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PCR detection of oxidative base modifications in promoters of pulmonary artery endothelial cell (PAEC) genes differentially regulated by hypoxia
Author(s) -
Pastukh V. M.,
Ruchko M.,
Gillespie M. N.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a923-b
Subject(s) - promoter , gene , microbiology and biotechnology , dna glycosylase , biology , gene expression , dna , hypoxia (environmental) , chemistry , dna repair , biochemistry , oxygen , organic chemistry
Oxygen radicals generated during hypoxia cause oxidative base modifications in the VEGF promoter that may be linked to induction of VEGF mRNA expression (FASEB J. 19: –394, 2005). To explore this idea, we searched for oxidative modifications in functionally relevant sequences of genes that are up‐, down‐, or non‐regulated by hypoxia. PCR was used to amplify selected DNA sequences before and after treatment with formamidopyrimidine‐DNA glycosylase (Fpg), an N‐glycosylase and AP‐lyase that releases damaged bases from DNA, under the premise that if oxidation products are present, then Fpg will reduce PCR amplification efficiency. Hypoxic, but not normoxic, PAECs displayed Fpg‐induced reductions in PCR amplification of the hypoxic response elements of the VEGF and HO‐1 promoters but not in functionally insignificant promoter sequences or in intronic or exonic coding sequences. Fpg‐detectable modifications were related in time to changes in VEGF and HO‐1 mRNA expression. Neither promoter nor coding sequences of the hypoxia down‐regulated ODC gene displayed Fpg sensitive modifications. Similarly, neither promoter nor coding sequences of the constitutively active actin gene were modified in hypoxia. These observations suggest that hypoxia‐induced oxidative base modifications are targeted to functionally significant sequences of hypoxia‐inducible genes.