RESUMEN

Collateral effects of exogenous sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca(2+) transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca(2+) transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.

Asunto(s)

ATPasas Transportadoras de Calcio/genética , Miocardio/citología , Adenoviridae/genética , Animales , Western Blotting , Calcio/metabolismo , ATPasas Transportadoras de Calcio/biosíntesis , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Fragmentación del ADN , ADN Complementario/metabolismo , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Cinética , Microscopía de Contraste de Fase , Fenilalanina/farmacología , ARN Mensajero/metabolismo , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/farmacología

RESUMEN

The large tumor antigen (TAg) of simian virus 40 is able to transform cells through interactions with cellular proteins, notably p53 and Rb. Among the other proteins that form complexes with TAg is TEF-1, a transcription factor utilized by the viral enhancer to activate expression of the early gene which encodes TAg. We show that fibroblasts contain several alternately spliced TEF-1 mRNAs, the most abundant of which encodes a protein with an additional four amino acid exon compared to the database entry for Hela cell TEF-1. Transformation by TAg induces alternate splicing, producing a more abundant form lacking this exon and matching the published sequence. Splicing variants lacking this exon were detected in mouse pancreatic tumors and in cell lines derived from human pancreatic cancers, in contrast to a single isoform with the exon in normal mouse pancreas. A total of eight splice variants were identified, with the loss of the four amino acid exon typical of transformed cells. These and other data presented suggest that TAg 're-models' host cell transcription factors that are used early in viral infection, and thereby mimics an event that naturally occurs during transformation. The data indicate that TEF-1 alterations may be a hallmark feature of tumorigenesis.

Asunto(s)

Antígenos Transformadores de Poliomavirus , Proteínas de Unión al ADN/genética , Proteínas Nucleares , Empalme del ARN , Factores de Transcripción/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Transformación Celular Viral , Proteínas de Unión al ADN/química , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Neoplasias Pancreáticas/genética , Isoformas de Proteínas/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de Dominio TEA , Factores de Transcripción/química , Transfección , Células Tumorales Cultivadas

RESUMEN

M-CAT elements mediate both muscle-specific and non-muscle-specific transcription. We used artificial promoters to dissect M-CAT elements derived from the cardiac troponin T promoter, whose regulation is highly striated muscle specific. We show that muscle-specific M-CAT-dependent expression requires two distinct components: the core heptameric M-CAT motif (5'-CATTCCT-3'), which constitutes the canonical binding site for TEF-1-related proteins, and specific sequences immediately flanking the core motif that bind an additional factor(s). These factors are found in higher-order M-CAT DNA-protein complexes with TEF-1 proteins. Non-muscle-specific promoters are produced when the sequences flanking the M-CAT motif are removed or modified to match those of non-muscle-specific promoters such as the simian virus 40 promoter. Moreover, a mutation of the 5'-flanking region of the cardiac troponin T M-CAT-1 element upregulated expression in nonmuscle cells. That mutation also disrupts a potential E box that apparently does not bind myogenic basic helix-loop-helix proteins. We propose a model in which M-CAT motifs are potentially active in many cell types but are modulated through protein binding to specific flanking sequences. In nonmuscle cells, these flanking sequences bind a factor(s) that represses M-CAT-dependent activity. In muscle cells, on the other hand, the factor(s) binding to these flanking sequences contributes to both the cell specificity and the overall transcriptional strength of M-CAT-dependent promoters.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Transcripción Genética , Troponina/biosíntesis , Troponina/genética , Animales , Secuencia de Bases , Sitios de Unión , Embrión de Pollo , Huella de ADN , Metilación , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos , Sondas de Oligonucleótidos , Especificidad de Órganos , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción de Dominio TEA , Troponina T

