Evidence for 30-40S RNA as Precursor of the 60-70S RNA of Rous Sarcoma Virus

Canaani, Eli; Helm, K. von der; Duesberg, Peter H.

doi:10.1073/pnas.70.2.401

Cited by 110 publications

(72 citation statements)

References 19 publications

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“…The network structure we observe for the 60-70S RNA is consistent with the following earlier observations (21)(22)(23)(24): Virus collected at 12-to 24-hr intervals yields a 60-70S RNA peak that is very similar to that obtained from virus collected at 3-to 5-hr intervals. Heating the 60-70S RNA of 12-to 24-hr virus, however, results in a wide distribution of molecules sedimenting between zero and 40 S; whereas heating the 60-70S RNA of virus collected at 3-to 5-hr intervals yields a relatively sharp peak of 30-40S RNA.…”

Section: Methodssupporting

confidence: 76%

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Mangel

Delius

Duesberg

1974

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

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The structure and molecular weight of the 60-70S RNA complex and the 30-40S RNA species of Rous sarcoma virus were analyzed in an electron microscope after treatment of the RNAs with the bacteriophage T4 gene-32 protein to stretch out the RNA strands. Although all RNA preparations treated with gene-32 protein showed considerable heterogeneity in length, a significant fraction of the RNA retained its original sedimentation coefficient after treatment to allow the following conclusions to be made: The 30-40S RNA was confirmed to be a linear polynucleotide with a molecular weight of about 3 X 106. The 60-70S RNA exhibited a network structure with a molecular weight predominantly of about 6 X 106. Therefore, the subunit hypothesis for the 60-70S RNA is confirmed. A model for the structure and molecular weight of the 60-70S RNA postulates that the complex consists of two 30-40S RNA subunits held together at many points. This model elucidates the biological observation that the infectivity of RNA tumor viruses is proportional to the amount of 30-40S RNA in a virus preparation and not to the amount of 60-70S RNA.The structure and the molecular weight of tumor virus RNA have been difficult to study experimentally. The molecular weight is thought to range between a minimum of 3 X 106 and a maximum of 12 X 106 (1-10). This uncertainty is due mainly to the complex structure of the viral RNA (3) and to the difficulty in determining the exact content of RNA in purified virions (10). Extraction of the nucleic acid from virions followed by sedimentation reveals a major RNA species with a sedimentation coefficient of 60-70 S. When this RNA is placed in an environment wherein its secondary structure should be destroyed, the sedimentation coefficient decreases to 30-40 S and, at the same time, its electrophoretic mobility in sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels increases about 2-fold (11). The molecular weight of the 30-40S RNA species has been determined under denaturing conditions, and is about 3 X 106 (11). Thus, the 60-70S RNA was thought to be segmented, containing two, three, or four 30-40S RNA subunits held together by basepairing involving short complementary sequences.Although there is good, indirect, evidence for a subunit structure of the 60-70S RNA, nothing is known about how these subunits are linked. Linkage among 30-40S RNA subunits may be at only one site between two subunits, compatible with a relatively extended 60-70S RNA complex; or it may occur at several sites, compatible with a more compact, network-type 60-70S RNA complex. Furthermore, the available evidence does not exclude unequivocally that the conversion of tumor virus RNA from 60-70S to 30-40S may be due to an extreme conformational change rather than to a dissociation into subunits. Although there is no precedent for a conformational change that decreases the sedimentation coefficient but increases the electrophoretic mobility of nucleic acids, it was desirable to obtain independent evidence for the postulated subunit structu...

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Section: Methodssupporting

confidence: 76%

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Mangel

Delius

Duesberg

1974

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

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“…It was recently shown that considerable conformational changes occur in the RNA of extracellular RSV during hours of contact with the cells; RSV RNA, separated from the cells at 3-min intervals, was 30-35 S without heating, and formed the 70S component only secondarily (18). It appeared to us that terminal dephosphorylation might also take place during the customary hours of incubation of released virus in the presence of cells and their medium, and that different results might be obtained if virus for end-group analysis was also collected at 3-min intervals.…”

Section: Resultsmentioning

confidence: 99%

“…The 30-35S RNA used was obtained by heating viral RNAs at 1000 for 45 sec, followed by another sucrose gradient fractionation. The RNA, together with carrier RNA of tobacco mosaic virus (TMIV) used as a 30S marker on sucrose gradients, was hydrolyzed in 50,ul of 0.3 M KOH for 18 hr at 370 in a sealed capillary tube, and then spotted directly with absorbance markers (nucleosides and nucleotides) 44.5 cm from the anode side, and 8 cm from one edge of a sheet of Whatman 3 MM paper of 95 X 48 cm. Electrophoresis was in a refrigerated two-phase electrophoresis system, with pyridine-acetate buffer at pH 3.5 (5% v/v glacial acetic acid, 0.5% v/v pyridine, and 1 mM EDTA) at about 3000 V for 4 hr.…”

