RESUMEN
The alpha component of the receptor for ciliary neurotrophic factor (CNTF) differs from other known growth factor receptors in that it is anchored to cell membranes by a glycosylphosphatidylinositol linkage. One possible function of this type of linkage is to allow for the regulated release of this receptor component. Cell lines not normally responsive to CNTF responded to treatment with a combination of CNTF and a soluble form of the CNTF alpha receptor component. These findings not only demonstrate that the CNTF receptor alpha chain is a required component of the functional CNTF receptor complex but also reveal that it can function in soluble form as part of a heterodimeric ligand. Potential physiological roles for the soluble CNTF receptor are suggested by its presence in cerebrospinal fluid and by its release from skeletal muscle in response to peripheral nerve injury.
Asunto(s)
Proteínas del Tejido Nervioso/farmacología , Receptores de Superficie Celular/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Factor Neurotrófico Ciliar , Clonación Molecular , Expresión Génica , Glicosilfosfatidilinositoles/metabolismo , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-6/farmacología , Factor Inhibidor de Leucemia , Linfocinas/farmacología , Ratones , Desnervación Muscular , Músculos/inervación , Músculos/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/metabolismo , Fosfotirosina , ARN Mensajero/genética , Ratas , Receptor de Factor Neurotrófico Ciliar , Receptores de Superficie Celular/química , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The lack of reagents or molecular probes specific for the ciliary neurotrophic factor (CNTF) receptor has hindered characterization of the molecular mechanism(s) by which CNTF influences the proliferation, survival, and differentiation of cells of the vertebrate nervous system. We have developed methods for the detection and separation of cells expressing CNTF receptors by using a variety of binding assays based on a genetically engineered CNTF molecule containing an "epitope tag" at its C-terminus. These assays have allowed us to identify several neuronal cell lines, as well as embryonic and adult neurons in primary cultures, that bind CNTF and functionally respond to CNTF by rapidly activating the transcription of immediate early primary response genes.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuronas/química , Receptores de Superficie Celular/análisis , Animales , Secuencia de Bases , Embrión de Pollo , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/metabolismo , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Receptor de Factor Neurotrófico Ciliar , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/metabolismo , Formación de Roseta , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The mechanism of inhibition of RNA polymerase-catalyzed synthesis of RNA by actinomycin D and the phenan-thridinium derivatives ethidium bromide and 3,8-diamino-6-ethylphenanthridinium bromide (DEMB) is examined. A general kinetic equation describing the dependence of RNA synthesis on DNA template concentration is derived and distinct expressions corresponding to various possible mechanisms of inhibition are subsequently obtained by introducing into the equations assumptions as appropriate for the individual mechanisms. The fitting of the experimental results of inhibition into the resulting equations suggested that the ethidium bromide and DEMB inhibit RNA polymerase by forming an inhibitor-template complex which interferes with enzyme recognition of, and binding to, appropriate sites on the template (binding inhibition). The fitting of the dependence of the rate of RNA synthesis on the bound-inhibitor to DNA ratios to the derived kinetic expressions also allows a tentative distinction to be made as to whether ethidium bromide and DEMB interfere with RNA synthesis by a mechanism of 'partial' or 'complete' inhibition.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Dactinomicina/farmacología , Etidio/análogos & derivados , Etidio/farmacología , Transcripción Genética/efectos de los fármacos , Colifagos , ADN Viral , Escherichia coli/enzimología , Cinética , Matemática , Moldes GenéticosRESUMEN
Commonly used expression vectors direct the synthesis of antibiotic-resistance proteins at unnecessarily high levels that complicate purification of the desired recombinant product. To overcome this problem, the promoter of the kanamycin-resistance gene (kanR) was altered by site-specific mutagenesis. As a result, synthesis of KanR protein was greatly reduced to the minimum required for host selection. At the same time, recombinant protein production was increased up to two-fold. Since the mutations did not alter any coding sequences and had no effect on plasmid copy number, the results suggest that plasmid-coded protein production can be limited, at least in part, by other genes expressed from the same plasmid. Because of the dependence of protein synthesis on gene dosage, an important aspect of minimizing antibiotic resistance is continuous selection for cells harboring the maximum vector-copy number, thus ensuring maximal product synthesis.
