The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells

1985 ◽  
Vol 5 (5) ◽  
pp. 1191-1194
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rat Embryo ◽  
Early Passage ◽  
Early Region ◽  
Embryo Cells ◽  
Small T Antigen ◽  
Late Region ◽  
Control Sequences

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.

scholarly journals The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1191-1194 ◽  
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rat Embryo ◽  
Early Passage ◽  
Early Region ◽  
Embryo Cells ◽  
Small T Antigen ◽  
Late Region ◽  
Control Sequences

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen

1991 ◽  
Vol 11 (4) ◽  
pp. 1996-2003 ◽  
Author(s):  
K H Scheidtmann ◽  
M C Mumby ◽  
K Rundell ◽  
G Walter
Keyword(s):  
Protein Phosphatase ◽  
P53 Protein ◽  
Protein Phosphatase 2A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
C Subunit ◽  
Phosphatase 2A ◽  
Small T Antigen

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.

Control of protein phosphatase 2A by simian virus 40 small-t antigen

1991 ◽  
Vol 11 (4) ◽  
pp. 1988-1995
Author(s):  
S I Yang ◽  
R L Lickteig ◽  
R Estes ◽  
K Rundell ◽  
G Walter ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Simian Virus 40 ◽  
Histone H1 ◽  
Simian Virus ◽  
T Antigen ◽  
Light Chains ◽  
B Subunit ◽  
Phosphatase 2A ◽  
A Subunit ◽  
Small T Antigen

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.

scholarly journals Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

1984 ◽  
Vol 4 (8) ◽  
pp. 1657-1660 ◽  
Author(s):  
A Tunnacliffe ◽  
L V Crawford ◽  
P Goodfellow
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cell ◽  
Embryonal Carcinoma Cells ◽  
Large T Antigen ◽  
Early Region

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.

scholarly journals Enumeration of the Simian Virus 40 Early Region Elements Necessary for Human Cell Transformation

2002 ◽  
Vol 22 (7) ◽  
pp. 2111-2123 ◽  
Author(s):  
William C. Hahn ◽  
Scott K. Dessain ◽  
Mary W. Brooks ◽  
Jessie E. King ◽  
Brian Elenbaas ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Human Tumor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Experimental Models ◽  
T Antigen ◽  
Human Cells ◽  
Sv40 Large T Antigen ◽  
Early Region ◽  
Intracellular Pathways

ABSTRACT While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.

scholarly journals Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts

2002 ◽  
Vol 76 (7) ◽  
pp. 3145-3157 ◽  
Author(s):  
Tina M. Beachy ◽  
Sara L. Cole ◽  
Jane F. Cavender ◽  
Mary J. Tevethia
Keyword(s):  
Amino Acids ◽  
Simian Virus 40 ◽  
Suppressive Effect ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Rat Embryo ◽  
Specific Amino Acid ◽  
Antigen Fragment ◽  
Transcriptional Transactivator

ABSTRACT Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

scholarly journals Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

1988 ◽  
Vol 8 (9) ◽  
pp. 3582-3590 ◽  
Author(s):  
X Y Fu ◽  
J D Colgan ◽  
J L Manley
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Sequence ◽  
Small T Antigen

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.

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