RESUMO
We report the draft genome sequence of Porphyromonas gingivalis strain 381 Okayama (381OKJP). The strain, obtained from the Socransky collection, has been used for experimentation since 1987. This sequence allows for comparisons to other sequenced 381 strains to observe acquisition of mutations and genome rearrangements in a commonly used laboratory strain.
RESUMO
Human immunodeficiency virus type 1 (HIV-1) replication is suppressed by a small guide RNA (sgRNA) that targets the packaging signal of HIV-1 RNA. We unintentionally produced a plasmid with the reverse sequence of the sgRNA and its terminator (pR-Ψ-sgRNA-ter). Both sgRNA and R-Ψ-sgRNA suppress HIV-1, but the mechanism by which R-Ψ-sgRNA suppresses HIV is not clear. To evaluate whether the suppressive effect is caused by an RNA interference or microRNA (miRNA)-like mechanism, R-Ψ-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important enzyme for RNA interference and miRNA. The RNA was cleaved into fragments of approximately 24 nucleotides (nt). We analyzed the sequence of the RNA fragments and predicted the RNA secondary structure of R-Ψ-sgRNA to determine the region recognized by the Dicer enzyme. The lengths of the R-Ψ-sgRNA fragments ranged from 48 to 140 nt, and were predicted to form double strands, including mismatches, in this region. An HIV-1 p24 assay indicated that the R-Ψ-sgRNA fragments suppressed HIV-1 replication. These findings suggest that R-Ψ-sgRNA acts as a miRNA to inhibit HIV-1.
Assuntos
HIV-1/fisiologia , MicroRNAs/metabolismo , Replicação Viral , Algoritmos , Sequência de Aminoácidos , Células HeLa , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Interferência de RNA , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Pequeno RNA não TraduzidoRESUMO
In this study, we investigated an RNA (R-Psi-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Psi-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Psi-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed.
Assuntos
HIV-1/fisiologia , MicroRNAs/metabolismo , Interferência de RNA , Replicação Viral , Vetores Genéticos , Proteína do Núcleo p24 do HIV/análise , Células HeLa , Humanos , Ribonuclease III/metabolismo , Pequeno RNA não TraduzidoRESUMO
In this study, we investigated an RNA (R-Psi-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Psi-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Psi-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed. We are now analyzing the sequences of the RNA fragments.
Assuntos
Fármacos Anti-HIV/química , HIV-1/fisiologia , Interferência de RNA , Replicação Viral , Fármacos Anti-HIV/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Ribonuclease III/metabolismo , Pequeno RNA não TraduzidoRESUMO
Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species. In this paper the effects of growth rate and bicarbonate on the leukotoxin production by a toxin-production-variable strain (301-b) during growth in a chemostat were examined. When the bacterium was grown in anaerobic fructose-limited chemostat cultures (pH 7.0 and 37 degrees C) at dilution rates (D) ranging from 0.04 to 0.20 h-1 in the absence and presence of 10 mM bicarbonate, it produced leukotoxin as a cluster of two polypeptides (M(r) 113,000 and 120,000) and complexed with nucleic acids on the bacterial cell surface. The relationship between leukotoxin production and specific growth rate was analysed by plotting the specific rate of leukotoxin production [qLT, in microgram (mg dry wt)-1 h-1] against D. The plots were approximated to the linear relationships qLT = 2.7D-0.058 and qLT = 9.3D-0.407 without and with bicarbonate, respectively. These relationships suggest that the apparent leukotoxin production is a result of both growth-rate-dependent production and growth-rate-independent decomposition. The cellular leukotoxin level was also followed after the change from chemostat to batch culture in the same fermenter. In batch culture leukotoxin production stopped immediately and the cellular toxin level rapidly decreased, suggesting toxin decomposition. From the slopes of the approximated linear relationships between qLT and D, a theoretical maximum leukotoxin yield (YLT) was estimated as 2.7 and 9.3 micrograms (mg dry wt)-1 in the absence and presence of 10 mM bicarbonate, respectively. The increased YLT value in the cultures containing bicarbonate indicated that the addition stimulated the efficiency of leukotoxin synthesis up to about threefold. Further increases of bicarbonate concentration to between 20 and 40 mM had no effect on the total leukotoxin production, but the amount of extracellular leukotoxin increased with higher bicarbonate concentrations.