Asunto(s)
Escherichia coli/genética , Vectores Genéticos , Resistencia a la Kanamicina/genética , Proteínas Recombinantes/genética , Mutación , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesisRESUMEN
Because of the palindromic character of most 6-bp restriction sites, filling-in and ligation of the protruding ends create symmetric sequences which include new 6-bp restriction sites. The old site is, in most cases, lost. After cleavage at the new palindromic site and removal of the protruding ends, a new center of symmetry is created which is often part of yet another 6-bp restriction site. A compilation of potential and available sites as presented should prove useful in genetic engineering.
Asunto(s)
Secuencia de Bases , Enzimas de Restricción del ADN , ADN de Cadena Simple/metabolismo , Vectores Genéticos , Especificidad por SustratoRESUMEN
Ciliary neurotrophic factor (CNTF) inhibits the production of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-treated mice and protects against LPS lethality when coadministered with its soluble receptor (sCNTFR alpha). Both of these activities are abolished in adrenalectomized (ADX) mice. LPS-induced pulmonary polymorphonuclear neutrophil (PMN) infiltration and nitric oxide (NO) production were also inhibited by CNTF + sCNTFR alpha but not by CNTF alone. sCNTFR alpha did not alter the clearance or tissue distribution of CNTF. Furthermore, CNTF variants coadministered with sCNTFR alpha protected against LPS toxicity in a manner related to their affinity for the beta components of CNTFR. Thus, inhibition of TNF production and protection against LPS lethality by CNTF/sCNTFR alpha require an intact hypothalamus-pituitary-adrenal axis (HPAA) and may be mediated by endogenous glucocorticoids. This protective effect is, at least in part, due to the inhibition of PMN infiltration and NO production, and appears to be mediated by cells displaying only beta-receptor subtypes.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas del Tejido Nervioso/uso terapéutico , Fármacos Neuroprotectores , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Choque Séptico/prevención & control , Adrenalectomía , Animales , Factor Neurotrófico Ciliar , Humanos , Lipopolisacáridos/toxicidad , Pulmón/enzimología , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/farmacocinética , Nitratos/sangre , Nitritos/sangre , Peroxidasa/metabolismo , Receptor de Factor Neurotrófico Ciliar , Proteínas Recombinantes , Distribución Tisular , Factor de Necrosis Tumoral alfa/biosíntesisAsunto(s)
Interferón Tipo I/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Tampones (Química) , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli/genética , Vectores Genéticos , Humanos , Interferón Tipo I/genética , Peso Molecular , PlásmidosAsunto(s)
Enzimas de Restricción del ADN , ADN , Oligodesoxirribonucleótidos/aislamiento & purificación , Oligonucleótidos/aislamiento & purificación , Oligorribonucleótidos/aislamiento & purificación , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , ADN/aislamiento & purificación , ARN de Transferencia , Especificidad por SustratoRESUMEN
ColE1 derivative plasmids were constructed in which the natural promoter that primes replication or, in addition, the region coding for the RNA I control element had been deleted. In all of these molecules priming of the origin was effected by read-through transcription from constitutive or inducible (lacUV5) promoters inserted farther upstream. In the latter case, regulation of lac repressor activity with IPTG resulted in controlled plasmid levels in vivo. These results indicated that, at least in the absence of other control elements, regulation of the priming promoter was sufficient to control DNA replication and determine plasmid copy number.
Asunto(s)
Replicación del ADN , Escherichia coli/genética , Operón , Plásmidos , Secuencia de Bases , Deleción Cromosómica , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
A plasmid carrying the bacteriophage T7.3 endonuclease gene under the control of the lacUV5 promoter could be maintained in the transcriptionally active state only in recA+ strains. In recA- strains, endonuclease induction resulted in extensive degradation of the genomic DNA and cell death. In sharp contrast, the plasmid DNA remained intact in the supercoiled form. In recA+ strains, the recA protein levels were increased and the SOS functions of the host were activated, as shown by measurements of recA protein synthesis and prophage induction. These results indicate that in normal undisrupted and non-irradiated cells, enzymatic nucleolytic damage can induce the SOS response and can be controlled by the DNA repair system of the host. In addition, the higher sensitivity of the genomic DNA to the single-strand-specific endonuclease relative to the plasmid suggests that the two molecules differ in their physiological states and most likely in their degree of single-stranded content.
