RESUMO
To investigate the requirement for CD2 expression in activation of T lymphocytes via the CD3-Ti antigen/MHC receptor complex, we produced and characterized a series of CD2- Jurkat variants. These mutants lack detectable surface CD2 as determined by indirect immunofluorescence, immunoprecipitation analysis, and specific radiolabeled antibody binding assay, but nevertheless, expressed normal numbers of CD3-Ti receptors. As expected, the combination of anti-CD2 antibodies, termed anti-T112 and anti-T113, which are mitogenic for resting T lymphocytes, failed to stimulate activation of these variants. In contrast, triggering of their CD3-Ti components resulted in the normal set of T lymphocyte-associated activation events, including phosphoinositide turnover, elevation in intracellular free calcium, early gene-induction events, and IL-2 production. Assuming that the Jurkat cell line is representative of normal cycling human T lymphocytes, we conclude that the presence of the CD2 molecule on the plasma membrane is not in itself a requirement for an operational CD3-Ti-alpha/beta receptor.
Assuntos
Antígenos de Diferenciação de Linfócitos T , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Northern Blotting , Cálcio/metabolismo , Linhagem Celular , Humanos , Mutação , Fosfatidilinositóis/metabolismo , Biossíntese de Proteínas , Linfócitos T/metabolismoRESUMO
We have fused an H-2- thymoma (BM5R.9) with an H-2+ thymoma (BW5147) and have found that many of the resulting hybrids exhibit an H-2- phenotype. In several hybrids that were analyzed in detail, this phenotype is related to the absence of steady-state H-2 mRNA and shows some instability, possibly related to the loss of chromosomes in segregants. We conclude from our studies that BM5R.9 cells display a trans-acting mechanism that can repress the expression of H-2 antigens, and that the gene(s) causing the repression are not located on chromosome 17. This mechanism is not sufficient to explain the H-2- phenotype of BM5R.9, for which an additional, cis-acting process, must be postulated. We discuss these results in the context of the regulation of expression of the major class I transplantation antigens.
Assuntos
Regulação da Expressão Gênica , Antígenos H-2/genética , Timoma/imunologia , Animais , Fusão Celular , Linhagem Celular , Antígenos H-2/análise , Células Híbridas , Camundongos , Fenótipo , Timoma/genética , Transcrição GênicaRESUMO
We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.
Assuntos
Genes MHC da Classe II , Antígenos H-2/genética , Camundongos Endogâmicos BALB C/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Camundongos , Polimorfismo Genético , RNA Mensageiro/análise , Distribuição TecidualRESUMO
Environmental chemicals such as dioxin adversely affect immune, neurological and reproductive functions and have been implicated in cancer development. However, the mechanisms responsible for dioxin toxicity are still poorly understood. Here, we show that dioxin and related pollutants trigger a marked morphological change in epithelial cells that remodel their cytoskeleton to increase interaction with extra cellular matrix while loosening cell-cell contacts. Furthermore, dioxin-treated cells show increased motility. These dioxin-mediated effects are mimicked by constitutive expression and activation of the intracellular dioxin receptor (aryl hydrocarbon receptor (AhR)). They correlate with activation of the Jun NH2-terminal kinase (JNK) and are reverted by treatment with a JNK inhibitor. Dioxin-induced effects occur 48 h post-treatment initiation, a time scale, which argues for a genomic effect of the AhR, linked to induction of target genes. This novel Ahr action on cell plasticity points to a role in cancer progression.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metilcolantreno/toxicidade , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Actinas/metabolismo , Benzo(a)Antracenos/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Ligantes , Metilcolantreno/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Fumaça , NicotianaRESUMO
The woodchuck intronless proto-oncogene N-myc2 was initially discovered as a frequent target site for hepadnavirus integration in hepatocellular carcinoma. N-myc2 possesses characteristics of a functional retroposon derived from the woodchuck N-myc gene. We have investigated the regulatory signals governing N-myc2 expression and found that a short promoter, including a variant TATA box and potential binding sites for several transcription factors, is localized in the N-myc2 sequences homologous to the 5' untranslated region of the second N-myc exon. The corresponding region in the intron-containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc genes suggests that they contain a cryptic N-myc promoter which may be unmasked in the particular context provided by the N-myc2 retroposon. Although N-myc2, like the woodchuck N-myc gene, contributes to an extended CpG island and was found constitutively hypomethylated, it presents a highly restricted expression pattern in adult animals. Whereas the intron-containing N-myc gene is expressed at low levels in different tissues, N-myc2 mRNA was detected only in brain tissue, raising questions about the functional significance of the maintenance of a second N-myc gene in the woodchuck genome.
