Amplification and expression of foreign genes in cells producing polyoma virus large T-antigen.
Polyoma virus (Py) large T-Antigen (LT) can promote the amplification of viral genomes integrated in the chromosomal DNA of rat fibroblasts, and this phenomenon requires the interaction of the LT protein with the viral origin of DNA replication. To compare the rate and the modality of selectable amplification events promoted by the Py LT with cellular-driven events, we constructed a double expression vector containing a murine dihydrofolate reductase (dhfr) cDNA and the bacterial chloramphenicol acetyl transferase (cat) gene controlled by the viral regulatory region. The plasmid was introduced into a rat cell line constitutively producing a temperature sensitive LT (cl 2), and clones were selected in low concentration of methotrexate (MTX). Three cl 2 transformants and one control cell line lacking LT were propagated at temperatures permissive (33 degrees C) and non permissive (39 degrees C) for LT function and, subsequently, challenged in one step with high MTX dosage at 39 degrees C. While the control line produced the same number of colonies irrespective of the temperature of propagation, the three LT positive lines yielded between 2 and greater than 100 times more colonies following propagation at permissive temperature, indicating that the presence of Py LT considerably increased the rate of amplification of integrated sequences linked to the viral origin of replication. In all cases the amplification event involved the exogenous and not the endogenous dhfr gene, and overexpression of the cat gene occurred as a result of co-amplification with the selectable dhfr sequences. Analysis of the structure of the amplified domain in the various resistant derivatives revealed that, in the presence of the viral protein, amplification occurred within the boundaries of the primary plasmid insert. In the absence of a functional LT protein, amplification both internal or involving adjacent host DNA were observed.
Pellegrini
S
S
Department of Pathology, New York University School of Medicine, New York 10016.
Basilico
C
C
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D000957:Antigens, Viral, Tumor; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate