Transcription of Adenovirus Genes in Productively Infected and in Transformed Cells

Green, M.; Parsons, J. Thomas; Piña, Magdalena; Fujinaga, Kei; Caffier, H.; Landgraf-Leurs, I.

doi:10.1101/sqb.1970.035.01.095

Cited by 117 publications

(94 citation statements)

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“…This observation has been interpreted to mean that mRNA, like ribosomal RNA and tRNA, is synthesized in the nucleus as large precursor molecules, which at some later stage (but before they become engaged in protein synthesis in the cytoplasm) are cleaved by specific mechanisms (28). This hypothesis was supported by studies of cells transformed by the DNA viruses, SV40 (30, 31) and adenovirus (32), where virus-specific RNA molecules as large as 2-4 X 106 daltons were identified in the nucleus, while their counterparts on the cytoplasmic polysomes all appeared smaller than 1.7 X 106 daltons. However, there is no direct evidence for the proposed cleavage mechanism.…”

Section: Size Distribution Of Polysomes and Mrnpsupporting

confidence: 55%

Characterization of Messenger Ribonucleoprotein and Messenger RNA from KB Cells

Kumar

Lindberg

1972

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

103

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Messenger ribonucleoprotein and mRNA from KB-cells were isolated under conditions designed to minimize nonspecific RNA-protein interaction and to minimize degradation by contaminating ribonucleases. A large fraction, 60-70%, of the messenger ribonucleoprotein from polysomes dissociated in vitro by either EDTA or puromycin sedimented faster than the large ribosome subunit. Messenger ribonucleoprotein 'particles with sedimentation coefficients up to 200 S were observed.Released mRNA was also large, with maximal molecular weights around 5 X 108.Efforts have been made to unravel the mechanisms of transcription, processing, and transport of mRNA' from nucleus to cytoplasm in eukaryotic cells. The mRNA appears to be associated with proteins (1-4), although the function of these messenger ribonucleoprotein (mRNP) complexes is unknown. Since these complexes could be artifacts of the cell fractionation procedures, their characterization depends on elimination of nonspecific interactions between RNA and proteins during their preparation (5, 6); elimination of RNase activities in cell extracts is also important.We have characterized the mRNP and mRNA from KB cells under conditions that minimized the presence of artifacts. We avoided nonspecific interactions between RNA and protein by preparing cytoplasmic extracts in isotonic buffer; subsequently, polysomes were prepared in either isotonic or hypertonic buffer (0.5 M KCl). mRNP was released from the polysomes in either of two ways, either by-EDTA (3, 4) or by puromycin treatment in vitro in a buffer of high salt concentration (7). The sedimentation rate and buoyant density of such mRNP were determined. Degradation by ribonucleases from the cytoplasmic extract was avoided as follows: the RNA from the polysomes was released from attached proteins by treatment with Sarkosyl and Pronase; RNA size was determined by centrifugation in a sucrose density gradient and by electrophoresis on polyacrylamide gel in Sarkosyl-containing buffers at 40C. Ribonucleases contaminating the reagents used were removed either by treatment with diethylpyrocarbonate (DEP) or by autoclaving.Our results indicate that both mRNA and mRNP are significantly larger than has been described. Furthermore, the buoyant density determination of mRNP from polysomes dissociated as described above inferred that mRNP contains different classes of proteins. MATERIALS AND METHODS

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Section: Size Distribution Of Polysomes and Mrnpsupporting

confidence: 55%

Characterization of Messenger Ribonucleoprotein and Messenger RNA from KB Cells

Kumar

Lindberg

1972

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

103

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“…It is not surprising, therefore, to find certain disagreements among these previous reports. Green et al (10) and Price and Penman (29) reported that host cell RNA synthesis continues late in adenovirus infection, based on the observation that only -14 to 18% of newly synthesized nuclear RNA was complementary to viral DNA. Philipson et al (27), in contrast, found 40%o of newly synthesized nuclear RNA and 70 to 80% of the poly(A)+ nuclear RNA to be viral, and they therefore concluded that cellular heterogeneous nuclear RNA synthesis production was severely depressed.…”

Section: Methodsmentioning

confidence: 99%

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

Babich¹,

Feldman²,

Nevins³

et al. 1983

Mol. Cell. Biol.

