RESUMO
Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human ß-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Bacteroides fragilis/metabolismo , Inversão de Sequência/genética , Antibacterianos/farmacologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/fisiologia , Membrana Celular/metabolismo , Farmacorresistência Bacteriana , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteômica , Inversão de Sequência/fisiologiaRESUMO
In the organelles of plants and mammals, recent evidence suggests that genomic instability stems in large part from template switching events taking place during DNA replication. Although more than one mechanism may be responsible for this, some similarities exist between the different proposed models. These can be separated into two main categories, depending on whether they involve a single-strand-switching or a reciprocal-strand-switching event. Single-strand-switching events lead to intermediates containing Y junctions, whereas reciprocal-strand-switching creates Holliday junctions. Common features in all the described models include replication stress, fork stalling and the presence of inverted repeats, but no single element appears to be required in all cases. We review the field, and examine the ideas that several mechanisms may take place in any given genome, and that the presence of palindromes or inverted repeats in certain regions may favor specific rearrangements.
Assuntos
Replicação do DNA/fisiologia , Instabilidade Genômica/genética , Organelas/genética , Inversão de Sequência/fisiologia , Animais , Humanos , Recombinação Genética , Moldes GenéticosAssuntos
Proteínas de Ciclo Celular/genética , Fusão Gênica , Janus Quinase 2/genética , Proteína de Leucina Linfoide-Mieloide/genética , Inversão de Sequência , Trombocitemia Essencial/genética , Idoso de 80 Anos ou mais , Substituição de Aminoácidos/fisiologia , Sequência de Bases , Crise Blástica/patologia , Fusão Gênica/fisiologia , Histona-Lisina N-Metiltransferase , Humanos , Masculino , Proteínas Nucleares , Fenilalanina/genética , Polimorfismo de Nucleotídeo Único/fisiologia , Proteínas de Ligação a RNA , Inversão de Sequência/fisiologia , Trombocitemia Essencial/patologia , Valina/genéticaRESUMO
Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3'-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.
Assuntos
Linfo-Histiocitose Hemofagocítica/genética , Proteínas de Membrana/genética , Células Cultivadas , Pré-Escolar , Croácia , Análise Mutacional de DNA , Dinamarca , Feminino , Finlândia , Humanos , Lactente , Recém-Nascido , Íntrons/genética , Linfo-Histiocitose Hemofagocítica/classificação , Masculino , Mutação/fisiologia , Inversão de Sequência/fisiologia , Suécia , UcrâniaRESUMO
Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.