Expression of the human insulin gene in an alternate mammalian cell and in cell extracts.

The functional regions of the human insulin gene have been characterized by in vitro and in vivo experiments. The locations of the insulin promoter and cap site have been predicted from the sequence of the gene, and these assignments are consistent with the results of in vitro transcription experiments using HeLa cell extracts. For studies of intracellular expression, the human insulin gene including its promoter and two intervening sequences was inserted into the late region of the virus, SV40, in a construction that eliminates the major splice junction of the late SV40 region. Permissive CV1 monkey kidney cells were infected with this recombinant and a complementing helper virus. During the course of the infection, insulin gene transcripts accumulate at about one-third the level of SV40 late transcripts originated from the same promoter. Analysis of the RNAs containing insulin sequences showed that the insulin introns are excised with precision. Furthermore, the insulin promoter (cap site) and polyadenylation sites are recognized but are less efficiently employed than the viral late promoter and terminator. The mRNA is translated and immunoreactive human proinsulin is secreted into the extracellular medium.