RESUMO
The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.
Assuntos
Bacteriófago M13/genética , Inovirus/genética , Oligonucleotídeos/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular/métodos , Primers do DNA/genética , Primers do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Ligantes , Dados de Sequência Molecular , Oligonucleotídeos/genética , Concentração Osmolar , Iniciação Traducional da Cadeia Peptídica/genética , Ligação Proteica/genética , Sais , Análise de Sequência de DNA/métodos , Deleção de Sequência , Cloreto de Sódio , Proteínas Virais/genéticaRESUMO
Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments showed that it had strong anti-inflammatory effects. Tumour necrosis factor (TNF) is an important inflammatory mediator. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNF production from macrophages was altered by EsA under lipopolysaccharide (LPS) stimulated conditions. EsA was found to decrease both extracellular and cell associated TNF production in a dose dependent manner at concentrations higher than 1 mumol/l EsA. Previous studies have showed that EsA reduced the releasing of platelet activating factor (PAF) from rat macrophages. The reducing effects of EsA on the release of TNF and PAF may explain its anti-inflammatory effect.