Biomedical subjects

Luka A Clarke

Publications and source records attributed to Luka A Clarke.

5 recordsLinked to original sources

PubMed2005

The replication timing of CFTR and adjacent genes.

Correlations between transcriptional activity and replication timing have been observed for the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, as well as for other tissue-specific genes. In addition, the patterns of histone modifications and the nuclear positioning of chromosomal loci appear to be related to their replication timing. It is not understood why and how these different features are functionally linked. To address this problem, we investigated the replication timing of the human CFTR gene and of adjacent genes. Recently, we could show that CFTR and adjacent genes associate independently from each other with different nuclear regions and chromatin fractions, in accordance with their individual transcriptional regulation. Together, the results show that not the transcriptional activity, but rather the nuclear position of CFTR and adjacent genes appears to be a major determinant of their replication timing. Furthermore, the results imply a specific functional order of nuclear changes related to switches in replication timing.

Cell Cycle↗

PubMed2004

Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei.

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.

The scope of this article is to outline some of the basic methods for good quality RNA preparation from mammalian tissues and cells (including epithelial cells). Additionally, we give an outline of common techniques of measuring CFTR gene expression such as quantitative and semi-quantitative reverse transcription (RT) PCR and ribonuclease protection assay (RPA). These methods are designed to detect low abundance transcripts, which apply to CFTR mRNA in most cell types and tissues.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2004

Microarray analysis in cystic fibrosis.

DNA microarrays provide a versatile platform for applications including gene expression analysis and genotyping. In the case of cystic fibrosis (CF), DNA microarrays enable the measurement of gene expression levels of thousands of genes in parallel, and potentially therefore, to identify non-CFTR genes down- or up-regulated in CF, which could lead to insights into disease pathophysiology, as well as novel molecular markers and therapeutic strategies. Moreover, using optimised microarray protocols based on either primer extension analysis (i.e. minisequencing) or electronic hybridisation stringency control, the potential now exists to detect all relevant CFTR mutations on a single DNA microarray as a novel platform for CF screening.

Cystic Fibrosis↗

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↗