RESUMEN
We have cloned the full-length cDNA and genomic region of a human prostate specific G-protein coupled receptor with properties characteristic of an olfactory receptor. A partial cDNA sequence of this gene, called PSGR, was recently cloned. The gene contains two exons and one intron of 14.9 kb in its 5'untranslated region, and was mapped to human chromosome 11p15.2. A cluster of transcription initiation sites for the 2.8 kb PSGR mRNA was identified. Cloning of the homologous gene from the mouse revealed 93% amino acid homology between the human and mouse or rat (previously cloned as RA1c) proteins, and 99% identity between the rat and mouse homologs. Although northern analysis indicated expression of the human PSGR homolog was prostate specific, its mRNA could also be detected in the olfactory zone and the medulla oblongata of the human brain. In the mouse, the PSGR gene is predominantly expressed in the brain and colon. In the rat, the PSGR homolog is expressed in the liver in addition to the brain. These data add to the growing body of evidence suggesting that olfactory receptors may have functional roles in tissues other than the olfactory organ, and further, suggest that these functions may vary across species.
Asunto(s)
Secuencia Conservada/genética , Proteínas de Neoplasias , Receptores Odorantes/genética , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Clonación Molecular , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismo , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Distribución Tisular , Sitio de Iniciación de la TranscripciónRESUMEN
The chemokine RANTES/CCL5 is a proinflammatory agent produced by a variety of tissues in response to specific stimuli. In human monocytes, RANTES/CCL5 transcription is up-regulated rapidly and transiently in response to LPS. We describe here two regions that help control LPS-driven transcription from the human RANTES/CCL5 promoter in monocytic cells. These sites were analyzed by using DNase I footprinting, transient transfection assays, site-directed mutagenesis, and EMSA. RANTES site E (R(E), -125/-99) constitutively binds C/EBP proteins in monocytic Mono Mac 6 cells. Mutation of region R(E) led to a significant (40%-50%) reduction in LPS-induced promoter reporter activity. Region R(AB) is composed of tandem kB-like elements R(A) and R(B) (-73/-34). These sites working in concert act as an LPS-responsive promoter module. R(A) constitutively binds Sp1, and Rel p50/p65 following LPS stimulation. Either factor can mediate transcriptional effects at R(A). Induced Rel p50/p50 binding to site R(B) is required for LPS regulation of RANTES/CCL5 transcription. A series of computer models based on the RANTES/CCL5 promoter were generated to represent the organization of these functional elements. The models could identify LPS-regulated promoters in human, other vertebrate, and viral sequences in various databases.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Quimiocina CCL5/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Regiones Promotoras Genéticas/genética , Línea Celular , Quimiocina CCL5/genética , Simulación por Computador , Dimerización , Genes Reporteros/genética , Humanos , Monocitos/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , TransfecciónRESUMEN
The chemokine RANTES is produced by a variety of tissues, including cells of the monocyte/macrophage lineage. RANTES expression is rapidly and transiently up-regulated in primary monocytes and the monocytic cell line Mono Mac 6 in response to stimulation by the bacterial product lipopolysaccharide (LPS). Transient transfection of Mono Mac 6 cells with RANTES reporter-promoter deletion constructs, in conjunction with DNase I footprinting and heterologous reporter gene assays, allowed identification of an LPS-responsive region within the RANTES promoter. Electrophoretic mobility shift assays (EMSA), methylation interference and EMSA supershift experiments were used to characterize sequences and transcription factors responsible for this LPS inducibility. The region, termed RANTES site G [R(G)], contains consensus sites for Ets and CRE/AP-1-like elements. Site-directed mutagenesis of the Ets site resulted in a loss of only 15 % of promoter activity, while mutation of the CRE/AP-1 site led to a loss of 40 % of LPS-induced promoter activity. The Ets site constitutively binds the Ets family member PU.1. LPS stimulation leads to an induction of ATF-3 and JunD factor binding to the CRE/AP-1 site. Thus, LPS induction of RANTES transcription is mediated, in part, through the activation and selective binding of ATF and Jun nuclear factors to the R(G) promoter module.