146

107

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We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (>90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.The interaction of a virus with its host cell often leads to an alteration in the synthesis and metabolism of macromolecules in the cell. The study of these alterations offers the opportunity to gain information concerning the normal course of events within the cell. For example, many different cytolytic virus infections result in decreased host protein synthesis at the same time that viral protein synthesis increases (22). The molecular basis for this viral "takeover" is best understood for poliovirus (9,19,39,42) and reovirus (33) infections, where differentiation between capped and uncapped mRNAs allows discrimination between viral and host mRNAs. However, the long-standing observation that host protein synthesis is greatly depressed late in adenovirus infection (2, 4, 5) cannot be easily explained; adenovirus mRNAs are all capped (12,23,35,45) and polyadenylated [poly(A)+] (28) and therefore would appear to be indistinguishable from host mRNAs on a gross level (6,32). Virtually all of the newly labeled mRNA that reaches the polyribosomes is virus specific (5,20,27), suggesting that transcription of cellular genes may be inhibited. However, neither the rate of synthesis of specific cellular mRNAs nor the fate of preexisting cellular mRNA has been examined with cloned cell DNA sequences.Beltz and Flint (5) approached the question of host cell mRNA manufacture and host protein synthesis by using cDNA hybridization to total cellular mRNA...

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“…Before viral DNA synthesis (6 hr after infection) virus-specific RNA is transcribed from 15-20% of the Ad 2 genome (2); most of this RNA is detected as two major viral species, with sedimentation coefficients of 23S and 17S (4,5). Late after infection (16-18 hr), virus-specific RNA is transcribed from 85 to 95% of the adenovirus genome (3).…”

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confidence: 99%

Biochemical Studies on Adenovirus Multiplication, XIX. Resolution of Late Viral RNA Species in the Nucleus and Cytoplasm

Parsons

Gardner

Green

1971

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

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Late after infection of cultured human cells (KB) with adenovirus type 2, the nucleus contains heterogeneous viral RNA species ranging in size from 10 to 43 S. Four viral RNA species found in the nucleus (36, 38, 40, and 43 S) Cultured human cells infected with adenovirus type 2 (Ad 2) transcribe specific adenovirus gene sequences during productive infection (1-3). Before viral DNA synthesis (6 hr after infection) virus-specific RNA is transcribed from 15-20% of the Ad 2 genome (2); most of this RNA is detected as two major viral species, with sedimentation coefficients of 23S and 17S (4, 5). Late after infection (16-18 hr), virus-specific RNA is transcribed from 85 to 95% of the adenovirus genome (3). The virus-specific RNA species synthesized late after infection code for the synthesis of the 8-10 known capsid polypeptides, which range in molecular weight from 15,000 to 130,000 (6-9). We report here an analysis of the late-viral RNA species, present at 16-18 hr after infection, which reveals that high molecular weight viral RNA species sedimenting at 36-43 S are found in the nucleus and appear to be precursors of the 10-29S viral-specific RNA species found in the cytoplasm.MATERIALS AND METHODS Infection, cell fractionation, and RNA isolation Suspension cultures of KB cells, at 2-3 X 105 cells/ml, were infected with Ad 2 (strain 38-2) at an input multiplicity of 50 plaque-forming units (PFU)/cell, as described previously (3).[3H]uridine (4 yCi/ml, 20 Ci/mmol) was added to infected suspension cultures, cells were harvested at various times, and These measurements were performed with viral DNA immobilized on nitrocellulose filters as described (10, 11). Ad 2 DNA was prepared from highly purified virus (12).Polyacrylamide gel electrophoresis and zone centrifugation in sucrose density gradients Labeled RNA from infected cells was resolved on either 2.8 or 3.1% polyacrylamide gels, the gels were sliced, the RNA was eluted from each slice, and the viral RNA was quantitated by hybridization with viral DNA (4). RESULTSAd 2 late RNA species in the cell nucleus Nuclear RNA species from KB cells labeled with [3H]uridine for 15 min and 60 min, at 18 hr after infection with Ad 2, were resolved by electrophoresis on 2.8% polyacrylamide gels. Radioactive RNA was eluted from the gel slices and annealed with Ad 2 DNA. As many as 10 virus-specific RNA species larger than 26 S can be detected after 15-and 60-min labeling periods (Fig. 1). Nr-NIv appear to be the major viral RNA species labeled during a 15-min pulse with [8H]uridine (Fig. 1A). Increasing proportions of Nvi-Nx, as well as at least three smaller RNA species, were evident after 60 min of labeling (Fig. 1B). Treatment of nuclear RNA, labeled for 60 min, with 8 M urea (Fig. 2) or95% dimethylsulfoxide (notshown) did not alter the number of RNA species nor their rate of migration. Since it has been demonstrated that such treatment is sufficient to disrupt much of the secondary structure of the 70S RNA of murine leukemia virus (13, 14), we conclude that the...

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Transcription of Adenovirus Genes in Productively Infected and in Transformed Cells

Cited by 117 publications

References 0 publications

Characterization of Messenger Ribonucleoprotein and Messenger RNA from KB Cells

Characterization of Messenger Ribonucleoprotein and Messenger RNA from KB Cells

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

Biochemical Studies on Adenovirus Multiplication, XIX. Resolution of Late Viral RNA Species in the Nucleus and Cytoplasm

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