RESUMO
A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design.
Assuntos
DNA Nucleotidiltransferases/genética , DNA Fúngico/genética , Sítios de Ligação , DNA Nucleotidiltransferases/química , DNA Fúngico/química , Modelos Moleculares , Mutação Puntual , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
The Vibrio cholerae (Vc) gene (tagA) coding for the TagA lipoprotein has been isolated. Sequencing of tagA revealed the presence of an open reading frame (ORF) of 568 amino acids with a characteristic signal peptidase II cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli (Ec) cells following T7 promoter/RNA polymerase-directed expression of tagA, revealed a closely migrating doublet of proteins corresponding to two species of TagA. Computer-generated alignment algorithms predict that a homology exists between Vc TagA and Ec K99 fimbriae biogenesis determinant FanD.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Genes Bacterianos , Lipoproteínas/genética , Vibrio cholerae/genética , Algoritmos , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Bacteriófago T7/genética , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/fisiologiaRESUMO
Intravenous vitamin E was associated with the deaths of 38 infants in the US in 1984. Because the vitamin E preparation used contained both vitamin E and a high level of polysorbate detergent, the etiology of the syndrome remains unknown. In this study, we determined the tissue disposition of an intravenous preparation of vitamin E solubilized with polysorbate (E-Ferol) in neonatal piglets. One to two-day-old piglets were injected daily with 50 IU/kg/d of vitamin E for a period of 13 days. Other groups were injected intramuscularly, or with a slow, 7 h intravenous infusion with 50 IU/kg/d vitamin E for six days. Massive splenic accumulation of vitamin E (16,004 micrograms/g vs 73 micrograms/g in controls) occurred following rapid injection, with far lesser concentrations in the liver and lung. Levels of vitamin E in the kidney and heart were only slightly above control. Tissue changes correlated with dosage and duration of vitamin E administration and suggested massive accumulation of vitamin E in cells of the mononuclear phagocyte system. Following slow intravenous infusion the highest levels of vitamin E occurred in the liver rather than spleen. Intramuscular injections at similar doses produced slight, but insignificant changes in tissue levels of vitamin E. We speculate that rapid intravenous injection of vitamin E emulsions produces massive accumulation in phagocytic cells of the spleen and to a lesser extent liver and lung, possibly leading to increased susceptibility to sepsis and/or abnormal pulmonary function. Slow infusions of vitamin E produce major accumulations in the liver rather than spleen.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Animais Recém-Nascidos/fisiologia , Vitamina E/análogos & derivados , alfa-Tocoferol/análogos & derivados , Animais , Animais Recém-Nascidos/metabolismo , Infusões Intravenosas , Injeções Intravenosas , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Baço/efeitos dos fármacos , Baço/patologia , Suínos , Tocoferóis , Vitamina E/administração & dosagem , Vitamina E/farmacocinética , Vitamina E/toxicidadeRESUMO
The toxin-coregulated pilus (TCP)-encoding gene cluster (tcp) specifies a type-IV pilus that is a major colonization determinant of Vibrio cholerae. We have identified a gene 200 bp upstream from the tcp cluster that requires ToxR for expression. We have designated this gene tagD (ToxR-activated gene) and have shown that tagD is encoded on a 600-nt transcript. The deduced tagD product is a 164-amino-acid polypeptide (20 kDa). Interestingly, TagD shares a high degree of similarity to a protein of Streptococcus sanguis 12 that is thought to play a role in fimbriae synthesis or assembly. The high degree of similarity between tagD and the Ss 12 protein provides preliminary evidence that tagD represents an additional member of the tcp cluster.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/fisiologia , Genes Bacterianos/genética , Peroxidases , Fatores de Transcrição/fisiologia , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Sequência de Bases , Escherichia coli/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Família Multigênica/genética , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Regulon/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Virulence gene activation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade. The ToxR regulon consists of genes required for toxin-coregulated-pilus (TCP) biogenesis, accessory colonization factor genes, cholera toxin genes, and ToxR-activated genes (tag) of unknown function. The tagB gene was isolated by using a tagB::TnphoA fusion junction to probe a V. cholerae )395 bacteriophage lambda library. Nucleotide sequence analysis revealed that tagB is identical to tcpI, a gene which encodes a protein that negatively regulates the synthesis of the major pilin subunit of TCP (TcpA). Our results show that the tcpI gene encodes a 620-amino-acid protein that shares extensive sequence similarity with the highly conserved signaling domain in methyl-accepting chemotaxis proteins. Expression of tcpI in Escherichia coli results in the synthesis of a 71-kDa polypeptide that becomes localized to the inner membrane. Similarly, TcpI-PhoA alkaline phosphatase activity is enriched in V. cholerae inner membrane preparations. Colonies of V. cholerae tcpI::TnphoA mutant cells display increased swarming on solid media when compared with those of the parental V. cholerae O395. Taken together, these observations suggest that TcpI may play a dual role in promoting vibrio colonization of the small bowel. In response to the appropriate environmental signal(s), TcpI permits maximum expression of tcpA while simultaneously reducing vibrio chemotaxis-directed motility. We believe coordinate regulation of colonization and motility determinants, in such a fashion, facilitates efficient V. cholerae microcolony formation.
