RESUMEN
In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G --> A hypermutants derived from a Delta(vif) virus. By using an RNaseH- form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN de Cadena Simple/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Proteínas/fisiología , Desaminasa APOBEC-3G , Secuencia de Bases , Fosfatos de Dinucleósidos/metabolismo , Nucleósido Desaminasas , Proteínas/metabolismo , Proteínas Represoras , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
We report here the complete sequence of the mitochondrial (mt) genome of the pathogenic yeast Candida glabrata. This 20 kb mt genome is the smallest among sequenced hemiascomycetous yeasts. Despite its compaction, the mt genome contains the genes encoding the apocytochrome b (COB), three subunits of ATP synthetase (ATP6, 8 and 9), three subunits of cytochrome oxidase (COX1, 2 and 3), the ribosomal protein VAR1, 23 tRNAs, small and large ribosomal RNAs and the RNA subunit of RNase P. Three group I introns each with an intronic open reading frame are present in the COX1 gene. This sequence is available under accession number AJ511533.
Asunto(s)
Candida glabrata/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Proteínas de Transporte de Membrana , Mitocondrias/genética , Adenosina Trifosfatasas/genética , Secuencia de Bases , Candida glabrata/patogenicidad , Codón , Citocromos b/genética , ADN Ribosómico , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/metabolismo , Orden Génico , Código Genético , Intrones , Proteínas de la Membrana/genética , Proteínas Mitocondriales , ATPasas de Translocación de Protón Mitocondriales/genética , Datos de Secuencia Molecular , ARN de Transferencia , Ribonucleasa P/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Transcripción GenéticaAsunto(s)
ADN Polimerasa I/genética , Biblioteca de Péptidos , ADN Polimerasa Dirigida por ARN/metabolismo , Thermus/enzimología , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Inovirus/química , Inovirus/metabolismo , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , ADN Polimerasa Dirigida por ARN/genética , Moldes GenéticosRESUMEN
A novel property of DNA is described: the capacity of some bacterial DNA sequences to induce electromagnetic waves at high aqueous dilutions. It appears to be a resonance phenomenon triggered by the ambient electromagnetic background of very low frequency waves. The genomic DNA of most pathogenic bacteria contains sequences which are able to generate such signals. This opens the way to the development of highly sensitive detection system for chronic bacterial infections in human and animal diseases.
Asunto(s)
ADN Bacteriano/metabolismo , Fenómenos Electromagnéticos , Nanoestructuras/química , Animales , Infecciones Bacterianas/etiología , Sistemas de Computación , Filtración , Humanos , Modelos Biológicos , Mycoplasma/metabolismo , Agua/química , Contaminantes del Agua , Purificación del Agua/métodosRESUMEN
Caveolin-1 is a scaffolding protein that organizes and concentrates specific ligands within the caveolae membranes. We identified a conserved caveolin-1 binding motif in the HIV-1 transmembrane envelope glycoprotein gp41 and designed several synthetic peptides, referred to as CBD1, corresponding to the consensus caveolin-1 binding domain in gp41. In rabbits, these peptides elicit the production of antibodies that inhibit infection of primary CD4(+) T lymphocytes by various primary HIV-1 isolates. Interestingly, gp41 exists as a stable complex with caveolin-1 in HIV-infected cells. Anti-CBD1 peptide antibodies, therefore, might be functional by inhibiting the potential interaction of gp41 with caveolin-1. Because of their capacity to elicit antibodies that inhibit the different clades of HIV-1, CBD1-based peptides may represent a novel synthetic universal B cell epitope vaccine candidate for HIV/AIDS. Moreover, such peptides could also have an application as a therapeutic vaccine since CBD1-specific antibodies are rare in HIV-infected individuals from several geographic origins.
Asunto(s)
Vacunas contra el SIDA/inmunología , Caveolinas/metabolismo , Epítopos de Linfocito B , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caveolina 1 , Caveolinas/química , Línea Celular , Anticuerpos Anti-VIH/biosíntesis , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Datos de Secuencia Molecular , ConejosRESUMEN
Mycoplasma penetrans is a mycoplasma with unique morphology, recently identified in urine samples collected from HIV-infected patients. This mycoplasma has been found to be statistically associated with HIV infection, and to be cytopathic in vitro. The dominant antigen recognized during natural and experimental infections is an abundant lipoprotein, P35, which, upon extraction, segregates in the Triton X-114 detergent phase. It is used as the basis of M. penetrans-specific serological assays. Although mycoplasma lipoproteins, including M. penetrans P35, are the main antigens recognized by the host humoral immune response, very little is known about the nature of the epitopes involved. Immunoelectron microscopy revealed that all P35 is exposed at the cell surface and is distributed all over the membrane. P35 linear B-epitopes were mapped by an ELISA approach based on a set of overlapping peptides covering the entire mature polypeptide. The immunoreactivity of the peptides was first tested with sera from immunized animals. The dominant B-epitopes were found at the C- and N-terminal regions, in partial agreement with algorithmic predictions. Patient sera were evaluated with the same assay. Only some reacted with linear epitopes whereas others did not, indicating the importance of P35 nonsequential epitopes. Statistical analysis of the results allowed the definition of a set of peptides which were clearly immunodominant. Finally, the P35-encoding gene was modified by in vitro mutagenesis to allow the production and purification of a recombinant protein (rP35delta0) in Escherichia coil. The antigenicity of rP35delta0 was tested by Western blotting and compared to that of another recombinant product, rP35delta3, a truncated P35 polypeptide. Although rP35delta0 reacted with the M. penetrans-seropositive patient sera tested, rP35delta3 was only immunoreactive with one of six sera. This result confirmed that P35-nonsequential epitopes dominate during M. penetrans infection. Our results have important implications for the understanding of lipoprotein antigenicity during mycoplasma infections. In addition, the P35-derived immunodominant synthetic peptides defined in this study, as well as the purified rP35delta0, provide the antigenic material for the necessary improvement of M. penetrans serological assays.