Nucleic Acids Research, 1993, Vol. 21, No. 16 3607-3614
© 1993
MOLECULAR BIOLOGY |
RNA polymerase I catalysed transcription of insert viral cDNA
Institut für Mikrobiologie und Molekularbiologie der Justus-Liebig-Universität Giessen Frankfurter Straße 107, D-35392 Giessen, Germany
*To whom correspondence should be addressed
Received June 18, 1993. Accepted July 12, 1993.
RNA polymerase I has been used for transcription of Influenza hemagglutinln (HA) cDNA precisely linked In the anti-sense configuration to both mouse rDNA promoter and terminator segments. In transcription reactions based on Ehrllch ascltes cell nuclear extracts, specific uniform RNA products are synthesized In high rates, that are comparable to original rDNA template transcriptions. Primer extension reactions show the 5' ends of these RNA transcripts to be located exactly at position +1 , corresponding to the 5' end of negative strand HA viral RNA. RNA 3' ends in a first series of constructs were found extended beyond the accepted location of pre-rRNA 3' ends, in using both hybrid cDNA and original rDNA templates. But upon deletion of six basepairs from the rDNA termination region RNA polymerase I transcription has been adapted to yield correctly terminated Influenza viral RNA in vitro. This result has been confirmed In an in vivo experiment via synthesis of an anti-sense viral RNA molecule containing the chloramphenlcol acetyltransferase (CAT) gene, which In turn is recognized at Its terminal sequence by viral RNA dependent RNA polymerase for plus strand mRNA synthesis and expression of CAT activity.
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