title: Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification.
abstract: Initiation of DNA synthesis occurs with high frequency at oribeta, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oribeta/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oribeta/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oribeta/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.
authors.affiliation: Department of Pathology and Program in Cell and Molecular Biology, University of Vermont College of Medicine, Soule Medical Alumni Building, Burlington, VT 05405, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Brinton
authors.forename: N H
authors.initials: NH
authors.lastname: Heintz
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification / Q000187:drug effects; D015321:Gene Rearrangement; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate