RESUMO
Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Non-conservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.
Assuntos
Transformação Celular Neoplásica , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Divisão Celular , Linhagem Celular , Expressão Gênica , Guanosina Trifosfato/metabolismo , Humanos , Fenilalanina , Ratos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Using molecular cloning techniques, human homologs of the known members of the trk family of neurotrophin receptors have been cloned and sequenced. Overall, there is a high degree of similarity between the human sequences and those from other mammals; however, there are differences in splicing patterns. There are two spliced forms of the extracellular domain of trkC in the human, a finding that has not been described in other species. In contrast, fewer spliced forms were detected of the intracellular domains of human trkB and trkC than has been described in other mammals. Northern analysis and in situ hybridization experiments indicate that the human trks are expressed in a similar pattern to that described in other mammals. Expression of the trk extracellular domains as fusion proteins with IgG heavy chain yields soluble molecules that mimic intact trks in their binding specificity and affinity. These soluble chimeras block the biological activity of their cognate neurotrophin(s) in vitro.
Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Espaço Extracelular/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , DNA Complementar/genética , DNA Recombinante , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização In Situ , Sondas Moleculares/genética , Dados de Sequência Molecular , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar , Receptor trkC , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Distribuição TecidualRESUMO
One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.
Assuntos
Processamento Alternativo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/biossíntese , Sequência de Bases , Linhagem Celular , Primers do DNA , Éxons , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Humanos , Íntrons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , RNA Nuclear Pequeno/genética , Radioimunoensaio , TransfecçãoRESUMO
C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products.
Assuntos
Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Sequência de Bases , Linhagem Celular , DNA , Eletroquímica , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas c-jun/química , Serina/metabolismo , Transcrição GênicaRESUMO
Homologous recombination was utilized to generate mice with a deletion in the coding sequence of the nerve growth factor (NGF) gene. Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia. Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. Examination of the central nervous system revealed that, in marked contrast with neurons of sensory and sympathetic ganglia, basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the null mutant mice. Thus, differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.
Assuntos
Fibras Colinérgicas/fisiologia , Gânglios Simpáticos/citologia , Fatores de Crescimento Neural/fisiologia , Neurônios Aferentes/fisiologia , Prosencéfalo/citologia , Animais , Feminino , Gânglios Espinais/citologia , Gânglios Simpáticos/embriologia , Deleção de Genes , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mosaicismo , Fatores de Crescimento Neural/genética , Limiar da Dor/fisiologia , Prosencéfalo/embriologiaRESUMO
Oncogenic Ras proteins transform animal cells to a malignant phenotype only when modified by farnesyl residues attached to cysteines near their carboxyl termini. The farnesyltransferase that catalyzes this reaction recognizes tetrapeptides of the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is a carboxyl-terminal methionine or serine. Replacement of the two aliphatic residues with a benzodiazepine-based mimic of a peptide turn generated potent inhibitors of farnesyltransferase [50 percent inhibitory concentration (IC50) < 1 nM]. Unlike tetrapeptides, the benzodiazepine peptidomimetics enter cells and block attachment of farnesyl to Ras, nuclear lamins, and several other proteins. At micromolar concentrations, these inhibitors restored a normal growth pattern to Ras-transformed cells. The benzodiazepine peptidomimetics may be useful in the design of treatments for tumors in which oncogenic Ras proteins contribute to abnormal growth, such as that of the colon, lung, and pancreas.
