Simian Virus 40 -- Chinese Hamster Kidney Cell Interaction. IV. Enhanced Virus Replication in Infected Cells upon Treatment with Mitomycin C

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

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Analysis of Surface Antigens on Simian Virus 40-Transformed Cells. I. Unique Antigenicity of Simian Virus 40-Transformed Outbred Hamster Kidney Cell Lines2

JNCI Journal of the National Cancer Institute ◽

10.1093/jnci/51.1.95 ◽

1973 ◽

Vol 51 (1) ◽

pp. 95-114 ◽

Cited By ~ 16

Author(s):

Jeffrey J. Collins ◽

Paul H. Black

Keyword(s):

Kidney Cell ◽

Simian Virus 40 ◽

Surface Antigens ◽

Simian Virus ◽

Transformed Cells

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Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2428-2434.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2428-2434

Author(s):

J M Treger ◽

J Hauser ◽

K Dixon

Keyword(s):

Uv Radiation ◽

Uv Irradiation ◽

Simian Virus 40 ◽

Simian Virus ◽

Viral Dna ◽

Repair Capacity ◽

Pyrimidine Dimers ◽

Infected Cells ◽

Replicative Intermediates ◽

Kinetics Of

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.

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Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1623-1628.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1623-1628

Author(s):

M Cartier ◽

M W Chang ◽

C P Stanners

Keyword(s):

Escherichia Coli ◽

Cell Lines ◽

Animal Cell ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

Asparagine Synthetase ◽

Translational Initiation ◽

Shuttle Vectors ◽

Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

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Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes

Molecular and Cellular Biology ◽

10.1128/mcb.3.9.1598-1608.1983 ◽

1983 ◽

Vol 3 (9) ◽

pp. 1598-1608

Author(s):

R J Kaufman ◽

P A Sharp

Keyword(s):

Dihydrofolate Reductase ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

Polyadenylation Signal ◽

Ovary Cells ◽

Dividing Cells ◽

Major Late Promoter ◽

The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

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Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1623 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1623-1628 ◽

Cited By ~ 18

Author(s):

M Cartier ◽

M W Chang ◽

C P Stanners

Keyword(s):

Escherichia Coli ◽

Cell Lines ◽

Animal Cell ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

Asparagine Synthetase ◽

Translational Initiation ◽

Shuttle Vectors ◽

Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

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