John Yates

The Epstein-Barr viral (EBV) genome of approximately equal to 170 kilobase pairs (kbp) is maintained as a plasmid in human B lymphoblasts transformed by the virus. We have identified a cis-acting element within 1.8 kbp of the viral genome that allows recombinant plasmids carrying it to be selected at high frequency and maintained as plasmids in cells latently infected by EBV. This functional element(s) requires a segment of DNA at least 800 bp and at most 1800 bp long, which contains a family of 30-bp tandem repeats at one end. Since this region confers efficient stable replication only to plasmids transfected into cells containing EBV genomes, its function probably requires trans-acting products encoded elsewhere in the viral genome.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.

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.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.