Asunto(s)
Quimiocina CCL5/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 3 , Secuencia de Bases , Células Cultivadas , ADN/genética , ADN/metabolismo , Huella de ADN , Desoxirribonucleasa I/metabolismo , Genes Reporteros/genética , Humanos , Monocitos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Eliminación de Secuencia/genética , Especificidad por Sustrato , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The chemokine RANTES is an important mediator of inflammatory processes. In this report, we describe the DNA sequence and transcription factor requirements for interleukin-1beta (IL-1beta) induction of the RANTES promoter in the human astrocytoma line CH235. RANTES promoter sequences between -278 and +55 are sufficient for IL-1beta-inducibility. In vitro DNA binding assays demonstrate constitutive binding of Sp1, HMG, Ets domain, and bZIP family members to their cognate sites in the RANTES promoter, whereas NF-kappaB and IRF-1 bind in an IL-1beta-inducible manner. IL-1beta-inducibility of the RANTES promoter requires both constitutive and inducible transcription factors. The formation of a higher order nucleoprotein complex, or 'enhanceosome', may be critical for IL-1beta induction of the RANTES promoter.
Asunto(s)
Astrocitoma/metabolismo , Quimiocina CCL5/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Astrocitoma/patología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Factores de Unión a la G-Box , Humanos , FN-kappa B/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
OCI-5/Glypican 3, a member of the glypican family of proteoglycans, is the defective gene in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 expression is developmentally regulated in the intestinal epithelium, and the mechanism of its regulation was studied in the rat intestinal epithelial cell line IEC-18. A large induction of OCI-5 transcript and protein was observed at high cell density. Among other glypican family members, kappa-glypican also exhibited a confluence-dependent induction in select cell types. Nuclear run-on analysis indicated that cell-density regulation of OCI-5 occurs at the level of transcription. The rat and mouse OCI-5 promoters were cloned and found to be highly conserved, located within CpG islands and contain regions of alternating purine and pyrimidine residues. No TATA-box or recognizable INR element was observed. Consensus binding sites for AP-2, SP-1, zeste and NF-1/CTF are conserved across human, mouse and rat promoters. 5' deletion mapping of the rat promoter identified regions which enhance and repress promoter activity, with no apparent confluence-dependence or tissue-specificity. Nuclear run-on analysis probing different regions of the gene suggests that elongation control plays a role in the induction of OCI-5 by confluence.
Asunto(s)
Regulación de la Expresión Génica , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Proteoglicanos/genética , Animales , Secuencia de Bases , Línea Celular , Glipicanos , Heparitina Sulfato/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ratones , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , Regiones Promotoras Genéticas , Proteoglicanos/metabolismo , Ratas , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
Immature CD4/CD8 double-positive (DP) thymocytes expressing self MHC-restricted TCR are positively selected in response to TCR signals to survive and differentiate into functionally competent CD4 or CD8 single positive (SP) T cells. In contrast, DP precursors expressing autoreactive TCR are clonally deleted in response to TCR signals. We show here that in vitro TCR engagement of TCR(low) DP thymocytes rapidly triggers a variety of events considered to be hallmarks of positive selection in vivo. These include increased expression of CD5 and Bcl-2, termination of RAG-1 and pre-T(alpha) gene expression, and a switch in lck promoter usage. We also demonstrate that CD4- or CD28-mediated signals synergize with TCR signals to induce these outcomes. Finally, we show that the response of DP thymocytes to TCR engagement is selective in that clonal deletion, CD4/CD8 lineage commitment, and other events associated with maturation, such as changes in expression of Thy-1, HSA, MHC class I, and CD45-RB, were not induced. Thus, only subsets of maturational processes associated with positive selection in vivo were shown to be directly coupled to TCR signaling pathways at the DP stage. These observations support conclusions from in vivo systems suggesting that multiple, temporally separated TCR engagements are required to effect the entire spectrum of developmental changes associated with positive selection, and provide a conceptual and experimental framework for unraveling the complexity of positive selection.
