contained greater amounts of poly(A). Values ranged from 44 to 248 mol of AMP/mol of mRNA, assuming 2200 total nucleotide residues for ovalbumin mRNA (Shapiro, D. J., and Schimke, R. T. (1975) J. Biol. Chem. 250, 1759-1764). Translational specific activities in a rabbit reticulocyte lysate system were essentiall constant for all fractions. From the binding of ethidium bromide it could be estimated that approximately 50% of the nucleotide residues in ovalbumin mRNA are base-paired.
Asunto(s)
ARN Mensajero/aislamiento & purificación , Animales , Pollos , Cromatografía de Afinidad , Etidio , Femenino , Calor , Cinética , Matemática , Ovalbúmina/biosíntesis , Ovalbúmina/inmunología , Oviductos/análisis , Oviductos/metabolismo , Poli A/análisis , Poli U/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismoRESUMEN
A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.