Linked Life Data

18797917

Source:http://linkedlifedata.com/resource/pubmed/id/18797917

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2008-10-13
pubmed:abstractText
For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. Although previous studies described methylation of isolated DNA extracted from cells and tissues using a combination of appropriate restriction endonucleases, no application to tissue cell level has been reported. Here, we report a new method, named histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequences in a tissue section. In this study, we examined changes in the methylation level of CCGG sites during spermatogenesis in paraffin-embedded sections of mouse testis. In principle, the 3'-OH ends of DNA strand breaks in a section were firstly labeled with a mixture of dideoxynucleotides by terminal deoxynucleotidyl transferase (TdT), not to be further elongated by TdT. Then the section was digested with Hpa II, resulting in cutting the center portion of non-methylated CCGG. The cutting sites were labeled with biotin-16-dUTP by TdT. Next, the section was treated with Msp I, which can cut the CCGG sequence irrespective of the presence or absence of methylation of the second cytosine, and the cutting sites were labeled with digoxigenin-11-dUTP by TdT. Finally, both biotin and digoxigenin were visualized by enzyme- or fluorescence-immunohistochemistry. Using this method, we found hypermethylation of CCGG sites in most of the germ cells although non-methylated CCGG were colocalized in elongated spermatids. Interestingly, some TUNEL-positive germ cells, which are frequent in mammalian spermatogenesis, became markedly Hpa II-reactive, indicating that the CCGG sites may be demethylated during apoptosis.
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0948-6143
pubmed:author
pubmed:issnType
Print
pubmed:volume
130
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
917-25
pubmed:dateRevised
2009-4-23
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
In situ detection of methylated DNA by histo endonuclease-linked detection of methylated DNA sites: a new principle of analysis of DNA methylation.
pubmed:affiliation
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan. tkoji@nagasaki-u.ac.jp
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't