DNase-Sensitive Chromatin Structure Near a Chromosomal Origin of Bidirectional Replication of the Avian α-Globin Locus

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Abstract
We used an in vitro nuclear run-off replication assay to analyze the direction of replication in a 13-kb region 5′ to the avian α-globin genes. Previous work from this laboratory suggested that the α-globin genes of 5-day chick red blood cells and avian MSB cells replicate in vivo in the transcriptional direction, possibly from a chromosomal origin near the απ-globin gene. Here we extend those studies by showing that replication forks move divergently from an 8-kb region of α-globin 5′-flanking DNA. One potential zone for the initiation of bidirectional replication was located approximately 2.5 kb 5′ to the απ-globin gene in both of these cell types. Additionally, a barrier to replication fork movement, which may be located in a second origin zone, was found approximately 5 kb farther upstream. Both in 5-day RBCs, where the α-globin genes are expressed, and in MSB cells where they are not expressed, DNase I hypersensitive structures were found approximately 5 kb 5′ to the απ-globin gene, in the putative replication initiation domain. Another DNase I hypersensitive site was confirmed to exist in 5-day RBC nuclei upstream of the transcribed απ-globin gene. These results suggest that replication of the α-globin genes initiates in the nearby 5′-flanking DNA of this locus in a region marked by distinct chromatin structures.