Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilization of nucleosomes over the entire gene.

The effects of transcriptional activation on the chromatin structure of the Saccharomyces cerevisiae HIS3 gene were addressed by mapping the precise positions of nucleosomes in uninduced and induced chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a predominant nucleosomal array. In wild-type chromatin, this array is disrupted, and several alternative overlapping nucleosomal arrays are formed. The disruption of the predominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization over the entire HIS3 gene. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring positions different from those preferred by the SWI/SNF complex. Both the SWI/SNF and Isw1 complexes are constitutively present in HIS3 chromatin, although Isw1 tends to be excluded from the HIS3 promoter. Despite the apparent disorder of HIS3 chromatin generated by the formation of multiple nucleosomal arrays, nucleosome density profiles indicate that some long-range order is always present. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure.

Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

OBJECTIVE: Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. DESIGN: Prospective and retrospective analysis of randomized clinical trial samples and reference standards. METHODS: To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. RESULTS: The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. CONCLUSION: The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia. This retesting strategy could save time, money, and anxiety for patients and their providers, as well as decrease follow-up clinic visits without increasing the risk of virologic failure and resistance development.