Biomedical subjects

Rebecca Rowntree

Publications and source records attributed to Rebecca Rowntree.

3 recordsLinked to original sources

PubMed2004

Quantitative methods for the analysis of CFTR transcripts/splicing variants.

In 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.

Cystic Fibrosis↗

PubMed2002

DNA polymorphisms in potential regulatory elements of the CFTR gene alter transcription factor binding.

Over one thousand mutations have been identified to date in the cystic fibrosis transmembrane conductance regulator gene ( CFTR); however, about 5-10% of mutations remain undefined. It is likely that some of these undefined mutations occur within regulatory elements for the CFTR gene. Tissue-specific regulatory elements for CFTR are located outside the basal promoter region and may be associated with DNase I hypersensitive sites (DHS). We previously described a DHS at +15.6 kb 3' to the CFTR gene, which showed tissue specificity in vivo and was evaluated as a candidate regulatory element for CFTR. Polymorphisms in regulatory elements may have a significant effect on their activity and hence on the levels of gene expression. Two C-->T polymorphisms were identified within the +15.6 kb region that occurred on both cystic fibrosis (CF) and non-CF alleles. Both of the polymorphisms altered DNA-protein binding, as shown by electrophoretic mobility shift assays (EMSA). These changes in transcription factor binding at a putative regulatory region could influence CFTR gene expression.

Base Sequence↗

PubMed2002

Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhan- cer/reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell specificity of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in introns 10 and 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of regulation of expression of the CFTR gene in which multiple elements contribute to tightly co-ordinated expression in vivo.

Cystic Fibrosis Transmembrane Conductance Regulato↗