Adenovirus Messenger RNA in Mammalian Cells: Failure of Polyribosome Association in the Absence of Nuclear Cleavage

Liquid RNA-DNA hybridization with separated strands of adenovirus type 2 DNA revealed that late nuclear RNA can hybridize to about 85% of the 1-strand and 10-15% of the h-strand, whereas late cytoplasmic RNA hybridizes to 65-70% and 25% of the 1-and h-strand, respectively. With separated strands from the six EcoRI fragments of adenovirus type 2 DNA as probes, it was shown that late nuclear RNA hydridizes to 85-90% of the 1-strand from all six EcoRI fragments. Since late cytoplasmic RNA hybridizes to 40-50% of the h-strand from both fragments EcoRI-B and EcoRI-C, complementary viral RNA sequences are synthesized during adenovirus infection. Complementarity between nuclear and cytoplasmic RNA could also be demonstrated by showing that late cytoplasmic RNA which had been preincubated with late nuclear RNA hybridized to a smaller fraction of the h-strand of fragment EcoRI-C than without preincubation. Double-stranded RNA which contains sequences that correspond to at least 60% of the viral genome was isolated from infected cells. However, less than 2% of the newly synthesized late RNA became double-stranded after incubation under annealing conditions, which suggests that RNA derived from one of the strands is present at a low concentration. Accordingly, it was shown that nearly all viral cytoplasmic RNA which is synthesized late after infection is derived from the 1-strand.

Section: Methodssupporting

confidence: 82%

mentioning

confidence: 99%

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

Pettersson

Philipson

1974

“…Several lines of evidence suggest that cytoplasmic Ad2 mRNAs are derived from larger nuclear precursors that lose part of their sequences during processing (6,(25)(26)(27)(28)(29)(30)(31). If processing occurs by removal of sequences not only at the 5' and 3' ends of the molecule but also internally, the resulting mRNA would be expected to generate displacement loops when hybridized with the template strand of DNA.…”

Section: Discussionmentioning

confidence: 99%

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

Kitchingman¹,

Lai²,

Westphal³

1977

100

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...

“…The present map can be resolved into considerably more detail by the use of separated strands of DNA fragments as well as fragments generated by other restriction enzymes. The cytoplasmic mRNAs that are mapped here represent the final product of viral transcription and processing; the identification and localization of the original unprocessed transcripts (8,(25)(26)(27) remains to be accomplished.…”

Section: Hybridization Of Early and Late Cytoplasmic Rna To Dna Fragmmentioning

confidence: 99%

Localization of Adenovirus 2 Messenger RNAs to Segments of the Viral Genome Defined by Endonuclease R·R1

Tal

Craig

Zimmer

et al. 1974

(11). Hybridization to the DNA fragments has enabled (1) the detection of several new viral mRNAs, (2) the separation of mRNAs which migrate at the same rate on acrylamide gels, and (3) the localization of individual early and late viral mRNAs to distinct portions of the genome. METHODSCultured human cells (KB) were infected with adenovirus 2 and harvested as previously described (13-15). For preparations of early RNA, infections were performed in the presence of cycloheximide (25 u.g/ml) to prevent the beginning of late RNA transcription (14). Radioactive early RNA was obtained by labeling cultures with [3H]uridine 2-6 hr after infection (14) and radioactive late RNA was obtained by labeling cultures for 3H-uridine 18-21 hr after infection (11). Cytoplasmic RNA was purified and fractionated by oligo(dT) cellulose chromatography as described previously (14). RNA was fractionated by electrophoresis on 3.2% ethylene diacrylate-acrylamide gels, and the RNA in each gel slice was solubilized by incubating at 600 in 6 X SSC containing 0.1% sodium dodecyl sulfate (5). The sedimentation values assigned to viral RNA size classes were obtained by comparing the migration of the viral RNAs and the 28S and 18S ribosomal RNA.Adenovirus 2 DNA was purified according to the method of Green and Pina (13). Fragments of viral DNA were prepared by digestion with the E. coli endonuclease R -R1 (16). DNA fragments were resolved by electrophoresis in 1.4% agarose gels using a buffer which contained ethidium bromide (17). DNA bands were visualized with UV light (17), and DNA was eluted from gel slices by high voltage electrophoresis. The final yield of DNA fragments was 60% as monitored with [32P]DNA.DNA membranes (6.5 mm) were prepared and hybridizations were performed in 6 X SSC, 0.1% sodium dodecyl sulfate as described previously (14,18). For all hybridizations to R1 fragments, six membranes, each containing DNA from a unique fragment, were incubated simultaneously with [3H]-RNA in 200,ul.