RESUMEN
Early detection of human immunodeficiency virus (HIV) in blood and blood products can be achieved by a sensitive nucleic acid amplification-based assay. We report on the performance of a PCR-based qualitative assay that detects both HIV type 1 (HIV-1) and HIV-2 with a sensitivity of 20 to 50 copies/ml. The assay has a specificity of 99.6% and an inhibition rate of 1.7%. One milliliter of sample is processed with a manifold system and Qiagen columns, and one-third of the extracted sample is used for PCR amplification. An internal control sequence, which is processed and amplified with each sample, monitors for amplification inhibition. Samples are reverse transcribed and are then amplified by reverse transcription-coupled PCR, after which HIV-1- and HIV-2-specific probes are hybridized to the amplified products. Following hybridization, samples are detected in the LCx instrument by microparticle enzyme immunoassay techniques. The detection system has an automated inactivation step that controls for PCR contamination. The HIV-1/2 qualitative RNA assay detects HIV-1 group M subtypes A, B, C, D, E, F, and G and group O. Testing of several HIV-1 seroconversion panels has demonstrated that the HIV-1/2 qualitative RNA assay detects HIV infection on the average of 6 days before p24 antigen can be detected and 11 days before antibodies can be detected.
Asunto(s)
Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Anticuerpos Anti-VIH/sangre , Antígenos VIH/análisis , Antígenos VIH/inmunología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , VIH-2/clasificación , VIH-2/genética , Humanos , Juego de Reactivos para Diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
The MHC-linked hsp70 locus consists of duplicated genes, hsp70.1 and hsp70.3, which in primary mouse embryo cells are highly heat shock-inducible. Several mouse cell lines in which hsp70 expression is not activated by heat shock have been described previously, but the basis for the deficiency has not been identified. In this study, genomic footprinting analysis has identified a common basis for the deficient response of the hsp70.1 gene to heat shock in four such cell lines, viz., the promoter is inaccessible to transcription factors, including heat shock transcription factor. Southern blot analyses reveal extensive CpG methylation of a 1.2-kilobase region spanning the hsp70.1 transcription start site and hypermethylation of the adjacent hsp70.3 gene, which is presumably also inaccessible to regulatory factors. Of four additional, randomly chosen mouse cell lines, three show no or minimal hsp70.3 heat shock responsiveness and CpG methylation of both hsp70 genes, and two of the three lines exhibit a suboptimal hsp70.1 response to heat shock as well. In all three lines, the accessibility of the hsp70.1 promoter to transcription factors is detectable but clearly diminished (relative to that in primary mouse cells). Our results suggest that the tandem hsp70 genes are concomitantly methylated and transcriptionally repressed with high frequency in cultured mouse cells.
Asunto(s)
Ligamiento Genético , Proteínas HSP70 de Choque Térmico/genética , Complejo Mayor de Histocompatibilidad , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Clonación Molecular , Islas de CpG , ADN/química , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Calor , Metilación , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.