Assuntos
Alquil e Aril Transferases , Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Proteínas Oncogênicas/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Transferases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Benzodiazepinonas/química , Células CHO , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Transformação Celular Neoplásica/efeitos dos fármacos , Cricetinae , Desenho de Fármacos , Farnesiltranstransferase , Dados de Sequência Molecular , Oligopeptídeos/farmacologiaRESUMO
The plasminogen activator urokinase (u-PA) mediates proteolysis by a variety of human tumor cells. Competitive displacement of u-PA from cellular binding sites results in decreased proteolysis in vitro, suggesting that the cell surface is the preferred site for u-PA-mediated protein degradation. We studied the effect of u-PA receptor blockade on the metastatic capacity of human PC3 prostate carcinoma cells, using transfectants which expressed chloramphenicol acetyl-transferase (CAT). Eight weeks after subcutaneous inoculation of these cells into nude mice, CAT activity was detected in regional lymph nodes, femurs, lungs, and brain, thereby mimicking the organ tropism observed for naturally occurring metastases of prostate cancer. In a second transfection, CAT-expressing PC3 cells received cDNA encoding a mutant u-PA (Ser356-->Ala) which lacks enzymatic activity but which retains full receptor binding affinity. Three mutant u-PA expressors, each with < 5% of wild-type cell-associated u-PA activity, were compared in vivo with independently derived controls. Primary tumor growth was similar in each group of animals and all tumors expressed comparable CAT activity. In contrast, metastasis (as assessed by CAT activity) was markedly inhibited when cell surface u-PA activity was blocked. Levels of CAT activity were reduced by a factor of > 300 in regional lymph nodes, 40-100 in brain tissue, and 10-20 in lung tissue. Metastatic capacity was inhibited similarly when animals were given intermittent intraperitoneal injections of a u-PA/IgG fusion protein capable of displacing u-PA activity from the tumor cell surface. Our results indicate that cell surface u-PA activity is essential to the metastatic process. In addition, the assay system employed in these experiments may be generally useful in testing other therapeutic modalities to limit the spread of primary tumors.
Assuntos
Adenocarcinoma/patologia , Metástase Neoplásica , Neoplasias da Próstata/patologia , Receptores de Superfície Celular/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Pulmão/enzimologia , Pulmão/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Linfonodos/enzimologia , Linfonodos/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , Invasividade Neoplásica , Transplante de Neoplasias , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.
Assuntos
Processamento Alternativo , Genes ras , Hormônio do Crescimento/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/genética , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Sequência de Bases , Linhagem Celular , Desoxirribonuclease I/genética , Expressão Gênica , Genes , Vetores Genéticos , Humanos , Íntrons , Canamicina Quinase , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA/genética , RNA/isolamento & purificação , Precursores de RNA/genética , RNA Nuclear Pequeno/genética , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , TransfecçãoRESUMO
Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes.
Assuntos
Divisão Celular/fisiologia , Transformação Celular Neoplásica , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/farmacologia , Linfocinas/biossíntese , Linfocinas/farmacologia , Transfecção , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Células Clonais , Cricetinae , Fatores de Crescimento Endotelial/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Hibridização In Situ , Cinética , Linfocinas/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/ultraestrutura , RNA Mensageiro/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We demonstrate an example of signal transduction by an integrin and have begun to define the pathway through which this signaling is achieved. We constructed a stably transfected derivative of 293 cells (ATCC 1573) that expresses the platelet integrin GPIIbIIIa (alpha IIb beta 3). This cell line, clone B, adheres to and spreads on fibrinogen, a ligand for alpha IIb beta 3, while the parent cell line does not. Stimulation of these cells either by adhesion to fibrinogen or with antiserum directed against alpha IIb beta 3 results in induction of calcium oscillations, followed by tyrosine phosphorylation of at least one protein of molecular weight approximately 125 kDa. We establish that this phosphorylation, as well as the morphological rearrangements, requires the mobilization of calcium.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Integrinas/metabolismo , Transdução de Sinais , Tirosina/metabolismo , Adesão Celular , Linhagem Celular , Tamanho Celular , Fibrinogênio , Periodicidade , Fosforilação , TransfecçãoRESUMO
The goal of the present study was to assess the relative importance of receptor-bound and secreted plasminogen activator urokinase (u-PA) in generating cell-surface plasmin and fostering destruction of normal tissue by tumor cells. We first showed that active site-inhibited u-PA could displace endogenous u-PA from the surface of the human colon adenocarcinoma cell line HCT 116. We then prepared expression vectors for u-PA and for a mutant molecule in which the codon for the active site serine residue was changed to encode alanine. Expression of non-functional mutant u-PA decreased the level of cell-bound active u-PA by more than 95% via a mechanism that involved competition for receptor sites. Decreased cell-surface u-PA activity was associated with a decrease in cell-bound plasmin activity to undetectable levels, suggesting that receptor-bound u-PA plays an important role in the generation of plasmin on the cell surface. Transfectants that secreted eightfold to 20-fold elevated levels of active wild-type u-PA showed approximately 50% increases in cell-associated u-PA and only twofold to fourfold increases in cell-associated plasmin, suggesting that the role of secreted u-PA in generating cell-surface plasmin activity was relatively minor. In parent cells and both types of transfectants there was a good correlation between the amount of plasmin bound to the tumor cell surface and the extent to which a basement membrane substrate was degraded. These studies show that receptor-bound u-PA provides an efficient mechanism for plasmin generation on the surface of tumor cells, which, in turn, contributes significantly to their degradative potential.
