title: A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components.
abstract: A genetic element of Epstein-Barr virus, oriP, when present on recombinant plasmids allows those plasmids to replicate and to be maintained in cells that express the Epstein-Barr virus-encoded nuclear antigen EBNA-1. Here we define the DNA sequences required for oriP activity. Two noncontiguous regions of oriP are required in cis for activity. One consists of approximately 20 tandem, imperfect copies of a 30-base-pair (bp) sequence. The other required region, approximately 1,000 bp away, is at most 114 bp in length and contains a 65-bp region of dyad symmetry. When present together on a plasmid, these two components supported plasmid replication even when the distance between them was varied or their relative orientation was altered, or both. When present alone on a plasmid that expresses a selectable marker, the family of 30-bp repeats efficiently conferred a transient drug-resistant phenotype in human 143 cells that is dependent on the presence of EBNA-1. This result leads us to suggest that EBNA-1 interacts with the 30-bp repeated sequence to activate oriP. To test whether the 30-bp repeats might cause the increased transient expression of drug resistance by enhancing transcription, the family of 30-bp repeats was tested for the ability to activate the simian virus 40 early promoter present in plasmid pA10CAT2 (Laimins, et al., Proc. Natl. Acad. Sci. U.S.A. 79:6453-6457). In this assay, the 30-bp repeats could activate the simian virus 40 early promoter in Raji cells, an EBNA-positive Burkitt's lymphoma cell line, but not detectably an EBNA-positive 143 cells in which oriP also functions.
mesh_terms: D000123:Acetyltransferases / Q000235:genetics; D002051:Burkitt Lymphoma; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells; D010957:Plasmids; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes