Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells.
In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ' sequence, carried by our vectors.
Stary
A
A
Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Sarasin
A
A
D002478:Cells, Cultured; D005784:Gene Amplification / Q000528:radiation effects; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000528:radiation effects*; D006801:Humans; D010957:Plasmids; D012093:Replicon; D013539:Simian virus 40 / Q000235:genetics; D014466:Ultraviolet Rays