Expression of the human insulin gene and cDNA in a heterologous mammalian system.
The human insulin gene or the corresponding cDNA has been inserted into the early region of a simian virus 40 vector in which all SV40 splice junctions were deleted while the early promoter and polyadenylation regions remained intact. The expression of insulin-coding sequences was tested in permissive monkey COS cells. The insulin cDNA was transcribed from the early promoter to produce a stable polyadenylated RNA which was translated, and immunoreactive human proinsulin accumulated in the medium. Thus RNA splicing is not obligatory for insulin expression in this system. The genomic insulin transcript was also initiated from the SV40 promoter and terminated at the SV40 polyadenylation site. S1 endonuclease mapping revealed that the transcript is processed via two alternative splicing pathways within the insulin gene. About one-third of the total transcripts are processed normally with removal of the two insulin-specific introns. This transcript is apparently translated normally since immunoreactive proinsulin accumulates in the medium. About two-thirds of the transcripts are processed via an alternative splicing pathway involving a new splice acceptor site located within the coding region of the insulin gene. This results in a codon frameshift such that translation would produce a novel chimeric peptide containing the insulin NH2-terminal B chain, but a different COOH terminus containing human and SV40 sequences. A peptide of the predicted size is detected in the COS cell extract.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics*; D007668:Kidney; D011384:Proinsulin / Q000096:biosynthesis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D007328:Insulin; D012333:RNA, Messenger; D004247:DNA; D011384:Proinsulin; D004262:DNA Restriction Enzymes