Assuntos
Fibrinolisina/biossíntese , Ativadores de Plasminogênio/metabolismo , Receptores de Superfície Celular/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adenocarcinoma , Sequência de Bases , Membrana Basal/metabolismo , Sítios de Ligação , Linhagem Celular , Neoplasias do Colo , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Ativadores de Plasminogênio/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Transforming growth factor beta 1 (TGF-beta 1) is a key regulator of cell growth and differentiation. Under normal physiological conditions, it is made as a biologically latent complex whose significance is unknown. Previous work has indicated that active TGF-beta 1 has a very short plasma half-life in rats (Coffey, R. J., L. J. Kost, R. M. Lyons, H. L. Moses, and N. F. La-Russo. 1987. J. Clin. Invest. 80:750-757). We have investigated the possibility that latent complex formation may extend the plasma half-life of TGF-beta 1 and alter its organ distribution. Radiolabeled latent TGF-beta 1 was formed by noncovalent association of 125I-TGF-beta 1 with the TGF-beta 1 precursor "pro" region from recombinant sources. TGF-beta 1 in this latent complex had a greatly extended plasma half-life (greater than 100 min) in rats compared with active TGF-beta 1 (2-3 min). Whereas active TGF-beta 1 was rapidly taken up by the liver, kidneys, lungs, and spleen and degraded, TGF-beta 1 in the latent complex was largely confined to the circulation, and was less than 5% degraded after 90 min. The pharmacokinetics of TGF-beta 1 in the latent complex were shown to be critically dependent on the degree of sialylation of the complex. The results suggest that formation of latent complexes may switch endogenous TGF-beta 1 from an autocrine/paracrine mode of action to a more endocrine mode involving target organs distant from the site of synthesis.
Assuntos
Fator de Crescimento Transformador beta/farmacocinética , Animais , Autorradiografia , Taxa de Depuração Metabólica , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Ratos , Proteínas Recombinantes/farmacocinética , Ácidos Siálicos/fisiologia , Relação Estrutura-Atividade , Distribuição Tecidual , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Using recombinant DNA technology, we have generated Chinese hamster ovary (CHO) cell lines that synthesize latent transforming growth factor beta 1 (TGF-beta 1) to study immune regulation by TGF-beta 1. In vitro, latent TGF-beta 1 synthesized by transfectants or added exogenously as a purified complex after activation inhibited CTL generation to a similar extent as seen with acid-activated recombinant human (rHu) TGF-beta 1. In vivo, serum from nu/nu mice bearing CHO/TGF-beta 1 tumors contained significant levels of latent TGF-beta 1 in addition to depressed natural killer (NK) activity in spleens which paralleled that seen in C3H/HeJ mice treated with acid-activated rHuTGF-beta 1. rHuTGF-beta 1 treatment of mice receiving heart allografts resulted in significant enhancement of organ graft survival. Because of possible regulated tissue-specific activation, administration of latent rather than active TGF-beta may provide a better route to deliver this powerful immunosuppressive agent in vivo.