Asunto(s)
Apoptosis/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Animales , Antígenos CD28/farmacología , Antígenos CD4/análisis , Antígenos CD4/inmunología , Antígenos CD4/farmacología , Antígenos CD8/análisis , Antígenos CD8/inmunología , Separación Celular , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/efectos de los fármacos , Timo/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The lymphoid-specific protein tyrosine kinase, p56lck which is essential for both T cell development and function, is aberrantly expressed in colon and small lung carcinoma lines. In this paper, we demonstrate p56lck is also expressed in colon tumour biopsies due predominantly or exclusively to the use of the lck type I promoter. In T leukaemia lines, the lck type I promoter requires binding sites for both Ets- and Myb-related transcription factors. In contrast, in colon tumour lines the activation of the lck type I promoter requires the Ets but not the Myb binding site. In these lines, a consensus binding site for HMG-related transcription factors, AACAAAG, is required for efficient lck type I promoter activity. Sox-4 is a candidate transcription factor for binding and activating the lck type l promoter in colon carcinoma cells. Co-expression of Ets-1 and Sox-4, but neither protein alone, was sufficient to activate the lck type l promoter in HeLa cells which do not normally express lck transcripts. These results suggest that aberrant expression of p56lck from the lck type l promoter in colon carcinoma arises from transcriptional activation mediated by Ets- and HMG-related transcription factors.
Asunto(s)
Empalme Alternativo , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Sitios de Unión/genética , Carcinoma/enzimología , Carcinoma/genética , Sinergismo Farmacológico , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/fisiología , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/biosíntesis , Ratones , Ratones SCID , Neoplasias Primarias Múltiples/enzimología , Neoplasias Primarias Múltiples/genética , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ets , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción SOXC , Transactivadores/fisiología , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
The lck gene, which encodes a lymphoid-specific Src family tyrosine kinase, is transcribed from two promoters that are differentially utilized during T cell development. We have shown previously that the human lck type I promoter, which is preferentially expressed in immature thymocytes, requires a binding site (-97 to -90) for the Ets family of transcription factors for its activity in Jurkat T leukemia cells. Three putative Myb binding sites (-86 to -82, -77 to -72 and -59 to -54) were analysed for their ability to activate the lck type I promoter. In vitro assays demonstrated specific binding of purified, bacterially expressed c-Myb DNA binding domain to the Myb (-59 to -54) site. Transient transfection assays using the site-directed mutants of the lck type I promoter in Jurkat cells revealed that mutation of the Myb (-59 to -54) site abolished transcriptional activity. In transiently transfected HeLa cells, the lck type I promoter was activated by co-transfection with a vector that expresses c-Myb. This c-Myb dependent activation required the presence of intact Myb and Ets binding sites, indicating that the expressed c-Myb functions with endogenous Ets related transcription factors to activate the lck type I promoter. This effect was further enhanced by co-transfection with vectors that express either Ets1 or Ets2. These results demonstrate that Myb and Ets related transcription factors synergistically activate the human lck type I promoter.
Asunto(s)
Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Células HeLa , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Proteínas Proto-Oncogénicas c-mybRESUMEN
The requirement for cis-acting DNA sequences for transcriptional activity of the human lck type I promoter was investigated in two human cell lines that express type I transcripts, the leukemic T-cell line, Jurkat, and the colon carcinoma line, SW620. Transient transfection assays in Jurkat and SW620 cells revealed negative and positive cis-acting regulatory elements in the lck type I promoter between -570 and -480 and between -128 and -63 respectively. For the latter, a triple point mutation of a sequence, GCAGGAAGT, from -99 and -91 resulted in complete loss of lck type I promoter activity in both Jurkat and SW620 cells. In vitro binding assays indicated that this sequence, denoted the ETS-binding element or EBE, can interact with the lymphoid-specific transcription factor ETS-1. Thus, a protein(s) in the ETS family appears to be required for transcription of the lck type I promoter in T cells and may be important for the activation of the lck gene in human colon carcinoma.
