Quantitative methods for the analysis of CFTR transcripts/splicing variants

AbstractIn cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here

A tomato strigolactone-impaired mutant displays aberrant shoot morphology and plant interactions

Strigolactones are considered a new group of plant hormones. Their role as modulators of plant growth and signalling molecules for plant interactions first became evident in Arabidopsis, pea, and rice mutants that were flawed in strigolactone production, release, or perception. The first evidence in tomato (Solanum lycopersicon) of strigolactone deficiency is presented here. Sl-ORT1, previously identified as resistant to the parasitic plant Orobanche, had lower levels of arbuscular mycorrhizal fungus (Glomus intraradices) colonization, possibly as a result of its reduced ability to induce mycorrhizal hyphal branching. Biochemical analysis of mutant root extracts suggested that it produces only minute amounts of two of the tomato strigolactones: solanacol and didehydro-orobanchol. Accordingly, the transcription level of a key enzyme (CCD7) putatively involved in strigolactone synthesis in tomato was reduced in Sl-ORT1 compared with the wild type (WT). Sl-ORT1 shoots exhibited increased lateral shoot branching, whereas exogenous application of the synthetic strigolactone GR24 to the mutant restored the WT phenotype by reducing the number of lateral branches. Reduced lateral shoot branching was also evident in grafted plants which included a WT interstock, which was grafted between the mutant rootstock and the scion. In roots of these grafted plants, the CCD7 transcription level was not significantly induced, nor was mycorrhizal sensitivity restored. Hence, WT-interstock grafting, which restores mutant shoot morphology to WT, does not restore mutant root properties to WT. Characterization of the first tomato strigolactone-deficient mutant supports the putative general role of strigolactones as messengers of suppression of lateral shoot branching in a diversity of plant species

Non-PCR methods for the analysis of CFTR transcripts

Cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex mechanism of tissue-specific and temporal regulation. CFTR mRNA detection and measurement are extremely difficult because of the low to very low levels of its endogenous expression. In this paper, we describe four different non-PCR methods optimized to analyze CFTR transcripts in epithelial cell lines, primary cell lines and native tissues that express significant amounts of CFTR transcript

HNF1alpha is involved in tissue-specific regulation of CFTR gene expression.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout.

International audienceIn most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells. We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE(cs)) by three base pairs. This non-palindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor alpha (hERalpha) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERalpha-ERE(cs) and hERalpha-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo. In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes

From Alpha to Omicron BA.2: New digital RT-PCR approach and challenges for SARS-CoV-2 VOC monitoring and normalization of variant dynamics in wastewater

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