RESUMO
The family of repeats (FR) is a major upstream enhancer of the Epstein-Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico/fisiologia , Origem de Replicação/genética , Fatores de Transcrição/metabolismo , Linfócitos B/virologia , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica , Humanos , Fator 1 de Transcrição de Octâmero , Fator 2 de Transcrição de Octâmero , Ligação Proteica , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, is a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis. Approximately 40-65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T-cell response against this yeast is found in atopic dermatitis patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli. The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the searched database. The proteins encoded by the complete cDNA sequences were expressed in E. coli as recombinant proteins. Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M. furfur extract could specifically inhibit this IgE binding.
Assuntos
Alérgenos/genética , Antígenos de Fungos/genética , Dermatite Atópica/imunologia , Imunoglobulina E/imunologia , Malassezia/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/biossíntese , Sequência de Aminoácidos , Antígenos de Fungos/biossíntese , Antígenos de Plantas , Sequência de Bases , Criança , Pré-Escolar , Clonagem Molecular , DNA Complementar/metabolismo , Dermatite Atópica/sangue , Escherichia coli/metabolismo , Feminino , Biblioteca Gênica , Humanos , Immunoblotting , Imunoglobulina E/metabolismo , Malassezia/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
We demonstrate the potential of cloning by homologous recombination as a rapid method to construct DNA molecules encoding newly developing hemagglutinins (HA) of influenza A virus. The variable parts of the HA genes were cloned into a basic construct containing the HA gene from an H3N2 strain. The recombinant DNAs thus created encode different variable domains with neutralising epitopes from four recently circulating influenza A H3 strains. The technology allows rapid production of DNA constructs for vaccines that can induce antibody and, particularly, cellular immune responses. These new constructs were also capable of conferring protection to challenge in mice. The technology may hence be a valuable tool for rapid adaptation of influenza vaccines to changes in the circulating influenza strains.