RESUMEN

M-CAT motifs mediate muscle-specific transcriptional activity via interaction with binding factors that are antigenically and biochemically related to vertebrate transcription enhancer factor-1 (TEF-1), a member of the TEA/ATTS domain family of transcription factors. M-CAT binding activities present in cardiac and skeletal muscle tissues cannot be fully accounted for by existing cloned isoforms of TEF-1. TEF-1-related cDNAs isolated from heart libraries indicate that at least three classes of TEF-1-related cDNAs are expressed in these and other tissues. One class are homologues of the human TEF-1 originally cloned from HeLa cells (Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M., and Chambon, P. (1991) Cell 65, 551-568). A second class represents homologues of the avian TEF-1-related gene previously isolated (Stewart, A. F., Larkin, S. B., Farrance, I. K., Mar, J. H., Hall, D. E., and Ordahl, C. P. (1994) J. Biol. Chem. 269, 3147-3150). The third class consists of a novel, divergent TEF-1 cDNA, named DTEF-1, and its preliminary characterization is described here. Two isoforms of DTEF-1 (DTEF-1A and DTEF-1B) were isolated as 1.9-kilobase pair clones with putative open reading frames of 433 and 432 amino acids whose differences are attributable to alternative splicing at the C terminus of the TEA DNA binding domain. Cardiac muscle contains high levels of DTEF-1 transcripts, but unexpectedly low levels are detected in skeletal muscle. DTEF-1 transcripts are present at intermediate levels in gizzard and lung, and at low levels in kidney. DTEF-1A is a sequence-specific M-CAT-binding factor. The distinct spatial pattern of expression, and unusual amino acid sequence in its DNA binding domain, may indicate a particular role for DTEF-1 in cell-specific gene regulation. Recent work also suggests that at least one more TEF-1-related gene exists in vertebrates. We propose a naming system for the four TEF-1 gene family members identified to date that preserves existing nomenclature and provides a means for extending that nomenclature as additional family members may be identified.

Asunto(s)

Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Pollos , Cartilla de ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes , Miocardio/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción de Dominio TEA , Factores de Transcripción/química , Factores de Transcripción/metabolismo

RESUMEN

M-CAT sites are required for the activity of many promoters in cardiac and skeletal muscle. M-CAT binding activity is muscle-enriched, but is found in many tissues and is immunologically related to the HeLa transcription enhancer factor-1 (TEF-1). TEF-1-related cDNAs (RTEF-1) have been cloned from chick heart. RTEF-1 mRNA is muscle-enriched, consistent with a role for RTEF-1 in the regulation of muscle-specific gene expression. Here, we have examined the tissue distribution of TEF-1-related proteins and of M-CAT binding activity by Western analysis and mobility shift polyacrylamide gel electrophoresis. TEF-1-related proteins of 57, 54 and 52 kDa were found in most tissues with the highest levels in muscle tissues. All of these TEF-1-related proteins bound M-CAT DNA and the 57- and 54-kDa TEF-1-related polypeptides were phosphorylated. Proteolytic digestion mapping showed that the 54-kDa TEF-1-related polypeptide is encoded by a different gene than the 52- and 57-kDa TEF-1-related polypeptides. A comparison of the migration and proteolytic digestion of the 54-kDa TEF-1-related polypeptide with proteins encoded by the cloned RTEF-1 cDNAs showed that the 54-kDa TEF-1-related polypeptide is encoded by RTEF-1A. High resolution mobility shift polyacrylamide gel electrophoresis showed multiple M-CAT binding activities in tissues. All of these activities contained TEF-1-related proteins. One protein-M-CAT DNA complex was muscle-enriched and was up-regulated upon differentiation of a skeletal muscle cell line. This complex contained the 54-kDa TEF-1-related polypeptide. Therefore, RTEF1-A protein is a component of a muscle-enriched transcription complex that forms on M-CAT sites and may play a key role in the regulation of transcription in muscle.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Músculos/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Pollos , Regulación de la Expresión Génica , Genes , Técnicas Inmunológicas , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Proteínas Nucleares/metabolismo , Mapeo Peptídico , Fosfoproteínas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Factores de Transcripción de Dominio TEA , Distribución Tisular

