Effects of isoleucine deficiency on nucleic acid and protein metabolism in cultured Chinese hamster cells. Continued ribonucleic acid and protein synthesis in the absence of deoxyribonucleic acid synthesis

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

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 94%

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

Ley¹

1975

“…Such cells have several features in common with "Go"-arrested cells in vivo (20), the most notable of which is arrest in the pre-DNA synthetic phase of the cell cycle for prolonged periods under conditions in which biosynthetic capacities remain at high levels without causing cells to enter a state of gross biochemical imbalance (21) . Reversal of arrest was accomplished by restoration of isoleucine to the culture .…”

Section: Timing Of Phosphorylation and Synthesis Of Histone F1mentioning

confidence: 99%

Cell Cycle-Specific Changes in Histone Phosphorylation Associated With Cell Proliferation and Chromosome Condensation

Gurley

Walters

Tobey

1974

221

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO) . Results showed that histone fl phosphorylation, absent in GI-arrested and early GI-traversing cells, commences 2 h before entry of traversing cells into the S phase . It is concluded that fl phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old fi before synthesis of new fl during the S phase . Results also showed that f3 and a subfraction of fl were rapidly phosphorylated only during the time when cells were crossing the G 2/M boundary and traversing prophase .Since these phosphorylation events do not occur in G I , S, or G 2 and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes . This conclusion is supported by loss of prelabeled 32 P04 from those specific histone fractions during transition of metaphase cells into interphase G, cells . A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of fi phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and fl phosphorylation-mediated chromosome condensation .

“…G1 arrest following isoleucine (Ile) deficiency has been particularly well studied in these cells (3) and was used here for purposes of contrast.…”

mentioning

confidence: 99%

Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

Weissfeld¹,

Rouse²

1977