Assuntos
Transformação Celular Neoplásica , Células Matadoras Naturais/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Sobrevivência de Enxerto , Transplante de Coração/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Ovário , Proteínas Recombinantes/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Transfecção , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Within the past three years there has been a rapid expansion in our knowledge of the role TGF-beta mediates in regulating immune responses in vitro. Whether the TGF-beta will be clinically useful to suppress immune responses to transplanted organs or autoimmune responses is unknown. However, now that highly purified quantities of TGF-beta are available through recombinant DNA technologies, questions concerning the in vivo immunosuppressive activities of TGF-beta can be answered.
Assuntos
Fatores de Crescimento Transformadores/fisiologia , Animais , Rejeição de Enxerto , Humanos , Ativação Linfocitária , Choque Séptico/fisiopatologiaRESUMO
Native and recombinant forms of transforming growth factor-beta 1 (TGF-beta 1) are synthesized predominantly as biologically latent complexes. Physicochemical analysis demonstrates that the more recently described TGF-beta 2 and TGF-beta 3 are also latent, and reveals a common series of sharply defined parameters for activation. Human recombinant latent TGF-beta's 1 and 2 show identical profiles of activation by acid and base; the transition from latency occurs between pH 4.1 and 3.1, and between pH 11.0 and 11.9. The profile for chicken recombinant latent TGF-beta 3 is slightly shifted with activation between pH 3.1 and 2.5, and between pH 10.0 and 12.3. Thermal activation of native and recombinant latent TGF-beta 1 occurs over the temperature ranges of 75-100 degrees C and 65-100 degrees C, respectively, with complete activation after 5 min at 80 degrees C. Temperatures above 90 degrees C result in thermal denaturation of TGF-beta 1 itself. Recombinant latent TGF-beta's 2 and 3 are also activated over this temperature range; however, maximum activation occurs at 100 degrees C. These results suggest common elements in latent complex structure despite differences between the TGF-beta subtypes in pro-region primary sequence.
Assuntos
Fatores de Crescimento Transformadores/metabolismo , Plaquetas/metabolismo , Glicosídeo Hidrolases/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Peptídeo Hidrolases/farmacologia , Proteínas Recombinantes/metabolismoRESUMO
Adenovirus VAI-RNA is a small virally encoded RNA that is required for efficient protein synthesis at late times of adenoviral infection. We show that in transient transfection assays VAI-RNA promotes not only an increased level of protein encoded by a co-transfected marker (CAT) plasmid, but also a marked accumulation of its transcript. The increases in CAT protein and RNA levels reflect an enhanced stability of the cytoplasmic RNA as shown by primer extension analyses of RNA isolated from transfected cells upon transcriptional arrest. Surprisingly, the ability of VAI to activate expression of CAT requires the translation of a substantial portion of the RNA: when translation is prevented by elimination of the initiator AUG codon or by introduction of stop codons 5' to codon 107, VAI-RNA is no longer capable of increasing CAT RNA levels; the introduction of stop codons 3' of codon-135, on the other hand, does not significantly impair VAI-RNA function. We conclude that in addition to its role as a specific activator of translation, adenovirus VA genes function to regulate the stability of ribosome-bound RNA.
Assuntos
Adenovírus Humanos/fisiologia , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA Viral/fisiologia , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica/genética , Vetores Genéticos , Biossíntese de Proteínas , TransfecçãoRESUMO
We previously demonstrated that a point mutation in the last intron of the human H-ras oncogene causes a significant increase in its expression and transforming efficiency. Here we establish the basis of this phenomenon. Using gene reconstruction experiments, we have identified a negative-acting element in the intron that is completely inactivated by the mutation. The effects of other nucleotide alterations introduced into this region suggested that the negative element might constitute an alternative exon. Transcripts containing this putative exon were identified and S1 nuclease analysis confirmed that the mutation prevents their synthesis. The abundance of these transcripts is low, apparently due to message instability and/or defective processing. The predicted product of the alternative transcript is suggested to lack transforming potential. Our findings demonstrate that alternative splicing normally operates to suppress p21H-ras expression and that this negative control is abolished by a variety of mutations that interfere with this process.