RESUMEN
Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Porphyromonas gingivalis/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Humanos , Ratones , Datos de Secuencia Molecular , Porphyromonas gingivalis/genética , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
To evaluate the possible role for receptor-based tyrosine phosphorylation in growth signaling induced by interleukin-2 (IL-2), a series of substitution tyrosine mutants of the IL-2 receptor beta and gamma c chains was prepared and analyzed. Concurrent mutation of all six of the cytoplasmic tyrosines present in the beta chain markedly inhibited IL-2-induced growth signaling in both pro-B and T cell lines. Growth signaling in a pro-B cell line was substantially reconstituted when either of the two distal tyrosines (Tyr-392, Tyr-510) was selectively restored in the tyrosine-negative beta mutant, whereas reconstitution of the proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function. Furthermore, at least one of the two cytoplasmic tyrosines that is required for beta chain function was found to serve as a phosphate acceptor site upon induction with IL-2. Studies employing a chimeric receptor system revealed that tyrosine residues of the beta chain likewise were important for growth signaling in T cells. In contrast, although the gamma c subunits is a target for tyrosine phosphorylation in vivo, concurrent substitution of all four cytoplasmic tyrosines of this chain produced no significant effect on growth signaling by chimeric IL-2 receptors. However, deletion of either the Box 1, Box 2, or intervening (V-Box) regions of gamma c abrogated receptor function. Therefore, tyrosine residues of beta but not of gamma c appear to play a pivotal role in regulating growth signal transduction through the IL-2 receptor, either by influencing cytoplasmic domain folding or by serving as sites for phosphorylation and subsequent association with signaling intermediates. These findings thus highlight a fundamental difference in the structural requirements for IL-2R beta and gamma c in receptor-mediated signal transduction.
Asunto(s)
Interleucina-2/farmacología , Receptores de Interleucina-2/fisiología , Transducción de Señal , Tirosina , Secuencia de Aminoácidos , Animales , Linfocitos B , División Celular , Clonación Molecular , Humanos , Interleucina-2/metabolismo , Cinética , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/metabolismo , Receptores de Interleucina-2/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T , TransfecciónRESUMEN
Binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. A novel transient assay of signal transduction leading to proliferation is now described which allows the rapid functional assessment of wild type and mutant receptors, including the IL-2R and other members of the cytokine receptor superfamily. This assay has been used to define domains and specific residues within the IL-2R beta intracellular region that contribute to growth signal transduction. In these studies, internal deletion of either the conserved "Box 1" or "Box 2" proximal cytokine receptor homology segments significantly impaired receptor function. Similarly, mutation of specific key residues within or between Box 1 and Box 2, or deletion of the C-terminal 94 residues of the IL-2R beta chain, impaired growth signaling. In contrast, either replacement of the transmembrane domain with that of the CD4 molecule or internal deletion of the 119 amino acids immediately downstream of Box 2 had no impact on growth signaling competence. These studies thus further define the functional architecture of the intracellular region of IL-2R beta, and reveal specific receptor domains that are dispensable, unique, or functionally redundant.
Asunto(s)
Interleucina-2/metabolismo , Interleucina-2/farmacología , Receptores de Interleucina-2/química , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Linfocitos B , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Reactivos de Enlaces Cruzados , Riñón , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Interleucina-2/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Succinimidas , TransfecciónRESUMEN
The Salmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of the S. typhimurium and Escherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. The in vitro and in vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. The cys M gene of S. typhimurium possessed a GUG initiation codon, like its E. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteine in vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.
Asunto(s)
Acetiltransferasas/genética , Animales Modificados Genéticamente , Proteínas Bacterianas/genética , Cisteína/biosíntesis , Genes Bacterianos , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Ovinos/genética , Transfección , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Biblioteca Genómica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Conejos , Mapeo Restrictivo , Reticulocitos/metabolismo , Serina O-Acetiltransferasa , Transcripción GenéticaRESUMEN
A number of mesenchymal cells produce hemopoietic growth factors in response to inflammatory mediators in vitro and in vivo. Induced transcription from the hemopoietic growth factor genes is at least partially responsible for their increased expression. We have previously identified a sequence, cytokine (CK)-1, in the granulocyte (G)-CSF gene promoter that responds to TNF-alpha and binds a transcription factor, NF-GMa. We report here that the CK-1 sequence responds in a time- and dose-dependent manner to IL-1 beta and that the mutations which affect NF-GMa binding correlate with decreased transcriptional activity after stimulation with either TNF-alpha or IL-1 beta. The CK-1 sequence also responds to the human T lymphotrophic virus-1 transactivator, tax, so that this promoter element may contribute to the overall response of the G-CSF gene to these various agents. Although NF-GMa binding is seen in a number of cell types, the ability of the G-CSF CK-1 sequence to act as a transcriptional enhancer is specific for fibroblasts and not T cells. Furthermore, we show that an identical sequence in the granulocyte macrophage CSF gene, although apparently binding the same protein in vitro, cannot respond to any of these stimuli in either fibroblasts or T cells. Modification interference experiments, using the CK-1 region in the context of the granulocyte macrophage-CSF and G-CSF genes, indicated that the contact points for NF-GMa differ in each case and suggest that differences in sequences flanking the 10-bp CK-1 region probably leads to an altered DNA:protein conformation, which may explain the differential response of this conserved promoter element.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/fisiología , Fibroblastos , Regulación de la Expresión Génica , Productos del Gen tax/genética , Interleucina-1/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-alpha (TNF-alpha) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-alpha-responsive enhancer in these cells. The NF-GMa protein is distinct from another TNF-alpha-responsive transcription factor, NF-kappa B, by several criteria. Firstly, several NF-kappa B-binding sites, although having sequence similarity with the CK-1 sequence, cannot compete efficiently for NF-GMa binding to CK-1. Secondly, the CK-1 sequence from both G-CSF and GM-CSF does not respond to phorbol ester treatment as would an NF-kappa B-binding element. These results demonstrate that NF-GMa is a novel transcription factor inducible by TNF-alpha and binds to a common element in HGF gene promoters.
Asunto(s)
Factores Estimulantes de Colonias/genética , Sustancias de Crecimiento/genética , Interleucinas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Bases , Sitios de Unión , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Hematopoyesis , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Homología de Secuencia de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We report the isolation and complete nucleotide sequence of genes encoding the two major high-(glycine + tyrosine) (HGT) keratins of sheep wool in separate genomic clones. The genes have negligible sequence homology except for an 18-bp conserved element immediately preceding the initiation codon. The same element has been found in the corresponding position of a number of co-expressed high-sulphur (HS) keratin genes and may therefore represent a common element between these wool intermediate-filament-associated proteins. As seen for the HS keratin genes, the HGT keratin genes also lack introns. Southern blot data show that both HGT genes are unique in the sheep genome, indicating that the observed heterogeneity of the HGT protein class may not be as complex as previously suggested.
Asunto(s)
Queratinas/genética , Ovinos/genética , Lana , Animales , Secuencia de Bases , Clonación Molecular , ADN , Genes , Glicina , Biosíntesis de Proteínas , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TirosinaRESUMEN
cDNA clones coding for the high-glycine + tyrosine (HGT) proteins of sheep wool keratin have been isolated and sequenced. Clones were identified using a 25-base synthetic oligonucleotide probe, deduced from the amino acid sequence of a HGT protein present at about 0.4% in the wool fibre. Southern and Northern blot analysis suggest that the gene is present as a single copy in the genome and is transcribed as an mRNA species, 600 bases in size.