Assuntos
Encéfalo/metabolismo , Elementos de DNA Transponíveis , Genes myc , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Marmota , Metilação , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Proto-Oncogene Mas , Homologia de Sequência do Ácido Nucleico , Regiões Terminadoras Genéticas , Transcrição Gênica , Células Tumorais CultivadasRESUMO
A chimeric receptor composed of the extracellular domain of the human T-cell antigen CD2 (T11) joined to the membrane-spanning segment and the intracellular tyrosine kinase domain of the human colony-stimulating factor 1 receptor (CSF-1R) was expressed in murine NIH 3T3 fibroblasts. Stimulation of these cells with monoclonal antibodies to CD2 induced phosphorylation of the chimeric glycoprotein on tyrosine, receptor downmodulation, and mitogenesis. In contrast, neither human CSF-1R nor the chimeric receptor was able to function in interleukin-2-dependent murine T cells. In fibroblasts, then, CSF-1 per se is not required for activation of the receptor kinase or for a biological response, whereas in T cells, CSF-1R may be unable to engage the downstream signal transduction machinery.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Divisão Celular , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Antígenos CD2 , Regulação para Baixo , Epitopos , Dados de Sequência Molecular , Fosfotirosina , Proteínas Tirosina Quinases/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
In most cases, Acute Promyelocytic Leukemia (APL) is associated with t(15;17) translocation which juxtaposes sequences from PML and retinoic acid receptor alpha (RAR alpha) genes. The generated PML-RAR alpha fusion interferes with wild type RAR alpha-mediated transcription and disrupts subnuclear compartments, known as PML bodies. Both defects are corrected by all trans retinoic acid (ATRA) therapy which induces differentiation of leukemic cells and clinical remission. In a rare APL syndrome associated with t(11;17), fusion of the RAR alpha gene with the PLZF gene, encoding a Zinc-finger protein produces two reciprocal RAR alpha chimeras. Although PLZF-RAR alpha and PML-RAR alpha are similar in their apparent dominant negative effects, t(11;17)-associated APL is refractory to ATRA therapy. In a yeast two-hybrid genetic screening, we isolated clones encoding the GAL4 transactivation domain fused to various parts of PLZF. Using these autonomously transactivating hybrids, similar in structure to the RAR alpha-PLZF fusion, we mapped the DNA-binding domain of PLZF to the last five Zinc-fingers, a region retained in RAR alpha-PLZF chimera and characterized a specific PLZF target sequence. Our data support the hypothesis that RAR alpha-PLZF chimera is not an inert product of reciprocal translocation and may thus contribute to ATRA unresponsiveness of t(11;17)-associated APL.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Sequência de Bases , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes/genética , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/genética , Ativação Transcricional , Translocação Genética , Leveduras , Dedos de Zinco/genéticaRESUMO
Mammalian hepatitis B viruses encode a unique regulatory protein termed X, which is essential for infection and likely plays a role in the carcinogenic process associated with hepadnaviral infection. Among the numerous properties ascribed to X protein, two have been widely documented: promiscuous transcriptional transactivation and proapoptosis. However, full understanding of the mechanisms underlying these activities requires the identification of the genuine X partners among the multiple X-binding host proteins. Here we show that (i) mutations in X protein, which markedly alter affinity for the host protein UVDDBp127, inactivate both transactivation and proapoptosis; (ii) ectopic fusion of a functional UVDDB-binding domain to a deficient binding X mutant restored its activity; (iii) in contrast to the loss-of-binding mutants, a mutant with a strong gain-of-binding exerted trans-dominant negative effects on wt X activity and localized in the nucleus and (iv) increase in intracellular UVDDB concentration enhanced both wt X-mediated transactivation and apoptosis. Taken together, our data provide strong evidence for a common upstream step in X mode of action, consisting of its productive interaction with UVDDB, via a structurally and functionally autonomous module. In addition, they underscore a nuclear location step of the viral protein that depends on its ability to bind UVDDB.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Apoptose/fisiologia , Transporte Biológico , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Vírus da Hepatite B da Marmota/química , Humanos , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Proteínas Virais Reguladoras e AcessóriasRESUMO
