RESUMEN
Basic fibroblast growth factor (bFGF) is an important mitogen and neurotrophic factor that binds and signals through the high-affinity receptor, fibroblast growth factor receptor 1 (FGFR1). However, only a limited amount of information is available concerning the molecular forms and anatomical distribution of fibroblast growth factors (FGFs) in the normal human brain. We found multiple bFGF and FGFR1 mRNA transcripts which vary in expression pattern across human brain regions. Using in situ hybridization and immunohistochemistry, we localized bFGF and FGFR1 mRNA and protein to cells in the normal adult human hippocampus and caudal entorhinal cortex (ERC). The majority of pyramidal neurons contained FGFR1 mRNA and protein in the mesial temporal lobe, with neurons in the CA2/CA3 region demonstrating the highest levels of FGFR1 mRNA. In contrast to FGFR1, bFGF mRNA expression was detected at very low levels in a small fraction of the neurons in the human hippocampus and caudal ERC. While bFGF mRNA may be expressed at low levels in neurons, bFGF-immunopositive cells with astrocytic features were detected throughout the mesial temporal lobe in rats, monkeys and humans. bFGF immunoreactive processes are found traversing the dentate gyrus, and bFGF immunoreactive cells are found in the neurogenic subgranular zone in all three mammalian species studied. The anatomical distribution of these two FGF family members suggests that bFGF is endogenously positioned to be involved in ongoing neurogenesis in the adult hippocampus, and that FGF trophic signaling to differentiated neurons could involve the release of astrocytic bFGF acting on neuronal FGFR1 in the normal adult human hippocampus.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Hipocampo/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Adulto , Autopsia , Población Negra , Femenino , Humanos , Hibridación in Situ , Masculino , Mesotelina , Persona de Mediana Edad , ARN Mensajero/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Valores de Referencia , Estados Unidos , Población BlancaRESUMEN
1. Although endotoxaemia induces kinin B(1) receptors in several animal models, this condition is not documented in primates. This study examined the up-regulation of haemodynamic and pro-inflammatory responses to the B(1) agonist des-Arg(10)-kallidin (dKD) in a non-human primate model. 2. Green monkeys (Cercopithecus aethiops St Kitts) received lipopolysaccharide (LPS; 90 microg kg(-1)) or saline intravenously. After 4 h, anaesthetized monkeys were cannulated via the carotid artery to monitor blood pressure changes following intra-arterial injections of dKD or the B(2) agonist bradykinin (BK). Oedema induced by subcutaneous kinin administration was evaluated as the increase in ventral skin folds in anaesthetized monkeys injected with captopril at 4 h to 56 days post-LPS. 3. LPS increased rectal temperature but did not affect blood pressure after 4 h. dKD reduced blood pressure (E(max): 27+/-4 mmHg; EC(50): 130 pmol kg(-1)) and increased heart rate (E(max): 33 b.p.m.) only after LPS. In contrast, the dose-dependent fall in blood pressure with BK was comparable in all groups. The selective B(1) antagonist [Leu(9)]dKD (75 ng kg(-1) min(-1), intravenously) abolished responses to dKD but not BK. 4. dKD injection induced oedema dose-dependently (2.4+/-0.1 mm at 150 nmol) only following LPS (at 4 h to 12 days but not 56 days). In contrast, BK-induced oedema was present and stable in all monkeys. Co-administration of [Leu(9)]dKD (150 nmol) significantly reduced oedema induced by dKD (50 nmol). 5. These results suggest LPS up-regulation of B(1) receptor effects in green monkeys. This non-human primate model may be suitable for testing new, selective B(1) antagonists with therapeutic potential as anti-inflammatory agents.
