A viral double‐stranded RNA associated with virulence attenuation (hypovirulence) in the chestnut blight fungus (Cryphonectria parasitica) has been shown by DNA‐mediated transformation to be responsible for transmissible hypovirulence. In addition to reduced virulence, the fungal strain harboring this virus exhibits a diverse array of characteristics, termed hypovirulence‐associated traits, which distinguish it from an isogenic virus‐free strain. We have investigated one of these traits, suppressed lac‐1 (laccase) transcript accumulation. Two different and opposing regulatory pathways appear to govern lac‐1 transcript levels in the virus‐free strain: a stimulatory pathway was found to be dependent on the inositol trisphosphate (IP3) and calcium second messenger systems. A second pathway limiting transcript accumulation was shown to require ongoing protein synthesis. Additionally, changes in the level of lac‐1 transcript accumulation were found to be related to modulation of promoter activity and this activity was shown to be suppressed in the virus‐containing strain. We conclude that this hypovirulence‐associated virus interferes with transduction of an IP3‐calcium‐dependent signal that is required for stimulation of lac‐1 transcription. The perturbation of such signal transduction pathways by hypovirulence‐associated viruses may account for the manifold symptoms associated with transmissible hypovirulence.
The gene lac‐1, encoding the enzyme laccase, is one of several genes of the chestnut blight fungus, Cryphonectria parasitica, that are suppressed by virulence‐attenuating mycoviruses of the hypovirus group. Two antagonistic regulatory pathways have been shown to govern the activity of the lac‐1 promoter: a positive pathway that stimulates transcription and a negative pathway that represses transcription. We now report that these two regulatory pathways respond independently to specific changes in the nutritional environment. These newly defined conditions were used to confirm that a hypovirus suppresses the activity of the positive regulatory pathway, and to implicate calmodulin and calcineurin as components of the signal transduction cascades regulating lac‐1 transcription. Significantly, lac‐1 transcript accumulation was shown to be affected by amino acid availability. Further analysis revealed that transcriptional repression mediated by the negative regulatory pathway is relieved under conditions of amino acid deprivation. Thus, by blocking the positive pathway, hypovirus infection prevents increased lac‐1 transcript accumulation in response to amino acid deficiency. These observations are consistent with the hypothesis that hypoviruses alter the transcriptional response of the host fungus to changes in nutrient availability.
2 Dozens of recurrent chromosomal rearrangements have been described in acute leukaemia, where they often result in the fusion of two genes and in the expression of hybrid proteins.These genetic markers often delineate distinct clinical entities, and have been incorporated into the World Health Organisation classification. Today, their evaluation at diagnosis thus provides crucial information for risk stratification, treatment decisions and residual disease evaluation 1,2 .Conventional cytogenetics is usually regarded as the gold standard method for the detection of these rearrangements. It is often considered mandatory at diagnosis but it requires a high level of expertise, is time consuming, and is not suitable for the detection of many cryptic rearrangements 3,4 . Complementary approaches such as fluorescent in situ hybridisation (FISH) and RT-PCR are thus often needed 5 , but the cost of a comprehensive analysis can rapidly become prohibitive. Many markers that may provide important clinical information are therefore never tested, lowering the chances for the patients to benefit from the optimal treatments. To address this issue, we have developed a rapid and parsimonious ligationdependant RT-PCR amplification assay (LD-RTPCR) 6 that allows for the detection of dozens of gene rearrangements (Figure 1).We created a mix of 153 LD-RTPCR probes to target more than 50 recurrent fusion genes and three NPM1 mutations ( Figure 1B and C). The procedure is detailed in supplemental methods, and representative results are provided in supplemental figure 1. As shown in supplemental figure 2A, the results inform on the identity of the two partner genes, but also on the localisation of the breakpoint, guiding the choice of an appropriate assay for residual disease evaluation. The analysis of serial dilutions showed that approximately 3.10 3 copies of transcripts are sufficient for the identification of the two partners (Supplemental figure 2B).To validate this method, we applied it to a retrospective cohort of 540 patients treated in our institutions ( Figure 2 and supplemental table 2). Sixty eight rearrangements were detected in the 180 childhood ALL cases we tested. Fifty two (76.5%) had been identified using RT-PCR at diagnosis and 20 (29.4%) using cytogenetics. Sixty seven of these rearrangements (98.5%) were detected using LD-RTPCR. As expected in this cohort, the most common were the ETV6-RUNX1 fusion (33 cases), rearrangements of the MLL gene (8 cases, four with AFF1, two with MLLT1, one with AFF4 and one with MLLT3) 7 , and the BCR-ABL1 (six cases), TCF3-PBX1 (five cases) and STIL-TAL1 fusions (four cases) 2 . The LD-RTPCR assay only failed to detect one t(4;11)(q21;q23), which was identified at diagnosis using cytogenetics but which could not be confirmed using RT-PCR. The breakpoint of this rearrangement may thus be unusual, or involve genes other than MLL and AFF1. It also validated four junctions which © 2015 Macmillan Publishers Limited. All rights reserved.3 were detected using cytogenetics, but which had not...
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