Asunto(s)
Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Adulto , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
IL-2 is one of the principal growth factors regulating the proliferation of T lymphocytes. Although two independent IL-2-binding molecules have been molecularly cloned and shown to participate in the formation of a high affinity receptor complex, their primary structures do not suggest a specific mechanism for IL-2 growth signal transduction across the cell membrane. Neither IL-2 receptor subunit contains an intrinsic kinase domain; nevertheless, tyrosine phosphorylation of various intracellular substrates is one of the first biochemical changes observed following activation of the IL-2 receptor (IL-2R). Both serine/threonine and tyrosine kinases can be co-precipitated as part of the IL-2R complex suggesting that the IL-2 signalling may involve the activation of non-covalently associated intracellular kinases. However, controversy exists as to which kinases are involved in IL-2 signal transduction; in particular, which kinase(s) mediates the first or proximal event(s) in the signalling process. Activation of the IL-2R leads to serine and threonine phosphorylation of the SRC tyrosine kinase family member, LCK, and an increase in LCK tyrosine kinase activity. Furthermore, LCK can be co-immunoprecipitated with the beta chain of the IL-2R indicating its association with the receptor complex. IL-2 has also been reported to increase FYN kinase activity and to alter its association with the 85 kDa subunit of phosphatidylinositol-3 kinase thus suggesting a role for FYN in IL-2 signal transduction. However, in this report, we now demonstrate that neither LCK nor FYN are obligatory for IL-2-induced growth of HTLV-I-infected human T cells. Lack of expression of LCK or FYN in the HTLV-I-infected T cell lines was demonstrated by a combination of Northern blotting, polymerase chain reaction, Western blotting, and in vitro kinase activity. Despite the absence of LCK or FYN, IL-2 induced similar patterns of rapid tyrosine phosphorylation. Similar results were observed in cell lines lacking expression of the LYN, FGR, HCK, and LTK tyrosine kinases. Thus, none of these tyrosine kinases alone appears to be required for growth signalling through the IL-2R in the HTLV-I-infected T cell lines analyzed. The findings raise the possibility that an, as yet, unidentified tyrosine kinase is involved. Alternatively, this biological signalling system may exhibit remarkable redundancy whereby several different tyrosine kinases may be capable of associating with the IL-2R complex and mediating intracellular signalling.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano , Interleucina-2/farmacología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , División Celular , Línea Celular , Inducción Enzimática , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-fyn , Receptores de Interleucina-2/efectos de los fármacos , Receptores de Interleucina-2/metabolismo , Linfocitos T/enzimología , Linfocitos T/microbiologíaRESUMEN
The human beta-actin promoter, including its 5' flanking region and 5' untranslated region, is ubiquitously active in mammalian cells in culture. In this report we investigated the transcriptional activity of, and the protein-DNA interactions that occur within, the proximal region of the human beta-actin promoter. Efficient beta-actin promoter activity in transfected human HeLa cells requires only 114bp of 5' flanking sequences. Two of the cis-actin regulatory elements within this region of the beta-actin promoter, the CCAAT box and proximal CCArGG box, are specific in vitro binding sites for the transcription factors, nuclear factor Y (NF-Y) and serum response factor (p67SRF), respectively. These two elements are required together to stimulate in vivo transcription from the homologous as well as a heterologous promoter. Finally, a particular spatial alignment between the CCAAT box and proximal CCArGG box is required for trans-activation in vivo. The above provides strong evidence for a functional interaction between NF-Y and p67SRF when bound to their respective binding sites in the beta-actin promoter.
Asunto(s)
Actinas/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Homología de Secuencia de Ácido Nucleico , Factor de Respuesta Sérica , Transfección/genéticaRESUMEN
The human lymphocyte-specific tyrosine kinase gene, lck, is transcribed from two distinct promoters, resulting in two classes of transcripts (type I and II) differing in their 5' untranslated regions. The steady-state levels of the type I and II lck transcripts were measured in a variety of lymphoid and non-lymphoid human tumor cell lines by S1 nuclease mapping and by a sensitive assay system using the polymerase chain reaction. Human thymocytes and all the leukemic T cell lines tested express both type I and II lck transcripts, albeit at different relative levels. Peripheral blood T cells express mainly type II lck transcripts, whereas two colonic carcinoma lines, COLO 201 and COLO 205, express exclusively type I lck transcripts. Treatment of the leukemic T cell lines, P30/OKUBO