DNASE-SENSITIVE CHROMATIN STRUCTURE NEAR A CHROMOSOMAL ORIGIN OF BIDIRECTIONAL REPLICATION OF THE AVIAN ALPHA-GLOBIN LOCUS

We used an in vitro nuclear run-off replication assay to analyze the d irection of replication in a 13-kb region 5' to the avian alpha-globin genes. Previous work from this laboratory suggested that the alpha-gl obin genes of 5-day chick red blood cells and avian MSB cells replicat e in vivo in the transcriptional direction, possibly from a chromosoma l origin near the alpha(pi)-globin gene. Here we extend those studies by showing that replication forks move divergently from an 8-kb region of alpha-globin 5'-flanking DNA. One potential zone for the initiatio n of bidirectional replication was located approximately 2.5 kb 5' to the alpha(pi)-globin gene in both of these cell types. Additionally, a barrier to replication fork movement, which may be located in a secon d origin zone, was found approximately 5 kb farther upstream. Both in 5-day RBCs, where the alpha-globin genes are expressed, and in MSB cel ls where they are not expressed, DNase I hypersensitive structures wer e found approximately 5 kb 5' to the alpha(pi)-globin gene, in the put ative replication initiation domain. Another DNase I hypersensitive si te was confirmed to exist in 5-day RBC nuclei upstream of the transcri bed alpha(pi)-globin gene. These results suggest that replication of t he alpha-globin genes initiates in the nearby 5'-flanking DNA of this locus in a region marked by distinct chromatin structures.