Regulation of Initiation of Dna Synthesis in Chinese Hamster Cells

Ley, Kenneth D.; Tobey, Robert A.

doi:10.1083/jcb.47.2.453

Cited by 246 publications

(48 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The isoleucine (lle) 1 deprivation technique for synchronizing Chinese hamster cells in G~ phase (13,24) has provided one means of determining some of the sequential biochemical events that occur during G~ progression (22).…”

mentioning

confidence: 99%

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

Ley¹

1975

The Journal of Cell Biology

View full text Add to dashboard Cite

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 GI phase of the cell cycle. Cells arrested in Ga by isoleucine (lie) deprivation were prelabeled with [l~C]Ile, induced to traverse G~ by addition of unlabeled lie, and labeled with [gH]Ile at hourly intervals. Cells were fractionated into nuclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G~ were similar to those from cells arrested in G~ except for the presence of a major peak of [3H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [SH]Ile incorporation were not detected in nuclear preparations. Cellular fractionation by differential centrifugation showed that 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 G, 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 GI 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.Elucidation of biochemical events which occur during the Go ~ G~ transition and subsequent traverse of the G~ phase of the cell cycle may contribute to our understanding of how initiation of DNA synthesis is regulated. The isoleucine (lle) 1 deprivation technique for synchronizing Chinese hamster cells in G~ phase (13, 24) has provided one means of determining some of the sequential biochemical events that occur during G~ progression (22).1 Abbreviations used in this paper." lie, isoleucine; SDS, sodium dodecyl sulfate; TdR, thymidine.Chinese hamster cells (line CHO) deprived of Ile accumulate in G~ until resupplied with adequate amounts of lie, at which time the cells resume traverse of GI, initiate nuclear and mitochondrial DNA synthesis (12), and divide in synchrony. The effect of lie deprivation on DNA synthesis is rapid; synthesis is reduced to 44% within 1 h, whereas synthesis of RNA and protein ts relatively unaffected (4). DNA synthesis in CHO cells is dependent upon a relatively sharp threshold concentration of lie. Upon fourfold reduction of the minimum concentration of lie required for all G~ cells to initiate DNA synthesis, virtually all cells are

show abstract

mentioning

confidence: 99%

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

Ley¹

1975

The Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…When the cells were synchronized by isoleucine deprivation with the method of Ley and Tobey [15], control and DMSO-treated cells grew synchronously for three cell generations in almost 50 h (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Changes in the glycoprotein composition of plasma membrane during the differentiation of friend erythroleukemia cells

Bosman

Boer

Steyn-Parvé

1982

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

“…increase in cell number because cells initially in S, G2, or M, when deprived of isoleucine, continued to traverse the cell cycle until they reached G I phase (Ley and Tobey, 1970) . Time-course patterns of incorporation of [3H]TdR into nuclear DNA and mit-DNA before and after release of cells from G I arrest by addition of isoleucine are presented in Fig.…”

Section: Effect Of Isoleucine Deprivation On Mit-dna Synthesismentioning

confidence: 99%

“…No PPLO were detected during these studies . CHO cells were synchronized by depriving them of isoleucine as previously described (Ley and Tobey, 1970 ;Tobey and Ley, 1971) . After 30-32 h in isoleucine-free F-10 medium which contained twice the usual concentration of glutamine and 15% dialyzed serum, the cells were released from Gt arrest either by adding 2 X 10 -5 M isoleucine or resuspending them in complete F-10 medium .…”

Section: Cell Culturementioning

confidence: 99%

“…Chinese hamster cells, line CHO, can be synchronized in suspension culture by depriving them of isoleucine (Ley and Tobey, 1970 ;Tobey and Ley, 1971) . These cells are arrested in the Gl phase of their cell cycle and are unable to initiate nuclear DNA replication until supplied with isoleucine, at which time the cells reinitiate cell cycle traverse, replicate their DNA, and divide in synchrony .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Synchronization of Mitochondrial Dna Synthesis in Chinese Hamster Cells (Line Cho) Deprived of Isoleucine

Ley

Murphy

1973

The Journal of Cell Biology

View full text Add to dashboard Cite

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 G i 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 .

show abstract

Regulation of Initiation of Dna Synthesis in Chinese Hamster Cells

Cited by 246 publications

References 10 publications

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

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

Changes in the glycoprotein composition of plasma membrane during the differentiation of friend erythroleukemia cells

Synchronization of Mitochondrial Dna Synthesis in Chinese Hamster Cells (Line Cho) Deprived of Isoleucine

Contact Info

Product

Resources

About