T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells

1984 ◽  
Vol 4 (2) ◽  
pp. 317-323
Author(s):  
L Dandolo ◽  
J Aghion ◽  
D Blangy
Keyword(s):  
Embryonal Carcinoma ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Viral Dna ◽  
Differentiated Cells ◽  
Independent Replication ◽  
Ec Cells ◽  
Early Transcription

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

scholarly journals T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

1984 ◽  
Vol 4 (2) ◽  
pp. 317-323 ◽  
Author(s):  
L Dandolo ◽  
J Aghion ◽  
D Blangy
Keyword(s):  
Embryonal Carcinoma ◽  
T Antigen ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Viral Dna ◽  
Differentiated Cells ◽  
Independent Replication ◽  
Ec Cells ◽  
Early Transcription

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

scholarly journals Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor.

1991 ◽  
Vol 115 (3) ◽  
pp. 655-664 ◽  
Author(s):  
R A Kinloch ◽  
S Mortillo ◽  
C L Stewart ◽  
P M Wassarman
Keyword(s):  
Acrosome Reaction ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Transfected Cells ◽  
Mouse Sperm ◽  
Large Numbers ◽  
Ec Cells ◽  
Sperm Receptor

Mouse and hamster sperm receptors, called mZP3 (approximately 83,000 Mr) and hZP3 (approximately 56,000 Mr), respectively, are glycoproteins located in the ovulated egg zona pellucida. Certain of the glycoprotein O-linked oligosaccharides are essential for sperm receptor activity. Here, we transfected mouse embryonal carcinoma (EC) cells with mZP3 and hZP3 genes placed under control of a constitutive promoter. Transfected cells synthesized and secreted large amounts of the glycoproteins, called EC-mZP3 and EC-hZP3. Although the primary structures of mZP3 and hZP3 polypeptides (44,000 Mr) are very similar to one another, EC-mZP3 (approximately 83,000 Mr) and EC-hZP3 (approximately 49,000 Mr) were glycosylated to very different extents, such that they resembled their egg counterparts. Like egg mZP3, EC-mZP3 inhibited binding of sperm to ovulated eggs and induced sperm to acrosome-react in vitro. In addition, large numbers of sperm bound to aggregates of mZP3-transfected EC cells in vitro. On the other hand, unlike egg hZP3, EC-hZP3 did not exhibit either sperm receptor or acrosome reaction-inducing activity, and sperm failed to bind to aggregates of hZP3-transfected EC cells. Thus, transfected EC cells not only express sperm receptor genes, but also discriminate between very similar polypeptides with respect to glycosylation and, in the case of mZP3, add specific oligosaccharides essential for biological activity. In addition, the results demonstrate that EC cells can serve as a source for large amounts of functional mouse sperm receptor.

New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells

1986 ◽  
Vol 6 (11) ◽  
pp. 3920-3927
Author(s):  
K Ariizumi ◽  
H Ariga
Keyword(s):  
Embryonal Carcinoma ◽  
Regulatory Region ◽  
Host Cells ◽  
Circular Dna ◽  
New Class ◽  
Differentiated Cells ◽  
Copy Numbers ◽  
Undifferentiated Cells ◽  
Ec Cells ◽  
F9 Embryonal Carcinoma Cells

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

scholarly journals Apoptosis of Human Embryonal Carcinoma Cells with in vitro Differentiation.

1996 ◽  
Vol 21 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Taketo Yamada ◽  
Nao Suzuki ◽  
Nobuyoshi Hiraoka ◽  
Kentaroh Matsuoka ◽  
Sachiko Fukushima ◽  
...  
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
In Vitro Differentiation

scholarly journals Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells

1993 ◽  
Vol 13 (9) ◽  
pp. 5538-5548
Author(s):  
Y C Choi ◽  
C B Chae
Keyword(s):  
Embryonal Carcinoma ◽  
Cpg Island ◽  
Cpg Islands ◽  
High Density ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cpg Dinucleotides ◽  
F9 Embryonal Carcinoma Cells ◽  
Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

Wheat germ agglutinin chromatography of GlcNacβ1-3(GlcNAcβl-6)Gal and GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans

1990 ◽  
Vol 68 (1) ◽  
pp. 44-53 ◽  
Author(s):  
Antti Seppo ◽  
Leena Penttilä ◽  
Anne Makkonen ◽  
Anne Leppänen ◽  
Ritva Niemelä ◽  
...  
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Embryonal Carcinoma ◽  
Periodate Oxidation ◽  
Carcinoma Cells ◽  
Teratocarcinoma Cell ◽  
Partial Acid Hydrolysis ◽  
In Vitro Synthesis ◽  
In Vitro Biosynthesis

GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Gal and GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N- acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.Key words: GlcNAcβ1-3(GlcNAcβ1-6)Gal, GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc, wheat germ agglutinin – agarose chromatography, in vitro biosynthesis, teratocarcinoma cell.

Differential effects of epidermal growth factor (EGF) on cell locomotion and cell proliferation in a cloned human embryonal carcinoma-derived cell line in vitro

1986 ◽  
Vol 86 (1) ◽  
pp. 47-55
Author(s):  
W. Engstrom
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Embryonal Carcinoma ◽  
Carcinoma Cells ◽  
Colony Diameter ◽  
Cell Locomotion ◽  
Epidermal Growth

The effects of epidermal growth factor (EGF) on clones from a human embryonal carcinoma-derived cell line (Tera-2) have been studied. Cells were plated at clonal densities, whereafter the effects of serum and EGF on cell locomotion and cell proliferation were examined. The addition of 50 ngEGF ml-1 resulted in increased migration, as judged by increased colony diameter in the presence of EGF. However, the effect of EGF on cell locomotion was rarely accompanied by any effect on cell proliferation. It was concluded that EGF exerts a preferential effect on cell migration in human embryonal carcinoma cells in vitro.

Control of beta-interferon expression in murine embryonal carcinoma F9 cells

1989 ◽  
Vol 9 (8) ◽  
pp. 3553-3556
Author(s):  
M K Francis ◽  
J M Lehman
Keyword(s):  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Biologically Active ◽  
Rnase Protection ◽  
F9 Cells ◽  
Differentiated Cells ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory ◽  
Tissue Culture Model

Murine embryonal carcinoma F9 cells, a tissue culture model for early embryonic development, do not produce interferon (IFN) in response to poly(I-C), as determined by an antiviral assay. RNase protection analyses were used to examine total RNA extracted from the cells for the presence of beta-IFN RNA. Whereas F9 cells differentiated in vitro with retinoic acid produced a biologically active protein as well as beta-IFN RNA in response to poly(I-C), undifferentiated F9 cells produced no detectable beta-IFN RNA even in the presence of cycloheximide, an IFN-superinducing agent. These results show that undifferentiated embryonal carcinoma cells do not accumulate beta-IFN RNA in response to an IFN-inducing agent, suggesting a transcriptional regulatory mechanism. However, this control mechanism is altered upon differentiation, since the gene can be transcriptionally activated in retinoic acid-differentiated cells.

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.

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