Nucleic Acids Research, Vol 27, Issue 15 3009-3017, Copyright © 1999 by Oxford University Press
L Phi-van and WH Stratling
We previously identified a broad initiation zone of DNA replication at the
chicken lysozyme gene locus. However, the existence of a highly preferred
origin of bidirectional replication (OBR), often found in initiation zones,
remained elusive. In order to re-examine this issue we used a competitive
PCR assay to determine the abundance of closely spaced genomic segments in
a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand
abundance occurred at the 3" end of the gene, where initiation events were
17 times more frequent than upstream of the gene. This primary initiation
site, active in lysozyme expressing myelomonocytic HD11 cells and
non-expressing hepatic DU249 cells, was found to reside within an unusually
located CpG island. While most CpG islands are found at the 5" end of
genes, the lysozyme gene island extends from the 3" end of the second
intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed
by methylation-sensitive restriction enzymes, the island is largely
non-methylated in HD11 cells, DU249 cells and inactive chicken
erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is
composed of two subsites separated by approximately 100 bp, was localised
very close to the segment with the highest initiation activity. Our results
suggest that the non-methylated CpG island and the HS provide an accessible
chromatin structure for the lysozyme gene origin of replication.
ARTICLES
An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene
Institut fur Tierzucht und Tierverhalten, Bundesforschungsanstalt fur Landwirtschaft, Dornbergstrasse 25-27, 29223 Celle, Germany. loc.phi- van@fal.de
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