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E Wintersberger

Publications and source records attributed to E Wintersberger.

At least 37 records · Page 2Linked to original sources
PubMed1993

A binding site for transcription factor E2F is a target for trans activation of murine thymidine kinase by polyomavirus large T antigen and plays an important role in growth regulation of the gene.

The promoter of the murine thymidine kinase gene contains a binding site for transcription factor E2F. Using cell lines (3T3-LT) conditionally expressing polyomavirus large T antigen from a hormone-responsive promoter and reporter gene constructs carrying the thymidine kinase promoter with intact or mutated E2F sites, we show that this E2F site is the target for trans activation by the viral protein. Transcription of the growth-regulated endogenous thymidine kinase gene can be activated in serum-starved, quiescent 3T3-LT cells by induction of T antigen. Activation of transcription from the thymidine kinase promoter requires an intact binding site for the retinoblastoma protein in the T antigen. The same promoter region was furthermore shown to play a major role in growth regulation of the gene. As several other DNA synthesis enzymes also carry E2F binding sites in their promoters, our observations suggest a common mechanism of growth regulation of these genes and that they all might be targets for trans activation by DNA tumor virus proteins.

3T3 Cells↗
PubMed1993

Distinct amounts of polyomavirus large T antigen are required for different functions of the protein.

Many cellular and some virally coded proteins regulating key events in higher cells have been found to be multifunctional. One example of such a protein is the polyomavirus large T antigen, which is involved not only in viral DNA replication and gene expression but also in the induction of the S phase in host cells, in the immortalization of various cell types and in the transactivation of some cellular genes. We recently constructed cell lines in which T antigen was synthesized under the control of a hormone-inducible promoter. This allowed us to induce different concentrations of the protein in the cell and to investigate the levels of large T antigen required for replication, S-phase induction and transactivation of a growth-regulated promoter. We found that significantly higher concentrations of large T antigen are required for the replication function of the protein than for either S phase induction or transactivation. These observations for the first time provide clear evidence for a potential regulation of different functions of a pleiotropic protein by the amount of that protein produced in the cell.

3T3 Cells↗
PubMed1992

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.

3T3 Cells↗
PubMed1991

Presence of regulatory sequences within intron 2 of the mouse thymidine kinase gene.

The intron 2 of the murine thymidine kinase (TK) gene was observed to contain two DNase hypersensitive site. In vitro footprinting experiments indicated specific binding sites for nuclear proteins which were characterized within the sequence of intron 2. Two GC boxes (binding sites for transcription factor SP1) and two new protein binding regions, one at the promoter proximal end of intron 2, the other one close to the border to exon 3 were found. Oligonucleotides were synthesized comprising the two new binding sites and were shown in gel mobility shift experiments to be capable of forming specific complexes with nuclear proteins. These proteins are present in growing as well as in quiescent cells suggesting that the sites described here do not contribute to growth regulation of TK expression. That they might play a role in upregulation of TK expression is, however, indicated by the results of CAT assays in which inclusion of downstream sequences of the TK gene containing parts or all of intron 2 were found to positively modulate the activity of the TK promoter.

Animals↗
PubMed1990

Bidirectional promoter activity of the 5' flanking region of the mouse thymidine kinase gene.

The 5' flanking region of the gene coding for cytoplasmic thymidine kinase (TK) in the mouse (a total of 490 bp upstream of the initiation codon) was tested for promoter activity using the chloramphenicol acetyltransferase gene as reporter. It was found that the region can be divided into two parts, one of which carries promoter activity in the direction of TK, whereas the 5'-half has promoter activity in the opposite direction. A fragment of 140 bp was sufficient for growth-dependent promoter activity in the direction of TK, although about 100 bp further upstream, enhanced the activity. Expression from the divergent promoter was independent of cell growth.

Animals↗
PubMed1990

The processed pseudogene of mouse thymidine kinase is active after transfection.

