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Biomedical subjects

E Wintersberger

Publications and source records attributed to E Wintersberger.

14 recordsLinked to original sources
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↗
PubMed1978

Yeast DNA polymerases: antigenic relationship, use of RNA primer and associated exonuclease activity.

Highly purified preparation of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained. 1. Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes. 2. DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalyzed by polymerase B to any significant extent. 3. Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3' end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.

DNA-Directed DNA Polymerase↗
PubMed1977

Nuclear localisation of DNA polymerase alpha and DNA synthesis in polyoma virus infected mouse cells.

Studies using inhibitors of DNA synthesis have shown that DNA polymerase alpha is located in nuclei of polyoma virus infected mouse cells to the same degree as these nuclei are engaged in DNA replication. These results indicate that either the enzyme is actively transported into nuclei concomitant with the onset of DNA synthesis, or that it is bound much more strongly in nuclei during DNA replication. In any case, these observations support the hypothesis that DNA polymerase alpha is involved in the replication of cellular and viral DNA.

Animals↗
PubMed1976

Synthesis of yeast histones in the cell cycle.

The yeast, Saccharomyces cerevisiae, contains four types of histones resembling histones H3, H2b, H2a, and H4 of animal cells. These proteins are synthesized primarily, if not exclusively, in the S-phase of the cell cycle. This result is discussed with reference to the insensitivity of ongoing DNA replication in yeast to inhibitors of protein synthesis.

Cell Division↗
PubMed1976

Induction of DNA polymerase in polyoma virus-infected mouse cells requires transcription and translation.

The induction of DNA polymerase activity and of DNA synthesis in polyoma virus-infected mouse kidney cells is inhibited by actinomycin D and cycloheximide, even in cells that are already T-antigen positive. This indicates that the induction of the DNA-synthesizing apparatus requires transcription and translation. The increase in cellular DNA polymerase activity and in the DNA synthesizing capacity follows the appearance of T-antigen only after a characteristic lag period of several hours.

Animals↗
PubMed1975

DNA polymerases in polyoma virus-infected mouse kidney cells.

Infection of arrested mouse kidney cells by polyoma virus results in the induction of the cellular 6-8S DNA polymerase activity. Levels of this enzyme increase two- to threefold in the cytoplasm but seven- to tenfold in nuclei and nuclear extract, suggesting an accumulation of the enzyme in the nucleus. Experiments using the inhibitor of DNA synthesis, fluordeoxyuridine, indicate that this accumulation is linked to active DNA synthesis. The activity and cellular distribution of the small 3.4S DNA polymerase remains unchanged.

Cell Fractionation↗