scholarly journals Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle.

1990 ◽  
Vol 10 (10) ◽  
pp. 5591-5595 ◽  
Author(s):  
J M Gudas ◽  
G B Knight ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Chinese Hamster ◽  
S Phase ◽  
3T3 Cells ◽  
Chinese Hamster Cells ◽  
Intervening Sequences ◽  
Intron Removal

Concomitant with the onset of S phase, a series of thymidine kinase (TK) splicing intermediates as well as mature TK mRNA accumulates in the nucleus of BALB/c 3T3 cells. Most of the TK splicing intermediates are retained by oligo(dT)-cellulose chromatography, and, therefore, 3' end formation and polyadenylation probably precede the splicing of TK pre-mRNAs. We have further characterized the TK pre-mRNAs that are present in the nuclei of S-phase cells by using specific probes derived from each of the six TK intervening sequences. Based on the sizes of the pre-mRNAs and their patterns of hybridization with these intron probes, we propose a pathway for intron removal from nascent TK transcripts. Intron excision occurred by a preferred, but not necessarily obligatory, order which appears to have been conserved in mouse and Chinese hamster cells.

Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle

1990 ◽  
Vol 10 (10) ◽  
pp. 5591-5595
Author(s):  
J M Gudas ◽  
G B Knight ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Chinese Hamster ◽  
S Phase ◽  
3T3 Cells ◽  
Chinese Hamster Cells ◽  
Intervening Sequences ◽  
Intron Removal

Concomitant with the onset of S phase, a series of thymidine kinase (TK) splicing intermediates as well as mature TK mRNA accumulates in the nucleus of BALB/c 3T3 cells. Most of the TK splicing intermediates are retained by oligo(dT)-cellulose chromatography, and, therefore, 3' end formation and polyadenylation probably precede the splicing of TK pre-mRNAs. We have further characterized the TK pre-mRNAs that are present in the nuclei of S-phase cells by using specific probes derived from each of the six TK intervening sequences. Based on the sizes of the pre-mRNAs and their patterns of hybridization with these intron probes, we propose a pathway for intron removal from nascent TK transcripts. Intron excision occurred by a preferred, but not necessarily obligatory, order which appears to have been conserved in mouse and Chinese hamster cells.

Control of thymidine kinase mRNA during the cell cycle

1987 ◽  
Vol 7 (8) ◽  
pp. 2925-2932
Author(s):  
D L Coppock ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Gene Transcription ◽  
Half Life ◽  
Mammalian Cells ◽  
Mrna Degradation ◽  
S Phase ◽  
3T3 Cells

To investigate the mechanism which controls the onset of DNA synthesis, we examined the regulation of thymidine kinase (TK) and its mRNA in the cell cycle. TK activity provides a useful marker for the onset of the S phase in mammalian cells. The present analysis of regulation of TK mRNA in BALB/c 3T3 cells showed that (i) the increase in TK activity depended on the availability of TK mRNA, (ii) the level of TK mRNA between G0 and S increased more than 20-fold, (iii) the rate of run-on TK transcription increased at most 2- to 4-fold between the G0 and S phases, (iv) the half-life of TK mRNA was greater than 8 to 12 h in the S and M phases and decreased as cells entered quiescence, (v) the TK mRNA increase was fully blocked by inhibition of protein synthesis by only 60%, (vi) this inhibition was completely effective for up to about 10 h following serum addition and progressively much less effective when the drugs were added later. These results suggest that the appearance of TK mRNA at the beginning of the S phase in serum-stimulated 3T3 cells is controlled not only by the rate of gene transcription but importantly also by the decreased rate of mRNA degradation. Similar mechanisms may be involved in regulation of the onset of DNA synthesis and the increase in TK mRNA since both are controlled in a manner consistent with a requirement for a labile protein.

scholarly journals Control of thymidine kinase mRNA during the cell cycle.

1987 ◽  
Vol 7 (8) ◽  
pp. 2925-2932 ◽  
Author(s):  
D L Coppock ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Gene Transcription ◽  
Half Life ◽  
Mammalian Cells ◽  
Mrna Degradation ◽  
S Phase ◽  
3T3 Cells

To investigate the mechanism which controls the onset of DNA synthesis, we examined the regulation of thymidine kinase (TK) and its mRNA in the cell cycle. TK activity provides a useful marker for the onset of the S phase in mammalian cells. The present analysis of regulation of TK mRNA in BALB/c 3T3 cells showed that (i) the increase in TK activity depended on the availability of TK mRNA, (ii) the level of TK mRNA between G0 and S increased more than 20-fold, (iii) the rate of run-on TK transcription increased at most 2- to 4-fold between the G0 and S phases, (iv) the half-life of TK mRNA was greater than 8 to 12 h in the S and M phases and decreased as cells entered quiescence, (v) the TK mRNA increase was fully blocked by inhibition of protein synthesis by only 60%, (vi) this inhibition was completely effective for up to about 10 h following serum addition and progressively much less effective when the drugs were added later. These results suggest that the appearance of TK mRNA at the beginning of the S phase in serum-stimulated 3T3 cells is controlled not only by the rate of gene transcription but importantly also by the decreased rate of mRNA degradation. Similar mechanisms may be involved in regulation of the onset of DNA synthesis and the increase in TK mRNA since both are controlled in a manner consistent with a requirement for a labile protein.

scholarly journals INTERMITTENT DNA SYNTHESIS AND PERIODIC EXPRESSION OF ENZYME ACTIVITY IN THE CELL CYCLE OF WI-38

1973 ◽  
Vol 58 (3) ◽  
pp. 564-573 ◽  
Author(s):  
Robert R. Klevecz ◽  
Leon N. Kapp
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Generation Time ◽  
Chinese Hamster ◽  
S Phase ◽  
Automated System ◽  
Chinese Hamster Cells ◽  
Synchronous Cultures ◽  
Ldh Activity ◽  
S Period

Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G1 between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.

Polyploidy Induced by X-Rays during the Cell Cycle of Chinese Hamster Cells in Vitro

1972 ◽  
Vol 52 (3) ◽  
pp. 509 ◽  
Author(s):  
C. K. Yu ◽  
W. K. Sinclair
Keyword(s):  
Cell Cycle ◽  
Chinese Hamster ◽  
X Rays ◽  
Chinese Hamster Cells

scholarly journals Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

1975 ◽  
Vol 66 (1) ◽  
pp. 95-101 ◽  
Author(s):  
K D Ley
Keyword(s):  
Time Course ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Protein S ◽  
S Phase ◽  
Supernatant Fraction ◽  
G1 Phase ◽  
Differential Centrifugation ◽  
Chinese Hamster Cells ◽  
Soluble Supernatant

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

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