A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome

Bourgaux, Pierre; Delbecchi, Louis; Yu, K; Herring, Elizabeth; Bourgaux-Ramoisy, Danielle

doi:10.1016/0042-6822(78)90291-x

Virology

1978

DOI: 10.1016/0042-6822(78)90291-x

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A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome

Pierre Bourgaux

Louis Delbecchi

K Yu

et al.

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1979

1986

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Cited by 29 publications

(27 citation statements)

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“…Firstly, do any of these molecules behave in Cyp cells as plasmids which, while maintained at a low copy number at 39°C, are readily amplified at 33°C? Secondly, is RmII produced by cells which are also capable of yielding P155 (2). Passage 11 of the colony yielded a series of clones (C10, Cll, C12, and C13) which all generate genomic viral DNA (P155) upon induction (6,18).…”

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“…Cyp cell cultures are routinely propagated at 39°C and undergo a massive cytopathic effect within 48 h of being transferred to 33°C (2). After the temperature downshift, free viral DNA accumulates from often less than 0.1 copy to as many as 104 copies per cell (6).…”

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“…The Cyp cell line resulted from the immortalization of mouse embryo cells by the tsP155 mutant of polyomavirus (Py) (2). Cyp cell cultures are routinely propagated at 39°C and undergo a massive cytopathic effect within 48 h of being transferred to 33°C (2).…”

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Alternative excision products originating from a single integration of polyomavirus DNA.

Huberdeau¹,

Sylla²,

Herring-Gillam³

et al. 1985

Mol. Cell. Biol.

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The Cyp cell line consists of mouse cells transformed by a thermoseasitive polyomavirus (Py) genome and routinely propagated at 39°C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 330C. In one subclone (C12/al/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmlI. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39°C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33°C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 390C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984).The Cyp cell line resulted from the immortalization of mouse embryo cells by the tsP155 mutant of polyomavirus (Py) (2). Cyp cell cultures are routinely propagated at 39°C and undergo a massive cytopathic effect within 48 h of being transferred to 33°C (2). After the temperature downshift, free viral DNA accumulates from often less than 0.1 copy to as many as 104 copies per cell (6). This free viral DNA has been analyzed in several clones, all derived from the same Cyp cell line. In most instances, it has been found to consist almost exclusively of genome-sized molecules of tsP155 DNA (P155) (6, 18). In one derivative (C12/al) of clone C12, however, a cyclic molecule called RmI is also produced at 33°C, generally in a 20:1 ratio to P155 (18). RmI is a hybrid made of 1.03 copies of viral DNA and a 1,628-base-pair (bp) segment of mouse DNA called Ins (3,17). Ins originates from sequences flanking the left end of the integrated viral DNA (4), which consists of a little over two copies of Py DNA arranged in a head-to-tail tandem (3, 4). RmI is the product of a recombination event occurring between the integrated viral DNA and adjacent nonhomologous cellular sequences (19).Recloning of the C12/al cells yielded subclones which were all able to produce RmI and P155 after a temperature downshift (Fig. 1A). It also led to the isolation of one subclone (S48) whose induction at 33°C generated three, instead of two, species of low-molecular-weight DNA: P155, RmI, and RmII (Fig. 1B) (Part of this work will be included in a thesis submitted by D.H. in partial fulfillment of the requirements for an M.S. degree in Microbiology from the Universitd de Sherbrooke.) MATERIALS AND METHODSCells and viruses. The origin of the Cy...

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confidence: 99%

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Alternative excision products originating from a single integration of polyomavirus DNA.

Huberdeau¹,

Sylla²,

Herring-Gillam³

et al. 1985

Mol. Cell. Biol.

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“…In these cells grown at 39°C, a transfer to 33°C resulted in the production of an active large T antigen which induced the excision-and subsequently the amplification-of DNA molecules inclusive of the viral origin (2,15). In some clones of the Cyp cell line, the predominant excision products were cyclic chimeric molecules made of specific sets of viral and cellular sequences, such as RmI and RmII (8,15).…”

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confidence: 99%

An excision event that may depend on patchy homology for site specificity.

Bourgaux-Ramoisy¹,

Gendron²,

Chartrand³

et al. 1986

Mol. Cell. Biol.