and Jurkat, by the phorbol esters tetradecanoylphorbol acetate (TPA) or phorbol dibutyrate (PDB) results in the down-regulation of the type I, and the up-regulation of the type II, lck transcript levels. The effect of PDB on the in vitro differentiation of Jurkat cells, and the expression of lck transcripts, is reversible. The modulation of lck transcript levels in TPA-treated Jurkat cells is not due to differential RNA stability, suggesting that the two lck promoters are utilized differentially during T cell differentiation. The leukemic T cell line, Jurkat, may thus serve as a model for the elucidation of molecular mechanisms that regulate lck transcription and T cell differentiation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Línea Celular , Células Cultivadas , Neoplasias del Colon , Células HeLa , Humanos , Cinética , Leucemia , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Proteínas Oncogénicas Virales/genética , Oncogenes/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Linfocitos TRESUMEN
The human T-cell- or lymphocyte-specific gene, lck, encodes a tyrosine kinase and is a member of the src family. In this report we demonstrate that there are two classes of human lck transcripts (types I and II), containing different 5'-untranslated regions, which are expressed from two distinct promoters. No apparent sequence similarity was observed between the 5'-flanking regions of the two promoters. The expression of lck in human T-cell leukemia and carcinoma cell lines and in human peripheral blood T lymphocytes was examined by S1 nuclease and primer extension mapping and by Northern (RNA) blot analysis of total cellular RNA. The following results were obtained. (i) Two RNA start sites in the downstream promoter were used to generate type I transcripts. (ii) The major human type I start site has not been described for the mouse. (iii) At least five RNA start sites in the upstream promoter were used to generate type II transcripts. (iv) In T cells and in two colon carcinoma cell lines, type II transcripts were present in higher amounts than type I transcripts. (v) In T cells treated with phytohemagglutinin, tetradecanoylphorbol acetate, and cyclosporin A, the modulation of lck expression was associated primarily with changes in levels of type II transcripts. The above results suggest that the two human lck promoters are utilized differentially and may be regulated independently during certain physiological states.
Asunto(s)
Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Transcripción Genética , Secuencia de Bases , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , ADN/genética , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Mapeo Restrictivo , Linfocitos T/citología , Linfocitos T/enzimología , Linfocitos T/inmunología , Células Tumorales Cultivadas/enzimologíaRESUMEN
A series of point mutants were generated in the human c-fos dyad symmetry element (DSE), found within the c-fos serum response element, to study the sequence requirements for its interaction with the human HeLa cell serum response factor (SRF). Plasmids that contain base substitutions within a core CC(A/T)6GG motif in the center of the DSE did not compete, or competed very poorly, with the wild-type c-fos DSE for formation of a specific SRF-DSE complex in vitro. The CC(A/T)6GG motif is not sufficient for maximal binding of SRF, as several plasmids that contain base substitutions in the sequences flanking this core motif competed either poorer or better than the wild-type c-fos DSE for SRF binding. Evidence is presented that supports the idea that SRF binds in a symmetrical fashion. Results of in vivo transient expression assays in HeLa cells suggest that negative regulation of c-fos transcription observed in serum-deprived cells is mediated through SRF binding to the DSE.
Asunto(s)
Secuencia de Bases , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Unión Competitiva , Fenómenos Fisiológicos Sanguíneos , Análisis Mutacional de ADN/métodos , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas c-fos , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Respuesta SéricaRESUMEN
We have identified a CCAAT box element that is required for the efficient transcription of the human beta-actin gene. Both in vivo transient transfection assays in cultured HeLa cells and in vitro run-off transcription assays in HeLa whole cell extracts demonstrated the requirement of this element for efficient promoter activity. A gel mobility shift assay revealed a Hela nuclear factor that specifically interacted with the beta-actin CCAAT element in vitro; mutation of the first three base pairs of the CCAAT pentanucleotide abolished binding of this factor. Competition gel shift experiments revealed that three sequence elements located within the beta-actin promoter, each containing a CC(A/T)6GG motif similar to that contained within the c-fos serum response element, were able to bind a different nuclear factor, serum response factor (SRF). One of these CC(A/T)6GG motifs is contained within a first intron fragment that enhanced transcription from a heterologous promoter in vivo.
