DNA strand selection during the transcription of the adenovirus 2 genome in infected and transformed cells

Landgraf-Leurs, Martina; Green, Maurice

doi:10.1016/0005-2787(73)90070-1

Cited by 13 publications

(8 citation statements)

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“…Of the total virus-specifie RNA, 32% was homologous to the H strand, and 68% was homologous to the L strand. This agrees with the findings of Landgraf-Leurs and Green (8) that both DNA strands are transcribed early in adenovirus 2 infection. The fact that twice as much viral RNA associated with polyribosomes early after infection is transcribed from the L strand as from the H strand leads to an interesting problem in the control of transcription.…”

Section: Discussionsupporting

confidence: 82%

“…The use of the separated strands of adenovirus DNA facilitates resolution of individual viral mRNA species. Landgraf-Leurs and Green (7) developed a procedure for the preparative separation of the adenovirus tylpe 2 DNA strands, and showed that both strands were transcribed early as well as late after infection (8). Viral RNA isolated by hybridization to DNA by the usual annealing conditions is thermally degraded and cannot be used for further characterization.…”

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confidence: 99%

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Isolation of DNA Strand-Specific Early Messenger RNA Species in Cells Infected by Human Adenovirus 2

Büttner

Veres-Molnár

Green

1974

Proc. Natl. Acad. Sci. U.S.A.

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Hybridization to the separated light (L) and heavy (H) strands of adenovirus 2 DNA in 50% formamide at 37°was used to isolate undegraded virus-specific RNA molecules from the polyribosomes of cycloheximidetreated human KB cells early after infection with adenovirus 2. About 20% of polyribosomal RNA labeled with [3Hluridine from 4 to 7 hr after infection was virus-specific. Twice as much labeled RNA was homologous to the L strand as to the H strand. Polyacrylamide gel electrophoresis of RNA selected with unfractionated adenovirus DNA resolved a major component of virus-specific RNA in the 19-20 S region of the gel and smaller amounts of viral RNA in two heterogeneous fractions migrating at 15-18 S and 21-26 S. Selection with individual DNA strands showed that the 19-20 S main size class of early mRNA consists of two homogeneous RNA species with slightly different mobilities, the transcripts from the L and H strand having molecular weights of 7.4 X 105 and 7.7 X 105, respectively. The 15-18 S RNA hybridized with the L strand and the 21-26 S RNA with the H strand.Only one of the two complementary DNA strands in any region of a duplex DNA molecule can be transcribed to a functional messenger RNA for protein synthesis. With some DNA viruses transcription occurs exclusively from one strand of the viral genome (1), while with others regions from both strands are transcribed (2, 3). The human adenoviruses, especially the well-studied adenovirus type 2, form excellent models for study of the regulation of transcription in mammalian cells. Of special interest is the transcription of early adenovirus 2 genes that occurs in the absence of protein synthesis and thus probably reflects the transcril)-tion controls of the host cell (4). Early after infection only a few viral genes are transcribed to stable viral mRNA species (5), and these include those adenovirus genes expressed in transformed cells (6). It would be of interest, therefore, to be able to isolate individual adenovirus mRNA molecules in an undegraded form for studies on structure, sequence, and translation. The use of the separated strands of adenovirus DNA facilitates resolution of individual viral mRNA species. Landgraf-Leurs and Green (7) developed a procedure for the preparative separation of the adenovirus tylpe 2 DNA strands, and showed that both strands were transcribed early as well as late after infection (8). Viral RNA isolated by hybridization to DNA by the usual annealing conditions is thermally degraded and cannot be used for further characterization.Abbreviations: L and H strand, light and heavy strands as defined by the relative buoyant density of the DNA strand. poly(U,G) complex; EDTA, ethylenediaminetetraacetate. 2951Although undegraded adenovirus-specific RNA can be isolated on a preparative scale on the basis of its content of poly-(adenylic acid) (9), this approach is useful only for the isolation of late viral RNA species which contain relatively little cell mRNA. Molecular hybridization to viral DNA is required to separate early vir...

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Section: Discussionsupporting

confidence: 82%

mentioning

confidence: 99%

Isolation of DNA Strand-Specific Early Messenger RNA Species in Cells Infected by Human Adenovirus 2

Büttner

Veres-Molnár

Green

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…In both cases, large RNA molecules can be detected in the nucleus which are polyadenylated and cleaved before entering the cytoplasm (1,(3)(4)(5)(6). Messenger RNA both early and late after infection with adenovirus type 2 (ad2) hybridizes to both strands of the viral DNA (7,8). The shift from early to late phase takes place at the onset of viral DNA replication (6-8 hr after infection) and leads to qualitative as well as quantitative changes of viral RNA synthesis.…”

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Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

Pettersson

Philipson

1974

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…For analysis of poly(A) tracts in viral RNA, [3H]ATP-labeled RNA was hybridized to Ad2 DNA on nitrocellulose filters. The filters were washed, and the poly(A) from the selected RNA was released by incubating filters with 5 ,ug of RNase A and 20 U of RNase T1 per ml in 200 mM NaCl, 10 mM Tris-hydrochloride, pH 7.8, and 1 mM EDTA for 30 min at 37°C. The released poly(A) tracts were phenol extracted and analyzed, after ethanol precipitation, on 15% polyacrylamide gels (12).…”

Section: Separation Of the Complementary Strands Of Ad2mentioning

confidence: 99%

“…Specificity of transcription in isolated nuclei. Late in the adenovirus productive cycle, newly synthesized mRNA is preferentially derived only from the r-strand of the viral genome (10,17). To establish whether this reflects a transcription control, isolated nuclei were labeled with [3H]GTP for 50 min.…”

Section: Downloaded Frommentioning

confidence: 99%

Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei

Vennström¹,

Philipson²

1977

J Virol

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An in vitro nuclear system from adenovirus type 2-infected cells was developed to study transcription of viral RNA. Nuclei isolated from adenovirusinfected HeLa cells late in the infectious cycle synthesized in vitro only RNA from the r-strand of adenovirus DNA. Around 15% of the virus-specific RNA in isolated nuclei was polyadenylated. Short pulse labeling of nascent RNA followed by hybridization of size-fractionated RNA to specific restriction endonuclease fragments of the genome suggested that the origin(s) for transcription is located on the r-strand in the left 30% ofthe adenovirus 2 genome at late times in the infectious cycle. Pulse-chase experiments were used to estimate the elongation rate of adenovirus high-molecular-weight RNA in isolated nuclei. An elongation of at least six nucleotides per second was observed in vitro. Viral RNA synthesis in in vitro nuclei showed several similarities to the in vivo system late in the infectious cycle.

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DNA strand selection during the transcription of the adenovirus 2 genome in infected and transformed cells

Cited by 13 publications

References 10 publications

Isolation of DNA Strand-Specific Early Messenger RNA Species in Cells Infected by Human Adenovirus 2

Isolation of DNA Strand-Specific Early Messenger RNA Species in Cells Infected by Human Adenovirus 2

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei

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