Localization and DNA sequence of a replication origin in the rhodopsin gene locus of Chinese hamster cells

Abstract A chromosomal origin of DNA replication has been localized within the single-copy rhodopsin gene locus in Chinese hamster (line CHO) cells using two methods. In the first method, single-copy segments were identified at 3 to 15 kb intervals within approximately 75 kb (kb = 10 3 bases) of cloned genomic DNA containing the early-replicating rhodopsin gene near its middle. The cloned single-copy segments were then used as hybridization probes to quantify the replication of their corresponding genomic segments as synchronized cells progressed into S phase. In the second method, genomic DNA synthesized in vivo or in permeabilized early S phase cells was hybridized with slot-blots of the cloned single-copy DNA segments to identify the earliest replicating part of the 75 kb mapped region. The first method indicates that the earliest replicating DNA is located within a 10 kb region beginning 4 kb upstream from and extending 1 kb beyond the rhodopsin gene. The second method confirms the location in the vicinity of the rhodopsin gene and indicates that the earliest replicating region is located within or very near the 4.5 kb rhodopsin gene itself. An extended region of 12 kb that encompasses the entire early-replicating region has been sequenced for analysis and comparison with currently characterized origin regions associated with the CHO dihydrofolate reductase (dhfr) and human c- myc genes. There are several sequence similarities between the dhfr rhodopsin origin regions, including common transcription promoter consensus sequences, rodent Alu repeats with their 3′-A + T rich flanking sequences, A + T-rich yeast ARS and Drosophila SAR concensus sequences, and simple (GA) n repeats, but there are no extended regions of direct similarity. The rhodopsin gene locus is the second sequenced CHO origin region.