RESUMEN

M-CAT elements mediate cardiac- and embryonic skeletal muscle-specific expression of the cardiac troponin T gene and a number of other cardiac-specific genes. M-CAT binding factor was shown to be related to cloned human TEF-1, a transcriptional regulator of the SV40 viral enhancer. Here we describe the cloning of TEF-1 from chick heart and the identification of several novel isoforms. We show that TEF-1 mRNA is considerably enriched in cardiac and skeletal muscle, consistent with a proposed role in muscle gene transcription. The predominant TEF-1 isoforms, TEF-1A and a novel isoform TEF-1B, bind M-CAT elements with high affinity and in a sequence-specific manner. We further demonstrate that the C-terminal portion of TEF-1B, which contains the 13-amino acid exon that distinguishes this isoform, can activate transcription when linked to a heterologous DNA binding domain, while the same domain of TEF-1A cannot. Therefore, isoforms of TEF-1 may play different roles in the regulation of M-CAT-dependent promoters in striated muscle cells.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Expresión Génica , Proteínas Musculares/genética , Músculos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Embrión de Pollo , Pollos , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Exones , Regulación de la Expresión Génica , Variación Genética , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción de Dominio TEA , Factores de Transcripción/biosíntesis

RESUMEN

In cultured rat cardiac myocytes, a 20-base pair sequence (-215/-196) of the rat beta-myosin heavy chain (MHC) promoter mediates induction by both alpha 1-adrenergic stimulation and a constitutively activated beta-protein kinase C (PKC), and binds cardiac myocyte nuclear factor(s) through an "enhancer core" element (5'-TGTGG-TATG-3') (Kariya, K., Karns, L. R., and Simpson, P. C. (1994) J. Biol. Chem. 269, in press). Here, we report identification of this enhancer core binding factor as the rat homologue of transcriptional enhancer factor-1 (TEF-1), a human transcription factor for viral enhancers. In gel mobility shift and immunoblot analyses, the myocyte factor and human TEF-1 were indistinguishable in terms of sequence recognition, mobility, and immunoreactivity. Furthermore, DNA binding activity for the beta-MHC enhancer core and TEF-1 immunoreactivity correlated closely. These results are the first to suggest a role for TEF-1 in transcriptional regulation by PKC. The data also provide direct evidence for interaction of TEF-1 with the beta-MHC promoter, supporting a function for TEF-1 in regulation of cellular gene expression, as well as viral, and outline a pathway for alpha 1-adrenergic regulation of beta-MHC gene transcription in cardiac myocytes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Miocardio/metabolismo , Miosinas/genética , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Miocardio/citología , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos , Unión Proteica , Ratas , Factores de Transcripción de Dominio TEA

RESUMEN

M-CAT binding factor (MCBF) governs the activity of the cardiac troponin T gene promoter. M-CAT motifs have also been implicated recently in the regulation of other contractile protein genes which like cardiac troponin T, do not require direct interaction with MyoD1 or related factors for activity. Mutational analysis of the M-CAT motif revealed that it can be functionally replaced by a regulatory motif of the SV40 enhancer which binds human transcription factor TEF-1. Biochemical analyses show that MCBF from muscle nuclei is indistinguishable from TEF-1 in terms of specificity of binding site recognition, fractionation on DNA-agarose, and apparent molecular weight. In addition, antibodies raised against amino- and carboxyl-terminal regions of human TEF-1 also bind to MCBF from chicken muscle. We conclude, therefore, that MCBF is closely related to TEF-1. Finally, MCBF/TEF-1 is highly enriched in the nuclei of striated muscle, as compared with other tissues, consistent with a role in muscle-specific promoter regulation.

