Induction of cellular thymidine kinase occurs at the mRNA level

1985 ◽  
Vol 5 (6) ◽  
pp. 1490-1497
Author(s):  
P Stuart ◽  
M Ito ◽  
C Stewart ◽  
S E Conrad
Keyword(s):  
Steady State ◽  
Thymidine Kinase ◽  
Cdna Clone ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Steady State Levels

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

scholarly journals Induction of cellular thymidine kinase occurs at the mRNA level.

1985 ◽  
Vol 5 (6) ◽  
pp. 1490-1497 ◽  
Author(s):  
P Stuart ◽  
M Ito ◽  
C Stewart ◽  
S E Conrad
Keyword(s):  
Steady State ◽  
Thymidine Kinase ◽  
Cdna Clone ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosomal Dna ◽  
Infected Cells ◽  
Steady State Levels

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

scholarly journals Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

1987 ◽  
Vol 7 (3) ◽  
pp. 1156-1163 ◽  
Author(s):  
C J Stewart ◽  
M Ito ◽  
S E Conrad
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
The Body ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Regulated Expression ◽  
Infected Cells

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889 ◽  
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis

1997 ◽  
Vol 110 (4) ◽  
pp. 489-495 ◽  
Author(s):  
I. Guenal ◽  
Y. Risler ◽  
B. Mignotte
Keyword(s):  
Steady State ◽  
Interleukin 1 ◽  
Simian Virus 40 ◽  
State Level ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Down Regulation ◽  
Actin Genes ◽  
Steady State Levels

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.

Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene

1987 ◽  
Vol 7 (3) ◽  
pp. 1156-1163
Author(s):  
C J Stewart ◽  
M Ito ◽  
S E Conrad
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
The Body ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Regulated Expression ◽  
Infected Cells

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene

1983 ◽  
Vol 3 (4) ◽  
pp. 643-653
Author(s):  
G M Santangelo ◽  
C N Cole
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Low Frequency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
African Green Monkey ◽  
Dna Fragments ◽  
Coding Region ◽  
Kinase Gene ◽  
Green Monkey

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

Gel Electrophoresis, Isoelectric Focusing, and Localization of Thymidine Kinase in Normal and Simian Virus 40-Infected Monkey Cells

Intervirology ◽  
1973 ◽  
Vol 2 (3) ◽  
pp. 137-151 ◽  
Author(s):  
Saul Kit ◽  
Wai-Choi Leung ◽  
David Trkula ◽  
Del Rose Dubbs ◽  
George Jorgensen
Keyword(s):  
Thymidine Kinase ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Infected Monkey
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