Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10905536 | Experimental Cell Research | 2005 | 12 Pages |
Abstract
The Chinese hamster dihydrofolate reductase (DHFR) origin consists of many inefficient initiation sites scattered throughout a 55-kb intergenic spacer, with the ori-β, ori-βâ², and ori-γ subregions being preferred. To test for the presence of genetic replicators within these subregions, fragments containing ori-β, ori-βâ², or, as a negative control, a fragment from the DHFR gene that never initiates in loco were placed at ectopic chromosomal positions. Two-dimensional gel analysis demonstrates that initiation occurs in all three fragments, including the fragment from the gene, and appears to occur at different positions within each fragment in different cells in the population. However, initiation is not detectable in the adjacent, transcribed, neomycin-resistance marker. When a cosmid containing an active DHFR gene was inserted into an ectopic chromosomal site, initiation could no longer be observed in the body of the gene, but was clearly evident in the transcriptionally-silent bacterial vector sequences. In the thirteen independent chromosomal positions into which the four different constructs were inserted, initiation occurred in early S-phase. Since these loci necessarily had to be permissive for transcription of the neor marker or DHFR gene used to select transfectants, it is possible that early-firing origin activity and local transcription are somehow linked. In total, these data suggest that potential initiation sites are distributed at very frequent intervals in the mammalian genome, with usage being regulated both positively and negatively by local transcription.
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Authors
H.-B. Lin, P.A. Dijkwel, J.L. Hamlin,