Asunto(s)
Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas Virales/metabolismo , Supervivencia Celular , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática , Operón Lac , Rec A Recombinasas/biosíntesis , Proteínas Virales/biosíntesisRESUMEN
T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.
Asunto(s)
ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Plásmidos , Enzimas de Restricción del ADN , ADN Recombinante , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/biosíntesis , Inducción Enzimática , Conformación de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript. In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger. It is proposed that the mode of cleavage within the RNase III site determines mRNA stability. Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation. In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times. This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels. In contrast, processing at the lambda int site reduces Int synthesis. Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. The end result appears to be determined by the mode of cleavage within the RNase III site.
Asunto(s)
Endorribonucleasas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Enzimas de Restricción del ADN/metabolismo , Humanos , Conformación de Ácido Nucleico , Plásmidos , Ribonucleasa III , Fagos T/enzimología , Transcripción GenéticaRESUMEN
A genetically engineered lac repressor/T7 endonuclease hybrid protein shows repressor-like binding toward restriction fragments carrying the lac operator. In addition, fragments carrying the operator near a particular pBR322 sequence are cleaved into specific products. Cleavage occurs at precise positions within that sequence, is independent of the orientation of the operator, is inhibited by isopropyl-1-thio-beta-D-galactopyranoside, and is observed when the target is separated from the operator by at least as few as 150 and as many as 240 base pairs. This evidence indicates that the hybrid protein is a site-directed nuclease that requires the following two structural elements for activity: the lac operator and a target. Repressor-like binding directs the enzyme to the operator and nearby single-stranded DNA targets. The discovery of an unusual target in a well-studied DNA sequence is evidence of the power of this approach for probing unusual structures in non-supercoiled duplex DNA.
Asunto(s)
Sondas de ADN , ADN Recombinante , Endodesoxirribonucleasas , Fagos T/enzimología , Unión Competitiva , Sondas de ADN/metabolismo , ADN Recombinante/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Desoxirribonucleasas de Localización Especificada Tipo II , Endodesoxirribonucleasas/metabolismo , Isopropil Tiogalactósido/farmacología , Plásmidos , Proteínas Represoras/metabolismo , Mapeo Restrictivo/métodos , Especificidad por Sustrato , Fagos T/genéticaRESUMEN
Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation.
Asunto(s)
ADN Recombinante , Genes Virales , Fagos T/genética , Secuencia de Bases , Enzimas de Restricción del ADN , PlásmidosRESUMEN
T7 late promoters have a 22-base pair sequence in common. The 22 base pairs are necessary and sufficient for recognition and initiation by T7 RNA polymerase.
Asunto(s)
Bacteriófago T7/genética , ADN Viral/genética , ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas/genética , Transcripción Genética , Bacteriófago T7/enzimología , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN Viral/química , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Proteínas ViralesRESUMEN
With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-alpha-d-[U-(14)C]galactose served as a precursor for a number of products. One of these products was characterized as a beta-(1-->4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of alpha-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The (14)C-labelled beta-(1-->4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [(14)C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [(14)C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive beta-(1-->4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1-->3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [(14)C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [(14)C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.
Asunto(s)
Galactosa/metabolismo , Plantas/enzimología , Polisacáridos/biosíntesis , Carbohidrato Epimerasas , Isótopos de Carbono , Cromatografía en Papel , Electroforesis en Papel , Hexosiltransferasas/metabolismo , Concentración de Iones de Hidrógeno , Azúcares de Nucleósido Difosfato , Oligosacáridos/análisis , Oxidación-Reducción , Ácido Peryódico , Polisacáridos/análisis , Solubilidad , Azúcares de Uridina Difosfato/metabolismoRESUMEN
The phage T7 endonuclease gene was fused to the 3' end of the lac repressor gene. The hybrid protein exhibits repressor and nuclease functions in a manner dependent on the conformation of the DNA. With supercoiled DNA, nuclease activity is directed to the major cruciform, whereas with linear DNA, the enzyme cleaves preferentially restriction fragments carrying the operator. These properties render the hybrid protein a unique probe of DNA conformation in vitro and in vivo.
Asunto(s)
Endodesoxirribonucleasas/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Represoras/biosíntesis , Factores de Transcripción/biosíntesis , ADN Superhelicoidal/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Regiones Operadoras Genéticas , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Especificidad por Sustrato , Fagos T/enzimología , Fagos T/genéticaRESUMEN
The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.