Asunto(s)
Actinas/genética , Genes , Intrones , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Mapeo Restrictivo , TransfecciónRESUMEN
We describe the ability of reovirus messenger RNA (mRNA) to serve as a template for translation and as an activator of protein phosphorylation in cell-free extracts prepared from untreated and from interferon (IFN)-treated mouse fibroblast L cells. In vitro transcribed reovirus mRNA was purified by column chromatography on CF-11 cellulose. This procedure removed trace amounts of double-stranded RNA (dsRNA) [0.01%-0.1%] present in mRNA preparations purified solely by extensive LiCl precipitation. In the absence of added dsRNA, CF-11 cellulose-purified reovirus mRNA did not detectably activate phosphorylation of either ribosome-associated protein P1 or the alpha subunit of protein synthesis initiation factor eIF-2 in S-10 extracts prepared from L cells; the CF-11 cellulose-purified reovirus mRNA was translated more efficiently than was LiCl-purified reovirus mRNA in these extracts. Highly purified CF-11 reovirus mRNA was, however, translated less efficiently by S-10 extracts prepared from IFN-treated L cells than by extracts prepared from untreated L cells, suggesting that the inefficient translation by IFN-treated extracts was an integral property of reovirus mRNA. Increasing the secondary structure of reovirus mRNA by substituting bromouridine (Br-uridine) for uridine in the mRNA caused an increased inhibition of mRNA binding to ribosomes in extracts prepared from IFN-treated as compared to untreated cells. The mechanism of inhibition of translation of CF-11 cellulose-purified reovirus mRNA in IFN-treated systems remains to be established.
Asunto(s)
Interferones/farmacología , Biosíntesis de Proteínas , Proteínas/metabolismo , Animales , Sistema Libre de Células , Centrifugación por Gradiente de Densidad , Cromatografía , Ratones , Fosforilación , ARN Mensajero/metabolismo , Reoviridae/genética , Ribosomas/metabolismoRESUMEN
Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus DNA in vitro and in vivo. We have examined the effect of natural and synthetic binding sites for NFI (FIB sites) on RNA synthesis in HeLa whole cell extracts. The natural binding site used is the 26bp FIB-2 site previously isolated from the human genome. When present upstream of the TATA box of the adenovirus major late promoter, the FIB-2 site stimulates RNA synthesis 3 to 5-fold. This stimulation occurs with either orientation of the FIB-2 site. A point mutation in FIB-2 that decreases NFI binding at least 100-fold reduces, but does not completely abolish, the stimulation of transcription. A number of synthetic binding sites for NFI were tested for the ability to increase RNA synthesis. The strongest binding sites stimulated transcription the most, while the weakest sites had the least effect. These studies strongly suggest a role for NFI and cellular FIB sites in the control of RNA synthesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Sitios de Unión , Unión Competitiva , Regulación de la Expresión Génica , Células HeLa , Humanos , Técnicas In Vitro , Factores de Transcripción NFI , Regiones Promotoras Genéticas , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Proteína 1 de Unión a la Caja YRESUMEN
Stimulation of in vitro transcription by the upstream element (UE) of the adenovirus-2 major late promoter (Ad2MLP) involves a specific trans-acting factor present in a HeLa whole-cell extract. By following its transcriptional stimulatory activity and its DNase I footprint on the Ad2MLP-UE, we have purified this factor to greater than 10% purity and separated it from RNA polymerase B and the general transcription factors required for transcription from the Ad2MLP.
Asunto(s)
Adenovirus Humanos/genética , Genes Virales , Genes , Regiones Promotoras Genéticas , Factores de Transcripción/aislamiento & purificación , Cromatografía/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Durapatita , Células HeLa/metabolismo , Humanos , Hidroxiapatitas , Transcripción GenéticaRESUMEN
Stimulation of in vitro transcription mediated by the upstream element of the adenovirus-2 major late promoter (Ad2MLP) involves its recognition by a specific trans-acting factor present in a HeLa whole-cell extract. DNase I footprinting and dimethylsulfate methylation protection experiments were used to determine, at the nucleotide level, upstream sequences which interact with this transcription factor. The ability of upstream element mutants to bind the transcription factor correlates directly with the efficiency of transcription from the corresponding Ad2ML promoters in vivo and in vitro. Competition footprinting experiments show that the transcription factor, which binds to the upstream element of the Ad2MLP, can also interact, but with a lower affinity, with the upstream elements of the Ad2E2a and rabbit beta-globin promoters, both of which display some sequence homology to the 'interacting' region of the Ad2MLP upstream element. The transcription factor does not, however, interact with the upstream elements of either the Ad2E2L, Ad5E3, SV40 early, herpes virus thymidine kinase or chicken conalbumin promoters.