mentioning

confidence: 99%

Characterization of the End Groups of RNA of Rous Sarcoma Virus

Keith

Gleason

Fraenkel‐Conrat

1974

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

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The 3' terminus of the 30-35S RNA of Rous sarcoma virus is adenosine. Its amount indicates an average molecular weight for that RNA of about 3 X 106. The 5' terminus of 30-35S RNA of Rous sarcoma virus was not di-or triphosphorylated, whether isolated by the standard procedure or from virus collected within 3 min of its release from the cells.The chemical structure of the RNA of the oncornaviruses is still in doubt. Although much evidence favors the existence of subunit RNA molecules of 30-35 S held together by basepaired segments to form the 65-70S genome of these viruses, this concept, as well as the size of the presumed component RNA molecules and the question of whether they are identical or different, are not yet firmly established.It appeared to us that a study of the end groups of the subunits would contribute significant answers to these questions. We previously reported preliminary results (at a meeting at the University of California, Los Angeles, in 1973) that adenosine appeared to be the predominant 3'-terminal residue of the RNA of Rous sarcoma virus (RSV), in contrast to earlier reports that various oncornaviral RNAs terminated in uridine (1-3). Adenosine has been found to be the predominant terminal nucleoside of the RNA of avian myeloblastosis virus (4-6), and the finding that the oncornaviruses contained poly(A) (7), 3'-terminal in avian myeloblastosis virus (5), made this the preferred and presumed end group also for RSV, notwithstanding the reports to the contrary. We have now refined our preparative and analytical procedures sufficiently to yield consistent and reproducible results for the ratio of 3'-terminal nucleosides to internal nucleotides. The results of these analyses are that there is one 3'-unphos- (11) with bicarbonate buffer, supplemented with 1% (v/v) calf and 1% (v/v) chick serum. Typically the label was administered to 40 dishes in a ratio of 2 mCi of adenosine (labeled in the 2 position) to 1 mCi of each of guanosine, cytidine, and uridine; 1 mCi of total label was used per 10-cm petri dish. Medium containing radioactive virus was removed after 4 hr and replaced with 6 ml of the same medium but without addition of more labeled nucleosides. Collections were continued at 4-hr intervals for a period of one or two days. Virus was purified and viral RNAs were extracted as described (10). The 30-35S RNA used was obtained by heating viral RNAs at 1000 for 45 sec, followed by another sucrose gradient fractionation. The RNA, together with carrier RNA of tobacco mosaic virus (TMIV) used as a 30S marker on sucrose gradients, was hydrolyzed in 50,ul of 0.3 M KOH for 18 hr at 370 in a sealed capillary tube, and then spotted directly with absorbance markers (nucleosides and nucleotides) 44.5 cm from the anode side, and 8 cm from one edge of a sheet of Whatman 3 MM paper of 95 X 48 cm. Electrophoresis was in a refrigerated two-phase electrophoresis system, with pyridine-acetate buffer at pH 3.5 (5% v/v glacial acetic acid, 0.5% v/v pyridine, and 1 mM EDTA) at about 3000 V for 4 hr. ...

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“…Stocks of vPN-1 and vPN-2 (virus harvested from VOL. 5,1985 on May 11, 2018 by guest http://mcb.asm.org/ Downloaded from cells containing pPN-1 and pPN-2 sequences, respectively) were recovered from transfected cells as RAV-1 pseudotypes after four cell passages; the delay between transfection and harvest allowed the cultures to become fully infected.…”

Section: Kbmentioning

confidence: 99%

Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.

Norton¹,

Coffin²

1985

Mol. Cell. Biol.

217

100

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We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, j-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of 0-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.Retrovirus replication and gene expression are interwoven in a complex fashion, as are the sequences which regulate these processes. As a consequence, an understanding of the viral genome requires not only dissection of isolated functional regions, but also analysis of the manner in which different regions interact, both structurally and functionally. Studies of deletion mutants have shown that the products encoded by the viral structural genes of gag, pol, and env may be supplied in trans, and have identified certain regions of the genome which act in cis. Regions required in cis for reverse transcription of genome RNA, integration of proviral DNA, and synthesis of RNA include the long terminal repeats (LTRs) and adjacent noncoding regions (45). The LTRs which flank an integrated DNA provirus are composed of sequences derived from both the 5' and 3' ends of the RNA genome (refered to as U5 and U3, respectively) and contain signals for transcription initiation and for generation of 3' ends of viral genome and mRNA. It is presumed that the genome contains sequences which modulate the splicing of mRNAs, as not all viral transcripts are spliced; still other sequences direct the encapsidation of full-length RNA genomes. There is genetic evidence that a sequence within the untranslated leader region near the 5' end of the genome is required for packaging of Rous sarcoma virus (RSV) genomes into virions (22,39). A similar function has also been attributed to 115 bases near the 3' end of the genome (44), as well as to a 28-base near-perfect palindrome whic...

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Evidence for 30-40S RNA as Precursor of the 60-70S RNA of Rous Sarcoma Virus

Cited by 110 publications

References 19 publications

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Characterization of the End Groups of RNA of Rous Sarcoma Virus

Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.

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