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Nucleic Acids Research, 1984, Vol. 12, No. 4 1925-1941
© 1984

MOLECULAR BIOLOGY

Expression of dihydrololate reductase, and of the adjacent Elb region, in an Ad5-dihydrofolate reductase recombinant virus

Kathleen L. Barkner and Phillip A. Sharp

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology Cambndge, MA 02139, USA

Received November 1, 1983. Revised January 23, 1984. Accepted January 23, 1984.

A gene with the Ad2 MLP end firat leader, and appropriate RNA processing signals (splicing, polyadenylation) positioned around a mouse DHFR cDNA clone was substituted for the EIa region of Ad5, and virus stocks of Ad5 (DHFR-I) were prepared on 293 cells. A DHFR RNA of the expected size and structure was expressed late after infection of 293 cells by Ad5 (DHFR-I), at levels comparable to that of other Ad5 late messages. Although this DHFR mRNA was translated as efficiently as other Ad late mRNAs in vitro it was only poorly translated in vivo.

The substitution of the DHFR gene for the Ad5 EIa region results in aberrant transcriptional activity in the adjacent EIb sequences. The transcriptional levels of the EIb 1kb message were down approximately 10-fold. In addition, a novel pIX-encoding mRNA was produced, generated by the splicing of the Ad first late leader onto sequences 14 bp upstream from the pIX initiation codon. This new mRNA was found to be potently efficient for translation both in vivo and in vitro.


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