Sequences involved in the regulated expression of the human interferon-beta1 gene in recombinant SV40 DNA vectors replicating in monkey cells.
The human genomic EcoRI fragment of 1.83 kb containing the interferon (IFN) gene IFN-beta1 with 285 nucleotides of 5'-flanking sequences was transfected into monkey kidney CV-1 cells as part of an SV40-pML2 vector. Induction of the monkey cells to produce IFN led to a rapid accumulation of IFN-beta1 RNA whose 5' ends were identical to the IFN-beta1 mRNA of human fibroblasts. This induction occurred with all recombinants tested. Expression from the SV40 late promoter was also seen in non-induced cells. We conclude that the regulation of the IFN-beta1 gene is retained in the replicating episomal SV40 vectors with high copy number, even when the gene is being transcribed from an external promoter. When the 5'-flanking sequences were deleted to leave only 40 bp before the presumed cap site of the IFN-beta1 gene, inducible formation of IFN-RNA with authentic 5' ends could still be demonstrated. However, inducibility and expression depended on the position of the deleted IFN-beta1 gene in the vector. We conclude that the sequences around the TATAA box and cap site on the IFN gene are involved in the regulation of its expression. Regulated short-term expression of the human IFN-beta1 gene in SV40 vectors provides a defined system in which the structures required to maintain the regulation and the influence of known external transcription signals can be examined.
Maroteaux
L
L
Department of Virology, The Weizmann Institute of Science, Rehovot, Israel.
Kahana
C
C
Mory
Y
Y
Groner
Y
Y
Revel
M
M
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004279:DNA, Viral; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics*; D024363:Transcription Initiation Site
D004279:DNA, Viral; D016899:Interferon-beta