Mapping unintegrated avian sarcoma virus DNA: Termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA

Shank, Peter R.; Hughes, Stephen H.; Kung, Hsing Jien; Majors, John; Quintrell, Nancy; Guntaka, Ramareddy V.; Bishop, J. Michael; Varmus, Harold

doi:10.1016/0092-8674(78)90063-6

Cited by 391 publications

(239 citation statements)

References 35 publications

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“…LTR are composed of a sequence derived from the 3' end and another one from the 5' end of the RNA genome. The linear DNA molecules mature as nonsegmented, fully doublestranded molecules and appear to be converted in the nucleus into two species of covalently closed circular viral DNAs (20,32), one shorter species harboring one LTR copy and the larger species harboring two LTR copies (31,42). Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins.…”

mentioning

confidence: 99%

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Richter¹,

Ozer²,

DesGroseillers³

et al. 1984

Mol. Cell. Biol.

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To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39°C) during the early part of the virus cycle (between 0-to 20-h postinfection) greatly inhibited virus production. This The replication of the RNA genome of retroviruses by reverse transcriptase is a complex process. Linear doublestranded DNA molecules with segmented (+) strand are first synthesized with a redundant sequence at each end, the long terminal repeats (LTR) (for review, see reference 36). LTR are composed of a sequence derived from the 3' end and another one from the 5' end of the RNA genome. The linear DNA molecules mature as nonsegmented, fully doublestranded molecules and appear to be converted in the nucleus into two species of covalently closed circular viral DNAs (20, 32), one shorter species harboring one LTR copy and the larger species harboring two LTR copies (31, 42). Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins. Indeed, infectious double-stranded linear viral DNA, as well as circular viral DNA with one LTR copy can be synthesized in vitro by purified virions (1,2,5,6,10). This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies. Most importantly, the infectivity of viral DNA made in vitro is several orders of magnitude lower than that of viral DNA made early during infection (10,23,28). This suggests that cell factors are required during the reverse transcription process. Other experiments also indicate that the cell provides essential * Corresponding author.factors during some steps of reverse transcription. Serum starvation has been shown to markedly affect the production of unintegrated viral DNA in newly infected avian (7,8,37) or murine (14) cells. Cycloheximide added early after infection prevented the formation of both species of supercoiled viral DNA, suggesting that newly synthesized proteins are required to complete this step (41). The treatment of newly infected murine (4, 13) or avian (16) cells with aphidicolin, an inhibitor of cellular DNA polymerase a (17) and 5 (24), also prevented the formation of supercoiled viral DNA, suggesting that cellular DNA polymerases may be involved at this step of the virus cycle.Although very suggestive, these experiments do not permit one to distinguish between a number of factors possibly affected by the inhibitors. Also, it is not clear whether the effect of these treatments is directly on retroviral DNA synthesis or only indirectly influencing it. It would therefore be helpful to identify the cellular factors required in the reverse transcription process by using cell mutan...

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mentioning

confidence: 99%

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Richter¹,

Ozer²,

DesGroseillers³

et al. 1984

Mol. Cell. Biol.

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“…These circular DNA molecules were initially described in nuclei of avian sarcoma virus-infected cells (19,(41)(42)(43)49). These closed circular forms that are formed upon nuclear entry of the linear DNA molecule are unnecessary for integration, although a recent study suggested that under some circum-stances the viral IN may use the two-LTR circle junction as a template for integration into the host genome particularly if it is the only, or predominant, species delivered into the nucleus (34).…”

Section: Discussionmentioning

confidence: 99%

“…One-and two-LTR circles are believed to result from homologous recombination between the LTRs or from direct end-to-end joining of the linear DNA, respectively (41). These circular forms are often defined as "dead-end" products because they were described as ineligible for integration (5).…”

mentioning

confidence: 99%

“…In addition to linear DNA, other forms of viral DNA were described, and at least two circular forms containing one and two LTRs resulting from the linear DNA circularization were found in the nucleus (41,42). One-and two-LTR circles are believed to result from homologous recombination between the LTRs or from direct end-to-end joining of the linear DNA, respectively (41).…”

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confidence: 99%

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Early Detection of a Two-Long-Terminal-Repeat Junction Molecule in the Cytoplasm of Recombinant Murine Leukemia Virus-Infected Cells

Serhan

Penaud

Petit

et al. 2004

J Virol

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We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-longterminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

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“…To estimate the amount of DNA that can be transferred stably to DBM-paper, trace amounts of 32P-labeled HindIII restriction fragments of A JaDZ-Vir were fractionated by electrophoresis in the presence or absence of [10][11][12][13][14][15] tested by using DNA from mutant hamster cells that have multiple copies of the gene encoding a multifunctional protein that catalyzes the first three steps of UMP synthesis (10). The probe was prepared from a hybrid plasmid containing a 2.3-kb insert complementary to the 3'-proximal region of the mRNA for this protein (10).…”

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confidence: 99%

Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

Wahl¹,

Stern²,

Stark³

1979

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

2,898

865

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We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [. G. Wetmur (1975) Biopolymers 14, [2517][2518][2519][2520][2521][2522][2523][2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.Southern's procedure (1) for transferring restriction fragments from agarose gels to nitrocellulose has been an essential part of recent advances in analyzing and purifying many DNA fragments and has also been used more recently to identify mutant globin genes in individuals with thalessemia (2) or sickle cell trait (3). However, several aspects of this method have not yet been optimized: (i) Large restriction fragments are not transferred from agarose gels efficiently, and fragments smaller than 0.3-0.5 kilobase (kb) do not bind well to nitrocellulose. (if) Some of the DNA bound noncovalently to nitrocellulose is removed by stringent posthybridization washes, reducing the intensity of the signal and limiting repeated uses of the transfer. (iii) The technique is relatively slow; up to 2 weeks can be required to detect fragments derived from unique genes of higher eukaryotes.One modification of Southern's technique follows from our previous work, which showed that RNA and single-stranded DNA could be linked covalently to diazobenzyloxymethyl (DBM)-cellulose and used in hybridization reactions (4), that RNA could be transferred from agarose gels to DBM-paper (5), and that small fragments of DNA could be transferred from composite agarose-acrylamide gels to DBM-paper (6). Transfer of DNA fragments of any size from agarose gels to paper has now been optimized. An important improvement follows from Wetmur's finding (7) that 10% dextran sulfate greatly accelerates hybridization reactions in solution. This anionic polymer gives an even larger acceleration in a two-phase system with double-stranded, randomly cleaved, denatured DNA as probe.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3683MATERIALS AND METHODS General Methods. Conditions for cleaving the DNA samples with restriction enzymes, for separating the fragments ,according to size by electrophoresis in agarose gels (8), and for preparing n...

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Mapping unintegrated avian sarcoma virus DNA: Termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA

Cited by 391 publications

References 35 publications

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Early Detection of a Two-Long-Terminal-Repeat Junction Molecule in the Cytoplasm of Recombinant Murine Leukemia Virus-Infected Cells

Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

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