RNA POLYMERASE-I CATALYZED TRANSCRIPTION OF INSERT VIRAL CDNA

RNA polymerase I has been used for transcription of influenza hemagglu tinin (HA) cDNA precisely linked in the anti-sense configuration to bo th mouse rDNA promoter and terminator segments. In transcription react ions based on Ehrlich ascites cell nuclear extracts, specific uniform RNA products are synthesized in high rates that are comparable to orig inal 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 a ccepted location of pre-rRNA 3' ends, in using both hybrid cDNA and or iginal rDNA templates. But upon deletion of six basepairs from the rDN A termination region RNA polymerase I transcription has been adapted t o 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 chloramphenicol acetyltransfer ase (CAT) gene, which in turn is recognized at its terminal sequence b y viral RNA dependent RNA polymerase for plus strand mRNA synthesis an d expression of CAT activity.