title: Discrete functional elements required for initiation activity of the Chinese hamster dihydrofolate reductase origin beta at ectopic chromosomal sites.
abstract: The Chinese hamster dihydrofolate reductase (DHFR) DNA replication initiation region, the 5.8 kb ori-beta, can function as a DNA replicator at random ectopic chromosomal sites in hamster cells. We report a detailed genetic analysis of the DiNucleotide Repeat (DNR) element, one of several sequence elements necessary for ectopic ori-beta activity. Deletions within ori-beta identified a 132 bp core region within the DNR element, consisting mainly of dinucleotide repeats, and a downstream region that are required for ori-beta initiation activity at non-specific ectopic sites in hamster cells. Replacement of the DNR element with Xenopus or mouse transcriptional elements from rDNA genes restored full levels of initiation activity, but replacement with a nucleosome positioning element or a viral intron sequence did not. The requirement for the DNR element and three other ori-beta sequence elements was conserved when ori-beta activity was tested at either random sites or at a single specific ectopic chromosomal site in human cells. These results confirm the importance of specific cis-acting elements in directing the initiation of DNA replication in mammalian cells, and provide new evidence that transcriptional elements can functionally substitute for one of these elements in ori-beta.
authors.affiliation: Department of Biological Sciences, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235-1634, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Gray
authors.forename: Guoqi
authors.initials: G
authors.lastname: Liu
authors.forename: Amy L
authors.initials: AL
authors.lastname: Altman
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Small
authors.forename: Ellen
authors.initials: E
authors.lastname: Fanning
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002877:Chromosomes, Human / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D018900:Dinucleotide Repeats; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018741:Replication Origin; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014981:Xenopus
chemical_list: D017931:DNA Primers; D014157:Transcription Factors; D013762:Tetrahydrofolate Dehydrogenase