Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Abstract

Separated strands of adenovirus DNA were annealed with ear1y cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, sin-tranded DNA. Loops have been observed and mapped in al four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression. The synthesis of mRNA in eukaryotic cells consists of a number of distinct but poorly understood steps. After transcription from the template DNA, the RNA transcript appears to undergo a series of nucleolytic cleavages, modification of the 5' end to form the "cap" structure, and addition of poly(A) to the 3' end (1, 2). We have been studying the RNAs synthesized after infection of KB cells by human adenovious type 2 (Ad2) in an attempt to understand these processes. The Ad2 system provides an excellent model in which to study these processes in that its DNA is far less complex than that of a eukaryotic cell and has few gene products but appears to share the same mechanism for RNA transcription and processing.The viral gene products can be placed into two separate classes: those produced early during infection (that is, before the onset of viral DNA synthesis); and those produced after its onset (late RNAs). We have mapped early and late cytoplasmic RNAs (3-5) as well as late nuclear RNAs (6) by using the R-loop electron microscope technique (7,8). In addition, use of quantitative electron microscopy has enabled us to determine the relative abundance of individual populations of cytoplasmic Ad2 RNA (9, 10). Hybridization of RNA with separated strands of Ad2 DNA revealed structures reminiscent of insertions previously observed in Drosophila melanogaster rDNA'RNA hybrids (7,11,12 RESULTSEarly cytoplasmic Ad2 RNA hybridizes to four regions of the genome (14, 15) which we (10) located at map positions 1.1-10.6, 61.6-68.1, 76.7-83.7, and 91.5-96.9. The rightward DNA strand contains regions 1 and 3; the leftward strand contains regions 2 and 4. Fig. 1A depicts a rightward Ad2 DNA molecule that carries RNA in region 1 (left end) and in region 3 (right of center). Arrows point to small loops of single-stranded nucleic acid that protrude, at two locations, from the DNA-RNA duplex structures. We have also observed loops in regions 2 and 4 on the leftward strand and a second loop occurring at a different site in region 1. Fig. 1 B and C shows enlargements of the two loops found in region 1. These RNAs have been mapped at positions 1.2-5.5 (region la) and 4.4-11.3 (region lb) with the loops mapping between points 2.9 and 4.0 and 5.9 and 9.7, respectively. The 3' terminus of region la RNA (5.5) overlaps with the 5' terminus of region lb (4.4) RNA. Both of these loops occur infrequently, with RNAs not generating a loop and mapping between positions 1.1 and 10.6 being the predominant RNAs found in this region.Two single-stranded DNA displacement loop...

In vitro RNA-RNA splicing in adenovirus 2 mRNA formation.

“…Because the cytoplasmic mRNA for the 72,000 Mr protein is mainly if not exclusively a poly(A)+ species (26,27,(29)(30)(31) and because the majority of the labeled poly(A)+ Labeled nuclear poly(A)+ RNA was eluted from 20S and 28S regions of gels similar to those shown in Fig. 4 and precipitated with ethanol.…”

“…The average size of these transcription units is approximately 2-8 kilobases (kb), considerably smaller than the major late Ad-2 transcription unit (12,13). However, like the late Ad-2 mRNAs, virtually all of the early Ad-2 mRNAs are composed of noncontiguously encoded sequences, some of which are at least 5 kb apart in the Ad-2 genome (29,30). Previously, Craig and Raskas (31) demonstrated that poly(A)-terminated nuclear RNA larger than mRNA was complementary to the DNA of two of these transcriptional units.…”

“…Previously, Craig and Raskas (31) demonstrated that poly(A)-terminated nuclear RNA larger than mRNA was complementary to the DNA of two of these transcriptional units. We chose to investigate further one of these, that which produces the mRNA for the 72,000 Mr DNA binding protein (26,29,30,32). Fig.…”

Adenovirus E1A: remodelling the host cell, a life or death experience

“…With the development of the calcium phosphate DNA transfection technique (Graham and van der Eb, 1973) and the use of de®ned Ad DNA fragments, it was convincingly shown that this region was responsible for the induction of transformation (Graham et al, 1974a,b). RNA mapping was carried out Flint et al, 1975) which de®ned the early region transcripts (reviewed by Shenk and Flint, 1991;Shenk, 1996) and later designated E1 (E1A+E1B) E2, E3 and E4 (Kitchingman et al, 1977). Viral RNA selected on speci®c Ad DNA fragments was then used in transcription/translation assays to identify the proteins expressed from these regions and their molecular weights (Lewis et al, 1976;Harter and Lewis, 1978;Halbert et al, 1979).…”

Nuclei of adenovirus 2-infected cells contain an RNA species that corresponds to an intron excised intact from mRNA precursors.

“…2, structure IV) is transcribed from the 1-strand (29,30) during the early phase of infection. The mRNA is produced by splicing two introns, thus linking together three exons (5,8,19). This mRNA encodes the 72,000-molecular-weight DNA-binding protein (13,17,24).…”