Aside of the gene coding for cytoplasmic thymidine kinase, the genome of mouse cells carries two pseudogenes. Both are inactive in situ. One of the pseudogenes is a processed pseudogene in which a two base pair deletion caused a shift of the reading frame and a shortening of the gene product from the 233 amino acids of thymidine kinase to 177 amino acids in the pseudogene product. We report here that introduction of this pseudogene into LTK- cells gave rise to cells with a thymidine kinase positive phenotype. The transformed cells carried multiple copies of the pseudogene the upstream region of which exhibited low but measurable promoter activity. Replacement of the upstream region of the pseudogene by a promoter of Simian virus 40 or of the mammary tumor virus resulted in high transfection efficiencies and in cell lines exhibiting high thymidine kinase activities.

Animals↗
PubMed1989

Mouse thymidine kinase: the promoter sequence and the gene and pseudogene structures in normal cells and in thymidine kinase deficient mutants.

The mouse genome carries one gene and two pseudogenes for cytoplasmic thymidine kinase. The overall structure of these genes was determined with the help of cosmids and lambda phage clones and the upstream sequence containing the promoter was determined. The data allow an allocation of bands seen in the complex patterns of genomic Southern blots obtained from the DNA of wild type cells and of thymidine kinase deficient mutants to the gene as well as to the two pseudogenes. The much used LTK cell line was found to lack the entire gene but to retain the pseudogenes. Two other TK cell lines had DNA patterns indistinguishable from the wild type. Whereas the LTK line did not produce any TKmRNA, the two other mutants had normal amounts of TKmRNA but no cytoplasmic TK activity.

Animals↗
PubMed1989

Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen.

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.

Antigens, Polyomavirus Transforming↗
PubMed1987

[Use of genetic technics in medical diagnosis exemplified by hemophilia A].

Recent years have seen the rapid development of molecular probes for the detection of defective human genes. These react either directly with the gene in question or with a DNA sequence which lies in close proximity to the gene and which exhibits polymorphism. In the case of haemophilia A two probes were found that belong to the latter category. We have used one of these probes to test its usefulness in the detection of carriers of haemophilia A. For these tests DNA was isolated from blood cells, but can, in principle, be obtained just as easily from other cells or tissues. The results of tests on several families show that the gene technological method is much more precise and sensitive than conventional methods used so far to detect carriers. In all cases a clear assertion was possible as to whether or not the person in question was a carrier of haemophilia A. The method should be applicable to prenatal diagnosis.

DNA↗
PubMed1987

Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA.

Animals↗
PubMed1986

Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts.

Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild-type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication.

Animals↗
PubMed1985

Poly ADP-ribosylation--a cellular emergency reaction?

We propose that the activation of poly(ADP-ribose) synthetase by DNA damage serves to decrease rapidly and transiently the cellular level of NAD (by production therefrom of poly ADP-ribose). The result is a slow-down of energy-requiring reactions, in particular of replicative DNA synthesis giving cells more time to repair the damage. We do not attribute any specific role to poly ADP-ribosylated proteins in this reaction beyond their action as acceptors for poly ADP-ribose.

DNA↗
PubMed1985

Homologous recombination of polyoma virus DNA in mouse cells.

We have produced nonviable deletion mutants of polyoma virus in order to study homologous recombination after DNA transfection into mouse cells. The frequency of recombination was determined by the formation of infectious virus. It was dependent on the amount of DNA transfected and the size of the region of homology between the mutations. Recombination frequencies were highest when both mutated genomes were transfected in closed circular form rather than after linearization of one or both of the recombination partners. The system described may be useful for a more detailed analysis of physiological and genetic conditions influencing the frequency of homologous recombination in mouse cells as well as to study enzymes involved and intermediates produced in this process.

Animals↗
PubMed1984

Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts.

Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control.

Animals↗
PubMed1983

Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts.

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistant cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2-4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.

Animals↗