scholarly journals No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

1981 ◽  
Vol 1 (8) ◽  
pp. 763-768
Author(s):  
A Oppenheim ◽  
A T Horowitz
Keyword(s):  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
3T3 Cells ◽  
Barr Virus ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Epstein Barr ◽  
Transformed Cell Lines

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus

1981 ◽  
Vol 1 (8) ◽  
pp. 763-768
Author(s):  
A Oppenheim ◽  
A T Horowitz
Keyword(s):  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
3T3 Cells ◽  
Barr Virus ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Epstein Barr ◽  
Transformed Cell Lines

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant

1990 ◽  
Vol 10 (11) ◽  
pp. 5626-5633
Author(s):  
R Wisdom ◽  
I M Verma
Keyword(s):  
Divalent Cations ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Ras Gene ◽  
Recessive Mutations ◽  
Activating Mutation ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Rat Fibroblasts

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.

scholarly journals Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant.

1990 ◽  
Vol 10 (11) ◽  
pp. 5626-5633 ◽  
Author(s):  
R Wisdom ◽  
I M Verma
Keyword(s):  
Divalent Cations ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Ras Gene ◽  
Recessive Mutations ◽  
Activating Mutation ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Rat Fibroblasts

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.

scholarly journals Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.

1981 ◽  
Vol 1 (8) ◽  
pp. 753-762 ◽  
Author(s):  
A Oppenheim ◽  
Z Shlomai ◽  
H Ben-Bassat
Keyword(s):  
Epstein Barr Virus ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lymphoid Cells ◽  
Track Length ◽  
Replication Forks ◽  
Constant Length ◽  
Barr Virus ◽  
Epstein Barr

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus

1981 ◽  
Vol 1 (8) ◽  
pp. 753-762
Author(s):  
A Oppenheim ◽  
Z Shlomai ◽  
H Ben-Bassat
Keyword(s):  
Epstein Barr Virus ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lymphoid Cells ◽  
Track Length ◽  
Replication Forks ◽  
Constant Length ◽  
Barr Virus ◽  
Epstein Barr

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid

1981 ◽  
Vol 1 (11) ◽  
pp. 1038-1047
Author(s):  
S Kawasaki ◽  
L Diamond ◽  
R Baserga
Keyword(s):  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Acid Synthesis ◽  
Simian Virus ◽  
S Phase ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Deoxyribonucleic Acid Synthesis ◽  
G1 Cells

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

scholarly journals Simian Virus 40 T/t Antigens and Lamin A/C Small Interfering RNA Rescue the Phenotype of an Epstein-Barr Virus Protein Kinase (BGLF4) Mutant

2010 ◽  
Vol 84 (9) ◽  
pp. 4524-4533 ◽  
Author(s):  
Qiao Meng ◽  
Stacy R. Hagemeier ◽  
Chad V. Kuny ◽  
Robert F. Kalejta ◽  
Shannon C. Kenney
Keyword(s):  
Small Interfering Rna ◽  
Epstein Barr Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lamin A ◽  
T Antigens ◽  
Barr Virus ◽  
293 Cells ◽  
Epstein Barr ◽  
Interfering Rna

ABSTRACT The Epstein-Barr virus (EBV)-encoded viral protein kinase, EBV-PK (the BGLF4 gene product), is required for efficient nuclear viral egress in 293 cells. However, since EBV-PK phosphorylates a number of different viral and cellular proteins (including lamin A/C), the relative importance of each target during lytic viral replication remains unclear. We show here that an EBV PK mutant (PKmut; containing stop codons at residues 1 and 5 in EBV-PK) is highly defective for release of infectious virus from 293 cells but not 293T cells. Furthermore, the phenotype of the PKmut in 293 cells is substantially reversed by expression of the simian virus 40 (SV40) large (T) and small (t) T antigens. Efficient rescue requires the presence of both SV40 T/t proteins. We show that 293T cells have a much higher level of constitutive lamin A/C phosphorylation than do 293 cells over residues (S22 and S392) that promote phosphorylation-dependent nuclear disassembly and that both large T and small t contribute to enhanced lamin A/C phosphorylation. Finally, we demonstrate that knockdown of lamin A/C expression using small interfering RNA also rescues the PKmut phenotype in 293 cells. These results suggest that essential roles of EBV-PK during lytic viral replication include the phosphorylation and dispersion of lamin A/C.

scholarly journals Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

1987 ◽  
Vol 7 (1) ◽  
pp. 379-387 ◽  
Author(s):  
R B DuBridge ◽  
P Tang ◽  
H C Hsia ◽  
P M Leong ◽  
J H Miller ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Human Cells ◽  
Missense Mutations ◽  
Barr Virus ◽  
Vector Systems ◽  
Epstein Barr

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

scholarly journals Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

1983 ◽  
Vol 3 (3) ◽  
pp. 325-339 ◽  
Author(s):  
M Kriegler ◽  
M Botchan
Keyword(s):  
Long Terminal Repeat ◽  
Recombinant Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Sv40 Virus ◽  
Transformed Cell ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

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