Asunto(s)
Endotoxinas/farmacología , Hemodinámica/efectos de los fármacos , Inflamación/patología , Calidina/análogos & derivados , Receptores de Bradiquinina/agonistas , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Temperatura Corporal/efectos de los fármacos , Bradiquinina/farmacología , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Edema/patología , Hemodinámica/fisiología , Calidina/farmacología , Lipopolisacáridos/farmacología , Masculino , Receptor de Bradiquinina B1RESUMEN
Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Regulación de la Expresión Génica , Plásmidos/genética , Northern Blotting , Southern Blotting , Calcio/metabolismo , Línea Celular Transformada , Quimiocina CCL22 , Quimiocina CXCL12 , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , ADN/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Dosificación de Gen , Vectores Genéticos , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Radioisótopos de Yodo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN/genética , Ensayo de Unión Radioligante , Receptores CCR4 , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores Opioides/agonistas , Receptores Opioides/genética , Receptores Opioides/fisiología , Proteínas Recombinantes de Fusión/genética , Transfección , Receptor de NociceptinaRESUMEN
Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.
Asunto(s)
Antagonistas de los Receptores de Bradiquinina , Bradiquinina/metabolismo , Línea Celular , Humanos , Biblioteca de Péptidos , Receptor de Bradiquinina B1 , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Several cell lines have become widely used in biotechnology, pharmaceutical and academic laboratories because of their desirable characteristics. Among these, the human embryonic kidney cell line HEK293 is one of the most versatile and powerful for expression of recombinant proteins, permitting the establishment of stable cell lines in just three weeks. Unfortunately, HEK293 cells adhere weakly to tissue culture grade plastic. Therefore, without cumbersome and sometimes expensive modifications to equipment, these cells are not suitable for use in a number of extremely important applications, including robot-based, high-throughput drug screening formats, growth and expansion in roller bottles and expression cloning experiments. We have cloned and transfected the human Class A macrophage scavenger receptor into 293EBNA cells (293EM) and found that expression of this receptor confers a sufficiently adherent property to the cells as to render them usable with automated equipment. In addition, the 293EM cells are now able to adhere to the surface of roller bottles and to untreated glass substrates, allowing growth of these cells in formats for which they are not normally well suited.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Macrófagos/metabolismo , Receptores Inmunológicos/fisiología , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Técnicas de Cultivo de Célula/instrumentación , División Celular , Línea Celular , Humanos , Riñón/citología , Macrófagos/citología , Macrófagos/fisiología , Fenotipo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Depuradores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Depuradores de Clase ARESUMEN
Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein alpha subunit (G alpha s) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.
Asunto(s)
Línea Celular , Diseño de Fármacos , Receptores de Hormona Liberadora de Corticotropina/genética , Animales , Células CHO , Cricetinae , Antígenos Nucleares del Virus de Epstein-Barr/genética , Expresión Génica , Vectores Genéticos , Herpesvirus Humano 4/genética , Humanos , Plásmidos/genética , ARN/genética , ARN/metabolismo , Receptores de Hormona Liberadora de Corticotropina/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/clasificación , Origen de RéplicaRESUMEN
Levels of expression of two reporter genes cloned into SV40 or Epstein-Barr virus (EBV) ori-containing plasmids were measured following transient transfection of cell lines constitutively expressing T-antigen or EBV nuclear antigen 1 (EBNA1). The TSA201 and COS7 cell lines stably produce T-antigen and support replication of the SV40 ori-containing constructs while the 293EBNA cell line produces EBNA1 and supports replication of EBV ori-containing plasmids. We found that 293EBNA cells express > 25-fold more beta-galactosidase (beta Gal) per mg protein than COS7 cells and 11-fold more beta Gal than TSA201 cells. We also demonstrate that 293EBNA cells are able to express 70-100-fold more angiotensin II type-1 receptor (AT1) per mg protein than COS7 or TSA201 cells. We examined the suitability of each cell line for use in expression cloning using a NaOH 'scrape' method as an improvement over emulsion autoradiography for detection. Measurable AT1 signals can be detected when reporter plasmids are diluted up to 1000-fold for COS7 and TSA201 cells, and up to 80,000-fold for 293EBNA cells. These data demonstrate that 293EBNA cells offer a significant improvement in expression cloning technology as compared to the conventionally used T-antigen-based cell lines.
