RESUMEN
Cooperative DNA binding by transcription factors that bind to separate recognition sites is likely to require bending of intervening sequences and the appropriate orientation of transcription factor binding. We investigated DNA bending in complexes formed by the basic region-leucine zipper domains of Fos and Jun with the DNA binding region of nuclear factor of activated T cells 1 (NFAT1) at composite regulatory elements using gel electrophoretic phasing analysis. The NFAT1-Fos-Jun complex induced a bend at the ARRE2 site that was distinct from the sum of the bends induced by NFAT1 and Fos-Jun separately. We designate this difference DNA bending cooperativity. The bending cooperativity was directed toward the interaction interface between Fos-Jun and NFAT1. We also examined the influence of NFAT1 on the orientation of Fos-Jun heterodimer binding using a novel fluorescence resonance energy transfer assay. The interaction with NFAT1 could reverse the orientation of Fos-Jun heterodimer binding to the ARRE2 site. The principal determinants of both cooperative DNA bending and oriented heterodimer binding were localized to three amino acid residues at the amino-terminal ends of the leucine zippers of Fos and Jun. Consequently, interactions between transcription factors can remodel promoters by altering DNA bending and the orientation of heterodimer binding.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteínas Nucleares , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-jun/química , Factores de Transcripción/química , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Factores de Transcripción NFATC , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.
Asunto(s)
Antígenos CD11/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Miocardio/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta/genética , Animales , Animales Recién Nacidos , Anticuerpos/uso terapéutico , Secuencia de Bases , Cartilla de ADN , Genotipo , Heterocigoto , Homocigoto , Inmunoterapia , Inflamación/genética , Recuento de Leucocitos , Activación de Linfocitos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Miocardio/patología , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
Null-mutant (knockout) mice were obtained through disruption of the sixth exon of the endogenous transforming growth factor-beta 1 allele in murine embryonic stem cells via homologous recombination. Mice lacking transforming growth factor-beta 1 (mutants) were born grossly indistinguishable from wild-type littermates. With time, mutant mice exhibited a wasting phenotype that manifested itself in severe weight loss and dishevelled appearance (between 15 and 36 days of age). Examination of these moribund mice histologically revealed that transforming growth factor-beta 1-deficient mice exhibit a moderate to severe, multifocal, organ-dependent, mixed inflammatory cell response adversely affecting the heart, stomach, diaphragm, liver, lung, salivary gland, and pancreas. Because of the known multifunctional nature of transforming growth factor-beta 1 on the control of growth and differentiation of many different cell types, it is important to determine the degree to which the inflammatory response interacts with or masks other deficiencies that are present. To this end, we examined the extent and nature of the inflammatory lesions in different ages of neonatal knockout mice (5, 7, 10, and 14 days of age) and older moribund mice (> 15 days of age) and compared them with the histology seen in wild-type normal animals. Mild inflammatory infiltrates were first observed in 5-day mutant mice in the heart, by day 7 in the lung, salivary gland, and pancreas, and by day 14 inflammatory lesions were found in almost all organs examined. Moderate to severe inflammation was not present until the mice were 10 to 14 days old. In the older animals, there was a slight increase in the severity of the inflammatory lesions as the mice aged.
Asunto(s)
Modelos Animales de Enfermedad , Inflamación/patología , Factor de Crecimiento Transformador beta/genética , Animales , Secuencia de Bases , Citometría de Flujo , Genotipo , Inflamación/genética , Inflamación/inmunología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Aumento de PesoRESUMEN
Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Fabaceae/microbiología , Plantas Medicinales , Purinas/metabolismo , Rhizobium/crecimiento & desarrollo , Ribonucleósidos/metabolismo , Simbiosis/fisiología , Aminoimidazol Carboxamida/metabolismo , Fabaceae/ultraestructura , Mutagénesis , Rhizobium/genética , Rhizobium/ultraestructura , Especificidad de la EspecieRESUMEN
Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional growth factor that has profound regulatory effects on many developmental and physiological processes. Disruption of the TGF-beta 1 gene by homologous recombination in murine embryonic stem cells enables mice to be generated that carry the disrupted allele. Animals homozygous for the mutated TGF-beta 1 allele show no gross developmental abnormalities, but about 20 days after birth they succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death. TGF-beta 1-deficient mice may be valuable models for human immune and inflammatory disorders, including autoimmune diseases, transplant rejection and graft versus host reactions.
Asunto(s)
Inflamación/genética , Factor de Crecimiento Transformador beta/fisiología , Animales , Secuencia de Bases , Citocinas/genética , Expresión Génica , Genes , Homocigoto , Inflamación/patología , Recuento de Leucocitos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Necrosis , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.
Asunto(s)
Canales de Calcio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Exones , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Miocardio/metabolismo , Oligodesoxirribonucleótidos/química , Empalme del ARN , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Mapeo RestrictivoRESUMEN
Several cDNAs encoding an isoform of the alpha 1 subunit of the voltage-dependent calcium channel were isolated from rat brain cDNA libraries. The complete nucleotide sequence of 6975 bp encodes a protein of 1634 amino acids, which corresponds to an Mr of 186,968. The protein exhibits 71% and 76% homology to skeletal and cardiac alpha 1 subunits, respectively. When compared with skeletal and cardiac alpha 1 isoforms, the rat brain protein is intermediate in size at the amino terminus and shorter at the carboxyl terminus. Multiple subtypes of this alpha 1 isoform cDNA were characterized. These are indicative of alternative splicing of a primary transcript and encode three variants between motif I and motif II and two within the S3 region of motif IV. Thus, multiple isoforms of this rat brain alpha 1 subunit are possible.