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Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

ABSTRACT: In mammalian genomes a sixth base, 5-hydroxymethylcytosine ((hm)C), is generated by enzymatic oxidation of 5-methylcytosine ((m)C). This discovery has raised fundamental questions about the functional relevance of (hm)C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare (hm)C against the far more abundant (m)C is technically challenging and to date no methods for direct sequencing of (hm)C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves (hm)C-containing sequences. We determined the consensus cleavage site of PvuRts1I as (hm)CN(11-12)/N(9-10)G and show first data on its potential to interrogate (hm)C patterns in mammalian genomes.

SUBMITTER: Szwagierczak A

PROVIDER: S-EPMC3130283 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

Szwagierczak Aleksandra A Brachmann Andreas A Schmidt Christine S CS Bultmann Sebastian S Leonhardt Heinrich H Spada Fabio F

Nucleic acids research 20110304 12

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ((hm)C), is generated by enzymatic oxidation of 5-methylcytosine ((m)C). This discovery has raised fundamental questions about the functional relevance of (hm)C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare (hm)C against the far more abundant (m)C is technically challenging and to date no methods for direct sequencing of (hm)C have been reported. Here, we report on a purified recombinant e ...[more]

PMID: 21378122

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Project description:BackgroundDystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.ResultsWe generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.ConclusionsThe novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.

Dataset Information

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

Publications

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

Similar Datasets

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