Asunto(s)
Clonación Molecular/métodos , Genes Reporteros , Vectores Genéticos/genética , Herpesvirus Humano 4/genética , Receptores de Angiotensina/biosíntesis , Línea Celular , Receptores de Angiotensina/genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
A DNA clone of HIV-1 containing the full-length infectious viral sequence was cleaved at a unique Nco I restriction site within the viral genome, and DNA fragments containing the 5' and 3' portions of the HIV genome were subcloned into separate plasmid vectors. The 5' 'half-virus' construct was further modified by incorporating a class IIS restriction site, Esp3I, near the 3' end of the protease gene of HIV. This site, in combination with a natural ApaI site near the 5' end of the protease gene, creates a convenient cassette shuttle vector in which the protease coding region can be easily replaced. Recombinant viruses containing protease genes either altered by site-directed mutagenesis or amplified from clinical or laboratory isolates can be reconstructed. The DNA fragment containing the protease gene is first subcloned into the 5' half-virus shuttle vector plasmid. Infectious recombinant virus is subsequently recovered by cotransfecting 5' and 3' half-virus plasmids linearized at their common Nco I sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in protease.
Asunto(s)
Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , ADN Viral/genética , Desoxirribonucleasas de Localización Especificada Tipo III , Genes Virales , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la PolimerasaRESUMEN
The binding interactions for the three primary reactants of the fibroblast growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the cofactor heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<==>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<==>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<==>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed growth factor and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glicosilación , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Poliéster Pentosan Sulfúrico/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , TermodinámicaRESUMEN
The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.
Asunto(s)
Receptores de Factores de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Pollos , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Expresión Génica , Variación Genética , Humanos , Pulmón/fisiología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/clasificación , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaAsunto(s)
Baculoviridae/genética , ADN Recombinante/análisis , ADN Viral/análisis , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas de la Matriz de Cuerpos de Oclusión , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Virales/genética , Proteínas Estructurales ViralesRESUMEN
A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.
Asunto(s)
Interleucina-1/genética , Ingeniería de Proteínas , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Dicroismo Circular , Escherichia coli/genética , Humanos , Interleucina-1/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Conformación Proteica , Ingeniería de Proteínas/métodos , Ensayo de Unión Radioligante , Proteínas Recombinantes/síntesis químicaRESUMEN
Determination of the size of a population of nucleic acids can be achieved by several distinct methods. Most of these methods are cumbersome and require complicated equipment or techniques. We demonstrate here the use of a differential pressure capillary viscometer for the rapid and simple determination of RNA molecular weight. This highly sensitive viscometer allowed single viscosity determinations on dilute solutions of RNA, providing a direct measure of the intrinsic viscosity without the need to extrapolate from several concentrations. The molecular weights and conformations of the linear single-stranded RNA homopolymer poly(inosinic acid) (poly(I] and the single-stranded RNA (ssRNA) copolymer poly(cytidylic acid:uridylic acid, 12:1) (poly(C12,U], were determined. The ssRNAs were synthesized in a range of sizes (100 to 100,000 bases). They were widely polydisperse. The Mandelkern-Flory equation (1952, J. Chem. Phys. 20, 212-214), which requires both the intrinsic viscosity and sedimentation coefficient of a macromolecule, was used to calculate the molecular weights. The molecular weights determined by agarose gel electrophoresis were compared to those determined by intrinsic viscosity plus sedimentation coefficient. The correlation between the molecular weights determined by these two methods was good, at R2 greater than or equal to 0.92. The conformations of the RNAs were determined by application of the Mark-Houwink equation. The Mark-Houwink exponents for poly(I) and poly(C12,U) intrinsic viscosities were 0.90 and 0.84, respectively. When compared to other nucleic acid polymers, for which the conformations have been established by several methods, poly(I) and poly(C12,U) are rigid, extended random coils, in a low-salt buffer (15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ARN/química , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Conformación de Ácido Nucleico , Poli I/química , Poli U/química , ViscosidadRESUMEN
We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.
Asunto(s)
Encéfalo/enzimología , Creatina Quinasa/genética , Genes , Músculos/enzimología , Animales , Secuencia de Bases , Desoxirribonucleasa I , Células HeLa/enzimología , Humanos , Datos de Secuencia Molecular , Mapeo Nucleótido , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , Ratas , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.