A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks, (ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/patogenicidade , Transativadores/metabolismo , Animais , Hepatite B/veterinária , Hepatite B/virologia , Vírus da Hepatite B da Marmota/metabolismo , Marmota , Mutação , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
Three hepatitis B viruses infecting humans, woodchucks and ground squirrels increase the risk of hepatocellular carcinoma in their respective hosts. The woodchuck hepatitis B virus (WHV), unlike the two other viruses, induces a rapid carcinogenic process characterized by direct activation of myc proto-oncogenes by insertion of viral DNA. The highly preferred target of insertional mutagenesis in woodchucks is N-myc2, an intronless N-myc gene. Strikingly, N-myc2 has no human homolog and the homologous N-myc2 locus previously detected in the ground squirrel genome, remains silent during hepatocarcinogenesis. Therefore, N-myc2 may represent a critical host determinant in the evolution of the disease associated with hepadnavirus infection. To address this question, we performed a structural and functional analysis of the ground squirrel N-myc2 locus. We show that ground squirrel N-myc2 is highly homologous to its woodchuck counterpart and is a functional proto-oncogene. Existence of a functional N-myc2 gene as a potential target for insertional activation by viral DNA is therefore not restricted to the woodchuck species. This suggests that viral rather than host factors determine the higher oncogenic phenotype of WHV as compared to the two other mammalian hepadnaviruses.
Assuntos
Genes myc , Hepadnaviridae/patogenicidade , Neoplasias Hepáticas Experimentais/virologia , Retroelementos , Sciuridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , DNA Viral , Neoplasias Hepáticas Experimentais/genética , Dados de Sequência Molecular , Proto-Oncogene Mas , Ratos , Ratos Endogâmicos F344 , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The role of hepatitis B virus HBx protein in the carcinogenesis associated with chronic viral infection remains ill-defined. Indeed, pleiotropic effects have been ascribed to HBx: in addition to its well-documented ability to indirectly stimulate transcription, the protein has been reported to affect cell growth, signal transduction, DNA repair and apoptosis. In this work, we generated Chang (CCL-13)-derived cell lines constitutively expressing wild type or mutant HBx, as a model of HBx-host cell interaction closer to the chronic infection setting, than the classically used transient expression systems. We document the potentiation by HBx of the apoptotic cell death pathway in the recipient cells. This effect is unlikely to rely on p53 activity since the protein is functionally inactivated in CCL-13. In addition, antioxidants and cyclosporin A failed to reduce the apoptotic response back to the normal level, suggesting that production of reactive oxygen species and calcineurin activation are not directly involved in the proapoptotic effect of HBx. In contrast, our data show that transactivation and stimulation of apoptosis are tightly linked HBx activities. Finally, expression of transactivation-active protein did not result in detectable change in the pattern of MAP kinases phosphorylation nor did it affect the ability of the host cell to repair in vitro irradiated plasmid DNA.
Assuntos
Apoptose/fisiologia , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/virologia , Ciclosporina/farmacologia , Reparo do DNA/genética , Etoposídeo/farmacologia , Humanos , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.
Assuntos
Genes MHC Classe I , Antígenos H-2/genética , Camundongos/genética , Animais , Regulação da Expressão Gênica , Fígado/embriologia , Camundongos/embriologia , RNA Mensageiro/genética , Distribuição Tecidual , Saco Vitelino/fisiologiaRESUMO
The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.
