Simple amplification-based assay: a nucleic acid-based point-of-care platform for HIV-1 testing.

BACKGROUND: A new nucleic acid-based assay (simple amplification-based assay [SAMBA]) for rapid visual detection of human immunodeficiency virus-type 1 (HIV-1) by dipstick is described. The assay was designed to be simple, stable, robust, self-contained, and capable of detecting a broad spectrum of HIV-1 subtypes and recombinant forms. METHODS: The performance of the SAMBA HIV-1 test (amplification and detection chemistry) was evaluated using the World Health Organization HIV-1 RNA Genotype Reference Panel, with clinical samples representing various viral subtypes and recombinant forms common in sub-Saharan Africa. Sixty-nine randomly selected and blinded clinical samples that had undergone HIV-1 genotypic resistance analyses in a large London teaching hospital were also tested. These samples included 14 different viral subtypes or recombinant forms with viral loads of 78-9.5 x 10(6) copies/mL. RESULTS: The sensitivity and viral subtype coverage of the SAMBA HIV-1 test were either comparable to or better than those of the commercially available nucleic acid-based HIV-1 diagnostic tests. CONCLUSIONS: The unique characteristics and competitive performance of the SAMBA HIV-1 test render it suitable for point-of-care and near-patient testing in both developed and developing countries.

The bromodomain protein GTE6 controls leaf development in Arabidopsis by histone acetylation at ASYMMETRIC LEAVES1.

The transition from the juvenile to the mature phase during vegetative development in plants is characterized by changes in leaf shape. We show that GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6) regulates differences in leaf patterning between juvenile and mature leaves in Arabidopsis. GTE6 encodes a novel small bromodomain-containing protein unique to plants. Mutations in GTE6 disrupt the formation of elliptical leaf laminae in mature leaves, whereas overexpression of GTE6 resulted in elongated juvenile leaves. GTE6 positively regulates the expression of ASYMMETRIC LEAVES1 (AS1), which encodes a myb-domain protein that controls proximodistal patterning of leaves. Using chromatin immunoprecipitation (ChIP) assays, we show that GTE6 is associated with the promoter and the start of the transcribed region of AS1 and up-regulates expression of AS1 through acetylation of histones H3 and H4. Genetic studies demonstrated that AS1 is epistatic to GTE6, indicating that GTE6 regulates AS1 during leaf morphogenesis. Chromatin remodeling at AS1 is a key regulatory mechanism in leaf development, which ensures the continual production of mature leaves following juvenile-adult transition, thereby maintaining the identity of the mature vegetative phase.

The transcriptional enhancer of the pea plastocyanin gene associates with the nuclear matrix and regulates gene expression through histone acetylation.

The influence of the transcriptional enhancer of the pea plastocyanin gene (PetE) on the acetylation of histones was examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognize acetylated or nonacetylated histones H3 and H4. In transgenic tobacco plants containing the pea PetE promoter fused to uidA, both acetylated and nonacetylated histones H3 and H4 were present on the integrated transgene. Linking the PetE enhancer to the transgene resulted in increased beta-glucuronidase activity and increased amounts of acetylated histones H3 and H4 present on the promoter, suggesting that the enhancer may increase transcription by mediating the acetylation of histones. Trichostatin A and sodium butyrate, which are potent inhibitors of histone deacetylases (HDAs), activated expression from the PetE promoter by fourfold, with a concomitant increase in the acetylation states of histones H3 and H4, as determined by ChIP, indicating that the acetylation of histones has a direct positive effect on transcription. The HDA inhibitors did not increase expression from the PetE promoter when it was linked to the enhancer, consistent with preexisting hyperacetylated histones on the transgene. Mapping of histone acetylation states along the reporter gene indicated that the histones H3 and H4 associated with the promoter and the 5' region of uidA were hyperacetylated in the presence of the PetE enhancer. The PetE enhancer bound to isolated tobacco nuclear matrices in vitro and was associated with the nuclear matrix in nuclei isolated from transgenic tobacco plants. These results suggest that the pea PetE enhancer activates transcription by associating with the nuclear matrix, mediating the acetylation of histones on the promoter and the nearby coding region and resulting in an altered chromatin structure.

Microarray analysis of chromatin-immunoprecipitated DNA identifies specific regions of tobacco genes associated with acetylated histones.

The acetylation states of histones present on the upstream, promoter, coding or intronic regions of 88 tobacco genes were examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognised acetylated histone H4. The DNA sequences enriched in the immunoprecipitates were amplified by ligation-mediated PCR, labelled with Cy-dUTP and hybridised to DNA microarrays. In green tobacco shoots, histone H4 acetylation was localised to 300-600-bp sequences in the promoters or coding regions of 31 genes, or occurred extensively over several kilobase-pair regions containing the upstream, promoter and/or coding regions of 25 genes. Genes associated with high histone H4 acetylation levels at promoters were actively expressed, whereas genes depleted in acetylated histone H4 were non-transcribed or expressed at very low levels, suggesting a correlation between histone H4 acetylation and gene activity. Trichostatin A (TA), an inhibitor of histone deacetylases (HDAs), did not alter histone H4 acetylation states globally but increased acetylation levels at specific tobacco sequences, suggesting that HDAs are targeted to particular nucleosomes. Genes that were upregulated by TA were associated with increased histone H4 acetylation at promoter or coding regions, indicating that acetylation of histones on coding regions may activate transcription. Increased histone H4 acetylation leading to elevated expression was observed on genes with diverse functions, suggesting that histone H4 acetylation is involved in regulation of many plant processes.