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In mouse cells transformed by a mutant polyomavirus genome, recombination between integrated viral DNA and flanking cellular DNA resulted in the excision of two readily amplifiable chimeras, designated RmI and RmII. The crossing-over that generated RmII was unique in that it involved a simple cellular sequence in which the triplet 5'-CTG-3' was repeated many times. We show that the sequence across the junction resulting from excision was identical in several molecules of RmII, as if the cross-over generating this junction always involved exactly the same two sites on the viral and cellular DNA. We also show that the cellular site mapped where the replacement of a G by an A in one of many successive 5'-CTG-3' triplets generated a homology of five nucleotides (5'-CTACT-3') with the viral site. Oligonucleotides on both sides of these sites are probably involved in matching the two DNAs prior to recombination.Animal viruses, tumor viruses in particular, have been used extensively for analyzing the mechanisms of recombination that operate in somatic cells. Thus, the structure of amplified, deleted, integrated, or excised forms of the DNA of papovaviruses has been the subject of much study (for a review, see reference 3). Generally, these studies focused on the sequences that cross over during recombination, which sometimes were found to display patchy homology and sometimes showed no homology at all (3). These findings raised the possibility that short homologies at or near the cross-over site were fortuitous and played no role in the recombination event.Recently, we have undertaken to study the recombination that occurs when cultures of mouse cells (Cyp cells) transformed by an early temperature-sensitive mutant of polyomavirus (Py) are subjected to temperature downshift. In these cells grown at 39°C, a transfer to 33°C resulted in the production of an active large T antigen which induced the excision-and subsequently the amplification-of DNA molecules inclusive of the viral origin (2, 15). In some clones of the Cyp cell line, the predominant excision products were cyclic chimeric molecules made of specific sets of viral and cellular sequences, such as RmI and RmII (8,15). These chimeras were not only characteristic of the clone of origin, but were also consistently produced after temperature downshift, as expected if the mechanism responsible for excision were specific (8,15). In one of the chimeras, RmII, this mechanism allowed the joining of a complex viral sequence to a cellular sequence in which the triplet 5'-CTG-3' was repeated many times (8).The occurrence of such a joint raises two main questions about the generation of RmII. First, do all RmII molecules include the same number of contiguous 5'-CTG-3' triplets linked to the same viral sequence? In other words, does the crossing-over that yielded RmII always involve the same two sites on the viral DNA and the simple sequence DNA, respectively? If so, what then is the molecular basis for the site specificity of the recombination event? We report here the resu...

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“…Moreover, R cells seemed to have developed a resistance to superinfection that the parent Cyp cell line did not exhibit. Therefore, virus production in R cell cultures probably reflects the occasional excision and replication of integrated viral genomes rather than a carrier state.The shedding of virus by papovavirustransformed cells is a well-documented yet poorly understood phenomenon [1][2][3][4], 10 years ago, many cell lines that were transformed by SV40 and that released virus either spontane- …”

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Induction of Virus Multiplication in Permissive Cells Transformed by a Temperature-Sensitive Polyoma Virus

Herring¹,

Bourgaux-Ramoisy²,

Bourgaux³

1980

Intervirology

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A fibroblastic cell line transformed by ts-P155, an early temperature-sensitive mutant of polyoma virus, has been isolated after infection of secondary mouse embryo cells at the restrictive temperature of 39°. These cells, designated Cyp, undergo a massive CPE accompanied by the production of infectious, temperature-sensitive virus when transferred to the nonrestrictive temperature of 33°. Several independent clones were established from cells which survived the induction of virus multiplication occurring at 33°. When routinely cultured at this temperature, these cells, termed R, displayed the growth properties of transformants and produced variable amounts of infectious, temperature-sensitive virus. This shedding of virus could not be ‘cured’ by the addition to the culture medium of antipolyoma serum which had already been found to effectively suppress virus production in Cyp cell cultures transferred to 33°. Moreover, R cells seemed to have developed a resistance to superinfection that the parent Cyp cell line did not exhibit. Therefore, virus production in R cell cultures probably reflects the occasional excision and replication of integrated viral genomes rather than a carrier state.

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A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome

Cited by 29 publications

References 34 publications

Alternative excision products originating from a single integration of polyomavirus DNA.

Alternative excision products originating from a single integration of polyomavirus DNA.

An excision event that may depend on patchy homology for site specificity.

Induction of Virus Multiplication in Permissive Cells Transformed by a Temperature-Sensitive Polyoma Virus

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