scholarly journals Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells

1985 ◽  
Vol 225 (2) ◽  
pp. 529-533 ◽  
Author(s):  
A J Strain ◽  
W A H Wallace ◽  
A H Wyllie
Keyword(s):  
Cell Cycle ◽  
Gene Transfer ◽  
Transfection Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
Sv40 Virus ◽  
G2 Phase ◽  
Cycle Dependent ◽  
Sv40 Dna

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.

scholarly journals Phosphorylation of Simian Virus 40 T Antigen on Thr 124 Selectively Promotes Double-Hexamer Formation on Subfragments of the Viral Core Origin

2000 ◽  
Vol 74 (18) ◽  
pp. 8601-8613 ◽  
Author(s):  
Brett A. Barbaro ◽  
K. R. Sreekumar ◽  
Danielle R. Winters ◽  
Andrea E. Prack ◽  
Peter A. Bullock
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large Tumor ◽  
Sv40 Origin ◽  
Cycle Dependent ◽  
Sv40 Dna

ABSTRACT Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr→Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.

scholarly journals Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

1990 ◽  
Vol 10 (12) ◽  
pp. 6586-6595 ◽  
Author(s):  
P A Hamel ◽  
B L Cohen ◽  
L M Sorce ◽  
B L Gallie ◽  
R A Phillips
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Large T Antigen ◽  
T Complex ◽  
Cycle Dependent

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.

Cell cycle-dependent expression of early viral genes in one group of simian virus 40-transformed rat cells.

1984 ◽  
Vol 3 (11) ◽  
pp. 2587-2591 ◽  
Author(s):  
J. Imbert ◽  
J.J. Lawrence ◽  
F. Coulier ◽  
E. Jeunet ◽  
V. Billotey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Genes ◽  
Cycle Dependent

scholarly journals Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

1987 ◽  
Vol 7 (3) ◽  
pp. 1156-1163 ◽  
Author(s):  
C J Stewart ◽  
M Ito ◽  
S E Conrad
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
The Body ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Regulated Expression ◽  
Infected Cells

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

scholarly journals The uptake and stability of simian-virus-40 DNA after calcium phosphate transfection of CV-1 cells

1984 ◽  
Vol 218 (2) ◽  
pp. 475-482 ◽  
Author(s):  
A J Strain ◽  
A H Wyllie
Keyword(s):  
Calcium Phosphate ◽  
Transfection Efficiency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Fraction ◽  
Optimum Conditions ◽  
Viral Plaque ◽  
Sv40 Dna ◽  
Calcium Phosphate Transfection

The uptake and fate of purified SV40 (Simian virus 40) DNA, transfected into permissive CV-1 cells by calcium phosphate precipitates, was examined. By using a viral plaque assay, optimum conditions for transfection were established and transfection efficiencies of up to 10(6) plaque-forming units/micrograms of SV40 DNA were obtained. After a 2h exposure to 3H-labelled SV40 DNA-calcium phosphate co-precipitates under basal conditions, up to 7% of the input DNA became cell-associated, with approx. 4% reaching the nuclear fraction. This value was never exceeded, even under conditions known to enhance significantly the ultimate transfection efficiency, such as increased exposure time, addition of carrier DNA or treatment with DMSO (dimethyl sulphoxide) or glycerol. Substantial degradation of this SV40 DNA occurred within a further 4h, apparently in both nucleus and cytoplasm. Degradation of form-II and form-III SV40 DNA, which have lower transfection efficiencies than form-I DNA, was no more rapid than degradation of form-I DNA. The results indicate that less than 0.5% of the transfected DNA which reached the nucleus is protected from nuclease attack. The mechanism of action of agents such as glycerol, DMSO or carrier DNA remains obscure, but they may be involved in conferring greater stability to the intracellular SV40 DNA rather than merely affecting its rate of entry into the cell.

scholarly journals Permanent expression of p53 in FR 3T3 rat cells but cell cycle-dependent association with large-T antigen in simian virus 40 transformants.

1985 ◽  
Vol 4 (13A) ◽  
pp. 3413-3418 ◽  
Author(s):  
F. Coulier ◽  
J. Imbert ◽  
J. Albert ◽  
E. Jeunet ◽  
J.J. Lawrence ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Cycle Dependent

scholarly journals Overproduction of Rb protein after the G1/S boundary causes G2 arrest

1993 ◽  
Vol 13 (11) ◽  
pp. 6640-6652
Author(s):  
V Karantza ◽  
A Maroo ◽  
D Fay ◽  
J M Sedivy
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Single Amino Acid ◽  
Permissive Temperature ◽  
Wild Type ◽  
Facs Analysis ◽  
Rb Protein ◽  
G2 Phase

The Rb protein is known to exert its activity at decision points in the G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of rapid inducible gene amplification to conditionally overexpress Rb protein during G2 phase. A cell line expressing a temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing the Rb cDNA linked to the simian virus 40 origin of replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDNA, a frameshift mutation close to the beginning of the Rb coding region, and a single-amino-acid deletion in the E1A/T-Ag binding pocket, respectively. Numerous independent cell lines were isolated at the nonpermissive temperature; cell lines displaying a high level of episomal amplification of an intact Rb expression cassette following shiftdown to the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activated cell sorting (FACS) analysis revealed that cultures induced to overexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB-fs or rearranged pRB-wt and did not express Rb protein had normal cell cycle profiles. Double-label FACS analysis showed that cells overexpressing Rb or Rb-Dra proteins were uniformly accumulating in G2, whereas cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 phase.

scholarly journals SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Fanghua Li ◽  
Emil Mladenov ◽  
Sharif Mortoga ◽  
George Iliakis
Keyword(s):  
Cell Cycle ◽  
Cell Survival ◽  
S Phase ◽  
Repair Pathway ◽  
End Joining ◽  
Alternative End Joining ◽  
G2 Phase ◽  
Dna End Resection ◽  
Cycle Dependent ◽  
End Resection

Abstract The cell cycle-dependent engagement of DNA-end resection at DSBs is regulated by phosphorylation of CTIP by CDKs, the central regulators of cell cycle transitions. Cell cycle transitions are also intimately regulated by protein degradation via two E3 ubiquitin ligases: SCFSKP2 and APC/CCDH1 complex. Although APC/CCDH1 regulates CTIP in G1– and G2-phase, contributions by SCFSKP2 have not been reported. We demonstrate that SCFSKP2 is a strong positive regulator of resection. Knockdown of SKP2, fully suppresses resection in several cell lines. Notably, this suppression is G2-phase specific and is not observed in S-phase or G1–phase cells. Knockdown of SKP2 inactivates SCFSKP2 causing APC/CCDH1 activation, which degrades CTIP. The stabilizing function of SCFSKP2 on CTIP promotes resection and supports gene conversion (GC), alternative end joining (alt-EJ) and cell survival. We propose that CDKs and SCFSKP2-APC/CCDH1 cooperate to regulate resection and repair pathway choice at DSBs in G2-phase.

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