Asunto(s)

Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Genes Virales , Músculos/fisiología , Proteínas Nucleares , Virus 40 de los Simios/genética , Transactivadores/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/fisiología , Embrión de Pollo , Proteínas de Unión al ADN/metabolismo , Molleja de las Aves , Corazón/fisiología , Cinética , Datos de Secuencia Molecular , Músculo Liso/fisiología , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Virus 40 de los Simios/metabolismo , TATA Box , Factores de Transcripción de Dominio TEA , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Troponina/genética , Troponina T

RESUMEN

Transcription of the prolactin (PRL) gene has been analyzed in wild-type D6, PRL-deficient B3, and revertant r16 GH3 cells. Levels of processed nuclear transcripts from the PRL gene were substantially reduced in the deficient line compared to wild-type cells and returned to greater than wild-type levels in the revertant line. Rare PRL transcripts in the deficient line contained the same 5' end found on transcripts in wild-type and revertant cells as judged by primer extension and S1 nuclease protection assays, implying that the cells are deficient in utilization of the normal wild-type promoter. Deficient cells also contained wild-type levels of the PRL- and growth hormone-specific transcription factor pit-1/GHF-1, and no difference was found in the ability of extracts from wild-type and deficient cells to retard various restriction fragments from both the proximal and the distal PRL promoter regions. The deficient and wild-type cells were equally competent in initiating transcription from a transfected rat PRL promoter containing both the distal and proximal promoter elements. These observations imply that PRL-deficient cells are not defective in a trans-activating factor functioning on these PRL promoter fragments (trans model). Rather, inefficient use of the PRL promoter in the variant cells may reflect an increased methylation state of the PRL gene itself (cis model).

Asunto(s)

Prolactina/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética , Animales , Azacitidina/farmacología , Northern Blotting , Línea Celular , Mapeo Cromosómico , ADN/efectos de los fármacos , Metanosulfonato de Etilo/farmacología , Regulación de la Expresión Génica , ARN Mensajero/análisis , Ratas , Activación Transcripcional , Transfección

RESUMEN

N7-Ethyldeoxyguanosine 5'-triphosphate (N7-Etd-GTP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at approximately 5% yield. N7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage. At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of greater than 48 h. An enzymatic method for placing an N7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3' to 5' exonuclease and 5' to 3' polymerase of the Klenow fragment of Escherichia coli DNA polymerase I. The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine, N6-methyladenine, and others).

Asunto(s)

Nucleótidos de Desoxiguanina/síntesis química , Guanina/análogos & derivados , Oligodesoxirribonucleótidos/síntesis química , Escherichia coli/enzimología , Guanina/análisis , Concentración de Iones de Hidrógeno , Radioisótopos de Fósforo , Piperidinas/análisis , Espectrofotometría Ultravioleta

RESUMEN

Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.

Asunto(s)

Enzimas de Restricción del ADN , Oligodesoxirribonucleótidos/síntesis química , Compuestos Organofosforados , Mapeo Restrictivo , Adenina , Composición de Base , Clonación Molecular/métodos , ADN/análisis , ADN/metabolismo , Metilasas de Modificación del ADN , Enzimas de Restricción del ADN/metabolismo , Guanina , Piperidinas , Mapeo Restrictivo/métodos , Especificidad por Sustrato , Timina

RESUMEN

Ethylation of poly(dC-dG).poly(dC-dG) with ethyl methanesulfonate (EtMes), a known carcinogen, at increasing molar ratios of EtMes/C X G base pairs progressively stimulated the methyl-accepting ability of the DNA during in vitro methylation by partially purified rat DNA (cytosine-5)-methyltransferase (EC 2.1.1.37). Maximum stimulation was 2-fold over mock-treated DNA when 2.7% of the guanines were modified at the N-7 position, the major site of ethylation by EtMes in DNA. If a CpG site "hemiethylated" at guanine N-7 mimics a hemimethylated CpG site, we calculate that the enzyme has a relative affinity for hemiethylated CpG 18-fold above unmodified CpG. If ethylation of a dioxyphosphate oxygen of the phosphodiester bond is responsible for stimulation, the relative affinity could be much higher, up to 370-fold.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metanosulfonato de Etilo/farmacología , Metiltransferasas/metabolismo , Animales , Técnicas In Vitro , Modelos Moleculares , Polidesoxirribonucleótidos , Ratas , Especificidad por Sustrato