Berberich S, Leffak M
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435.
DNA Cell Biol. 1993 Oct;12(8):703-14. doi: 10.1089/dna.1993.12.703.
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 alpha-globin genes. Previous work from this laboratory suggested that the alpha-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 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 initiation 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 second origin zone, was found approximately 5 kb farther upstream. Both in 5-day RBCs, where the alpha-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 alpha pi-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 alpha pi-globin gene. These results suggest that replication of the alpha-globin genes initiates in the nearby 5'-flanking DNA of this locus in a region marked by distinct chromatin structures.
我们使用体外核延伸复制分析来研究禽α-珠蛋白基因5'端13 kb区域内的复制方向。本实验室先前的研究表明，5日龄雏鸡红细胞和禽MSB细胞中的α-珠蛋白基因在体内沿转录方向复制，可能起始于靠近απ-珠蛋白基因的一个染色体起始位点。在此，我们通过研究发现复制叉从α-珠蛋白5'侧翼DNA的一个8 kb区域向两侧移动，从而扩展了这些研究。在这两种细胞类型中，双向复制起始的一个潜在区域位于απ-珠蛋白基因5'端约2.5 kb处。此外，在距离上游约5 kb处发现了一个可能位于第二个起始区域的复制叉移动障碍。在α-珠蛋白基因表达的5日龄红细胞以及不表达该基因的MSB细胞中，在假定的复制起始结构域内，于απ-珠蛋白基因5'端约5 kb处均发现了DNase I超敏结构。另一个DNase I超敏位点在转录的απ-珠蛋白基因上游的5日龄红细胞核中得到证实。这些结果表明，α-珠蛋白基因的复制起始于该基因座附近5'侧翼DNA中一个具有独特染色质结构的区域。
