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1.
Nucleic Acids Res ; 42(6): 3551-64, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24413563

RESUMO

CpG dinucleotides are known to play a crucial role in regulatory domains, affecting gene expression in their natural context. Here, we demonstrate that intragenic CpG frequency and distribution impacts transgene and genomic gene expression levels in mammalian cells. As shown for the Macrophage Inflammatory Protein 1α, de novo RNA synthesis correlates with the number of CpG dinucleotides, whereas RNA splicing, stability, nuclear export and translation are not affected by the sequence modification. Differences in chromatin accessibility in vivo and altered nucleosome positioning in vitro suggest that increased CpG levels destabilize the chromatin structure. Moreover, enriched CpG levels correlate with increased RNA polymerase II elongation rates in vivo. Interestingly, elevated CpG levels particularly at the 5' end of the gene promote efficient transcription. We show that this is a genome-wide feature of highly expressed genes, by identifying a domain of ∼700 bp with high CpG content downstream of the transcription start site, correlating with high levels of transcription. We suggest that these 5' CpG domains are required to distort the chromatin structure and to increase gene activity.


Assuntos
Ilhas de CpG , Elementos Reguladores de Transcrição , Sítio de Iniciação de Transcrição , Transcrição Gênica , Animais , Células CHO , Linhagem Celular , Quimiocina CCL3/genética , Cromatina/química , Cricetinae , Cricetulus , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Elongação da Transcrição Genética , Transgenes
2.
Proc Natl Acad Sci U S A ; 110(30): 12402-7, 2013 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-23836650

RESUMO

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.


Assuntos
Linfócitos B/metabolismo , Sinalização do Cálcio , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Linfócitos B/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica
3.
Immunobiology ; 213(6): 511-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18514753

RESUMO

Little is known about the VP1unique region (VP1u), a part of one major capsid protein of human parvovirus B19 (B19), concerning its involvement in viral replication and infection cycle. Showing a phospholipase A2 (PLA2)-like activity, which is discussed to be necessary for viral release from host cell, its precise function remains unclear. The purpose of this study was to generate multifunctional monoclonal antibodies (mabs) for different applications that may be useful in investigating VP1u's relevance. To establish antiVP1u antibodies, spleen cells from Balb/c mice immunized with purified recombinant viral protein were used for generating antibody-producing hybridoma cell lines. Usability of the antibodies was tested in enzyme-linked immunosorbent assay (ELISA), Western-blot analysis, immunofluorescence and an inhibition assay of enzymatic activity of PLA2. Three hybridoma cell lines secreting mab's specifically directed against the VP1u protein of B19 could be generated and functioned in every screening method used in this study. These antibodies are helpful tools for investigations in B19 research and diagnosis. Furthermore, the antibodies could help in gaining a deeper understanding of VP1u's role in viral replication and infection especially in the importance of its constitutive PLA2-like activity.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Parvovirus B19 Humano/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Proteínas do Capsídeo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Fosfolipase A2 , Fosfolipases A2/metabolismo , Proteínas Recombinantes/imunologia
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