RNA-SYNTHESIS IN ISOLATED-NUCLEI - IDENTIFICATION AND COMPARISON OF ADENOVIRUS-2 ENCODED TRANSCRIPTS SYNTHESIZED INVITRO AND INVIVO

Nuclei isolated from HeLa cells 16 hours post infection by adenovirus serotype 2 synthesize high levels of viral specific RNA in vitro. This RNA shares many features in common with nuclear viral specific RNA synthesized in vivo. The size of the RNA is heterogeneous, although the bulk of the transcripts are 10 × 103 to 25 × 103 bases in length. The 5′ ends of transcripts synthesized in vitro that are colinear with the viral DNA map primarily at two co-ordinates: 16.5, the site of the major late promoter, and 19.5, the location of the first splice-point used in synthesis of late viral mRNA. Colinear transcripts with 5′ ends which map at sites corresponding to the locations of other splice-points are also labeled but are much less abundant. In particular, only low levels of fully spliced messenger RNA are produced after two hours of incubation in vitro. A fraction of the transcripts synthesized in vitro appears to extend all the way to the end of the genome. However, discrete RNA species with 3′ ends at 38.5, 50.0, 61.5 and 78.5 map units are also synthesized in vitro. Molecules with these co-ordinates are also detected in steady-state nuclear RNA in vivo. These four species are all accurately polyadenylated in vitro, although the efficiencies of polyadenylation vary: RNA molecules with 3′ ends at position 61.5 or 78.5 only accumulate in a poly(A)+ form, while species terminated at co-ordinate 38.5 or 50.0 are either poly(A)+ or poly(A)-. An identical distribution also exists in nuclear steady-state RNA synthesized in vivo. Further analysis of Ad2 † † Abbreviations used: Ad2, adenovirus serotype 2; kb, kilobase (103 bases); EGTA, ethylene glycol-bis (β-aminoethyl ether) N,N′-tetraacetie acid.-specific nuclear RNA, synthesized either in vitro or in vivo, reveals that large RNA molecules containing incompletely spliced tripartite leader segments (presumptive processing intermediates) are present in both poly(A)- and poly(A)+ RNA. However, molecules with mature 5′ ends (presumably created by splicing of the tripartite leader segment onto main-body sequences) accumulate to detectable levels only as fully processed, polyadenylated mRNA. © 1979.