scholarly journals The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells

1993 ◽  
Vol 291 (1) ◽  
pp. 315-322 ◽  
Author(s):  
W D Rees ◽  
S M Hay
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Eukaryotic Expression ◽  
Cell Protein ◽  
3T3 Cells ◽  
Threonine Synthase ◽  
Homoserine Kinase ◽  
A Cell

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

scholarly journals Expression of Escherichia coli homoserine kinase in mouse 3T3 cells

1992 ◽  
Vol 281 (3) ◽  
pp. 865-870 ◽  
Author(s):  
W D Rees ◽  
S M Hay ◽  
H J Flint
Keyword(s):  
Escherichia Coli ◽  
Animal Cell ◽  
Intact Cell ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Eukaryotic Expression ◽  
3T3 Cells ◽  
E Coli ◽  
Homoserine Kinase ◽  
Neomycin Resistance

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

Energy requirement for degradation of tumor-associated protein p53

1984 ◽  
Vol 4 (3) ◽  
pp. 442-448
Author(s):  
R M Gronostajski ◽  
A L Goldberg ◽  
A B Pardee
Keyword(s):  
Energy Requirement ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Total Cell ◽  
Cell Protein ◽  
3T3 Cells ◽  
Protein P53 ◽  
Mouse Sarcoma ◽  
Tumorigenic Cells ◽  
Sarcoma Cells

A 53,000-dalton protein (p53) present in large amounts in several types of tumorigenic cells was rapidly degraded in nontumorigenic BALB/c 3T3 fibroblasts (t 1/2, approximately 0.5 h) but not in tumorigenic methylcholanthrene-induced mouse sarcoma cells (t 1/2, greater than 2 h). In 3T3 cells, dinitrophenol and 2-deoxyglucose, agents which reduce ATP production, inhibited the rapid degradation of p53 and the slower breakdown of total cell protein. After removal of these agents, the degradation of both p53 and total cell proteins resumed at their normal rates. Inhibitors of intralysosomal proteolysis (Ep475 and chloroquine) did not reduce the rate of degradation of p53. Thus, in 3T3 cells, p53 appears to be degraded by a nonlysosomal, ATP-dependent proteolytic system similar to that previously shown to degrade short- and long-lived proteins in growing fibroblasts. The immunoreactive p53 which remained in ATP-depleted cells had the same molecular weight as the p53 in the control cells. No intermediate products of p53 degradation were detected by immunoprecipitation in either ATP-depleted or control cells. Hence, ATP seems to be required for an initial step in the degradation of p53. Although the amount of labeled p53 was increased in simian virus 40-transformed and methylcholanthrene-induced mouse sarcoma cells, the amount of p53 labeled during a 3-h pulse in Moloney virus- and Rous sarcoma virus-transformed cells and untransformed 3T3 cells was similar. Thus, an increased net rate of p53 accumulation is not a common feature of transformed tumorigenic cells.

scholarly journals Energy requirement for degradation of tumor-associated protein p53.

1984 ◽  
Vol 4 (3) ◽  
pp. 442-448 ◽  
Author(s):  
R M Gronostajski ◽  
A L Goldberg ◽  
A B Pardee
Keyword(s):  
Energy Requirement ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Total Cell ◽  
Cell Protein ◽  
3T3 Cells ◽  
Protein P53 ◽  
Mouse Sarcoma ◽  
Tumorigenic Cells ◽  
Sarcoma Cells

A 53,000-dalton protein (p53) present in large amounts in several types of tumorigenic cells was rapidly degraded in nontumorigenic BALB/c 3T3 fibroblasts (t 1/2, approximately 0.5 h) but not in tumorigenic methylcholanthrene-induced mouse sarcoma cells (t 1/2, greater than 2 h). In 3T3 cells, dinitrophenol and 2-deoxyglucose, agents which reduce ATP production, inhibited the rapid degradation of p53 and the slower breakdown of total cell protein. After removal of these agents, the degradation of both p53 and total cell proteins resumed at their normal rates. Inhibitors of intralysosomal proteolysis (Ep475 and chloroquine) did not reduce the rate of degradation of p53. Thus, in 3T3 cells, p53 appears to be degraded by a nonlysosomal, ATP-dependent proteolytic system similar to that previously shown to degrade short- and long-lived proteins in growing fibroblasts. The immunoreactive p53 which remained in ATP-depleted cells had the same molecular weight as the p53 in the control cells. No intermediate products of p53 degradation were detected by immunoprecipitation in either ATP-depleted or control cells. Hence, ATP seems to be required for an initial step in the degradation of p53. Although the amount of labeled p53 was increased in simian virus 40-transformed and methylcholanthrene-induced mouse sarcoma cells, the amount of p53 labeled during a 3-h pulse in Moloney virus- and Rous sarcoma virus-transformed cells and untransformed 3T3 cells was similar. Thus, an increased net rate of p53 accumulation is not a common feature of transformed tumorigenic cells.

scholarly journals Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells

1983 ◽  
Vol 214 (3) ◽  
pp. 695-702 ◽  
Author(s):  
W Engström ◽  
A Zetterberg
Keyword(s):  
Dna Synthesis ◽  
Time Course ◽  
Simian Virus 40 ◽  
Complete Removal ◽  
Simian Virus ◽  
Phosphate Concentration ◽  
Serum Starvation ◽  
Cell Multiplication ◽  
Inhibitory Effects ◽  
3T3 Cells

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.

scholarly journals Simian Virus 40 Late Proteins Possess Lytic Properties That Render Them Capable of Permeabilizing Cellular Membranes

2006 ◽  
Vol 80 (13) ◽  
pp. 6575-6587 ◽  
Author(s):  
Robert Daniels ◽  
Nasser M. Rusan ◽  
Anne-Kathrin Wilbuer ◽  
Leonard C. Norkin ◽  
Patricia Wadsworth ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasma Membranes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cellular Membranes ◽  
Bacterial Membranes ◽  
Cell Death Pathways ◽  
Late Protein ◽  
Temporally Correlated ◽  
Infectious Cycle

ABSTRACT Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

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.

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