Assuntos
Genoma Viral , Hepacivirus/genética , Proteínas Virais/metabolismo , Glutationa Transferase/genética , Hepacivirus/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/genéticaAssuntos
Linfócitos B/enzimologia , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidiltransferases/metabolismo , Hibridomas/enzimologia , Plasmocitoma/enzimologia , Timo/enzimologia , Animais , Fusão Celular , Linhagem Celular , Células Clonais , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB CAssuntos
DNA Nucleotidilexotransferase/isolamento & purificação , DNA Nucleotidiltransferases/isolamento & purificação , Timo/enzimologia , Animais , Galinhas , Cromatografia de Afinidade , DNA Nucleotidilexotransferase/metabolismo , Imunofluorescência , Soros Imunes , Cinética , Peso Molecular , Especificidade por SubstratoRESUMO
Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Dioxinas/toxicidade , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.
Assuntos
Carcinoma Hepatocelular/microbiologia , DNA Viral/análise , Neoplasias Hepáticas/microbiologia , Orthohepadnavirus/genética , Integração Viral , Animais , Sequência de Bases , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , Dados de Sequência Molecular , Orthohepadnavirus/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , SciuridaeRESUMO
The T-cell receptor (TCR) is a molecular complex comprised of a clonally restricted, immunoglobulin-like heterodimer (Ti), responsible for specific antigen recognition, and a set of monomorphic polypeptide CD3 subunits, termed gamma, delta, epsilon, zeta, and eta, presumed to be involved in transmembrane signaling events. To investigate the role of the CD3 epsilon subunit in signal transduction, we have transfected a murine hybridoma T-cell line with either wild-type or variant human CD3 epsilon cDNA that encodes a protein lacking 49 of the 55 cytoplasmic amino acid residues. Both wild-type and truncated CD3 epsilon human proteins assemble with endogenous murine CD3/Ti subunits to form functional surface TCRs: Anti-human CD3 epsilon monoclonal antibodies bind exclusively to these chimeric TCRs and trigger interleukin 2 production from the murine cells. Thus, the CD3 epsilon cytoplasmic domain is not required for assembly of the multimeric TCR. Furthermore, it is dispensable for the transduction of a stimulus delivered to the external part of the molecule, suggesting that interaction between the transmembrane and/or external regions of the other TCR chains is a prerequisite for transmembrane signaling.
Assuntos
Antígenos de Diferenciação de Linfócitos T/genética , Genes , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Southern Blotting , Complexo CD3 , Linhagem Celular , Deleção Cromossômica , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Humanos , Substâncias Macromoleculares , MutaçãoRESUMO
HNF1 is a liver enriched atypical homeoprotein isolated from vertebrates which is involved in the transcriptional activation of liver, kidney, intestine and pancreas specific genes. HNF1 contains an N-terminal dimerisation and a POU-like domain both essential together with the homeodomain for DNA specific recognition. Using the yeast two-hybrid system we searched for proteins interacting with HNF1. We repeatedly obtained cDNA clones encoding DCOH/4-alpha-carbinolamine dehydratase, an enzyme involved in the oxidation of aromatic amino acids that was shown to bind to and stabilise HNF1 dimers. Using the yeast system, we show that the enzymatic activity of DCOH is not essential for HNF1 binding and that the HNF1 dimerisation domain is sufficient for DCOH binding. Furthermore we demonstrate that both proteins co-localise in co-transfected cells.
Assuntos
Hidroliases/metabolismo , Fígado/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Hidroliases/biossíntese , Mucosa Intestinal/metabolismo , Rim/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Pâncreas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Transfecção , VertebradosRESUMO
The H-2Kd gene, which encodes a mouse major transplantation antigen, was transfected into L TK- mouse fibroblasts. Two transcripts of the gene were detected by S1 nuclease mapping analysis. They correspond to two previously characterized cDNA clones isolated from DBA/2 mouse liver RNA, leading to the conclusion that the H-2Kd gene gives rise to two distinct transcripts through an alternate use of splicing sites. The non-canonical RNA potentially encodes a so far undescribed H-2Kd-like molecule. It is present in all tissues tested (liver, spleen, thymus, kidney) albeit in lower amounts (approximately 10-fold less) than the canonical RNA coding for H-2Kd.