scholarly journals BROMODEOXYURIDINE-INDUCED MUTATIONS IN SYNCHRONOUS CHINESE HAMSTER CELLS: TEMPORAL INDUCTION OF 6-THIOGUANINE AND OUABAIN RESISTANCE DURING DNA REPLICATION

Genetics ◽  
1978 ◽  
Vol 90 (2) ◽  
pp. 311-321
Author(s):  
H John Burki ◽  
Paul M Aebersold
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Nuclear Genes ◽  
Induced Mutations ◽  
Temporal Dependence ◽  
Chinese Hamster Cells ◽  
G2 Cells

ABSTRACT Mutations were induced in synchronous Chinese hamster cells by bromodeoxyuridine (BUdR) incorporated into cells for one-hour periods in the cell cycle. There was a very pronounced temporal dependence during the first half of the DNA synthesis period for the induction of damage leading to 6-thioguanine (6TG) and ouabain resistance. No mutants above background were induced by exposure to BUdR in G1 and G2 cells, and very few mutants were induced in the latter part of the DNA synthesis period. The peak for the induction of 6TG resistance occurs at about two hr in the DNA synthesis period; one hour later there is a peak for the induction of ouabain resistance. Both peaks occur before the time of maximum incorporation of BUdR into DNA. These results suggest that the mutagenesis by BUdR is associated with at least two nuclear genes, which replicate at two hr and three hr in the DNA synthesis period.

scholarly journals SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

1973 ◽  
Vol 58 (2) ◽  
pp. 340-345 ◽  
Author(s):  
Kenneth D. Ley ◽  
Marilyn M. Murphy
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Time Course ◽  
Nuclear Dna ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

scholarly journals REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS

1970 ◽  
Vol 47 (2) ◽  
pp. 453-459 ◽  
Author(s):  
K. D. Ley ◽  
R. A. Tobey
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
High Cell ◽  
Chinese Hamster Cells ◽  
Rapid Production ◽  
Cell Concentrations ◽  
G1 Cells ◽  
Growing Population

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G1 following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G1, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 105 cells/ml. Under similar conditions, about 4 x 10-6 M isoleucine was required for all G1-arrested cells to progress through cell division. In contrast, 1 x 10-4 M glutamine was necessary for maximum initiation of DNA synthesis in G1 cells, along with sufficient isoleucine. A technique for rapid production of G1-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G1 after 30 hr.

scholarly journals REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS

1970 ◽  
Vol 46 (1) ◽  
pp. 151-157 ◽  
Author(s):  
R. A. Tobey ◽  
K. D. Ley
Keyword(s):  
Cell Cycle ◽  
Life Cycle ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Simple Technique ◽  
Chinese Hamster ◽  
Suspension Cultures ◽  
Chinese Hamster Cells ◽  
Synchronized Cells ◽  
Biochemical Mechanisms

Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8–9 x 105 cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G1 Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.

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.

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