Temporal order of gene replication in Chinese hamster ovary cells

1989 ◽  
Vol 9 (7) ◽  
pp. 2881-2889
Author(s):  
J Taljanidisz ◽  
J Popowski ◽  
N Sarkar
Keyword(s):  
Cell Cycle ◽  
Nuclear Dna ◽  
Chinese Hamster Ovary Cells ◽  
Repetitive Sequences ◽  
Chinese Hamster ◽  
Nuclear Dna Content ◽  
Single Copy ◽  
S Phase ◽  
Specific Gene ◽  
Cell Permeabilization

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

scholarly journals Temporal order of gene replication in Chinese hamster ovary cells.

1989 ◽  
Vol 9 (7) ◽  
pp. 2881-2889 ◽  
Author(s):  
J Taljanidisz ◽  
J Popowski ◽  
N Sarkar
Keyword(s):  
Cell Cycle ◽  
Nuclear Dna ◽  
Chinese Hamster Ovary Cells ◽  
Repetitive Sequences ◽  
Chinese Hamster ◽  
Nuclear Dna Content ◽  
Single Copy ◽  
S Phase ◽  
Specific Gene ◽  
Cell Permeabilization

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

scholarly journals Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

1981 ◽  
Vol 1 (7) ◽  
pp. 594-599 ◽  
Author(s):  
J J Harada ◽  
D R Morris
Keyword(s):  
Cell Cycle ◽  
Chinese Hamster Ovary ◽  
Doubling Time ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Limited Growth ◽  
Ovary Cells ◽  
Index Analysis ◽  
Thymidine Autoradiography

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.

scholarly journals The Complex Cell Cycle of the Dinoflagellate Protoctist Crypthecodinium Cohnii as Studied In Vivo and by Cytofluorimetry

1991 ◽  
Vol 100 (3) ◽  
pp. 675-682 ◽  
Author(s):  
YVONNE BHAUD ◽  
JEAN-MARIE SALMON ◽  
MARIE-ODILE SOYER-GOBILLARD
Keyword(s):  
Cell Cycle ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
S Phase ◽  
Vegetative Cells ◽  
Crypthecodinium Cohnii ◽  
Daughter Cells ◽  
The Times

The complete cell cycle of the dinoflagellate Crypthecodinium cohnii Biecheler 1938 was observed in vivo in a synchronized heterogeneous population, after DAPI staining of DNA. In a given population, the relative nuclear DNA content in each class of cell was measured using a new numerical image-analysis method that takes into account the total fluorescence intensity (FI), area (A) and shape factor (SF). The visible degree of synchronization of the population was determined from the number of cells with a nuclear content of 1C DNA at ‘synchronization’, time 0. One method of synchronization (method 1), based on the adhesiveness of the cysts, gave no better than 50% synchronization of the population; method 2, based on swimming cells released from cysts cultured on solid medium, gave 73% of cells with the same nuclear DNA content. A scatter plot of data for FI versus A in the first few hours after time 0 showed that the actual degree of synchronization of the population was lower. The length of the C. cohnii cell cycle determined in vivo by light microscopy was 10, 16 or 24 h for vegetative cells giving two, four or eight daughter cells, respectively. Histograms based on the FI measurements showed that in an initially synchronized population observed for 20 h, the times for the first cell cycle were: G1 phase, 6 h; S phase, 1 h 30 min; G2+M, 1h 30 min, with the release of vegetative cells occurring 1 or 2h after the end of cytokinesis. The times for the second cell cycle were G1+S, 3h; G2+M, 2h. FI and A taken together, suggested that the S phase is clearly restricted, as in higher eukaryotes. A and SF, taken together, showed that the large nuclear areas were always in cysts with two or four daughter cells. FI and SF, taken together, showed that the second S phase always occurred after completion of the first nuclear division. Our data concerning the course of the cell cycle in C. cohnii are compared with those from earlier studies, and the control of the number of daughter cells is discussed; this does not depend on the ploidy of the mother cell.

Patterns of protein synthesis during the cell cycle of Chinese hamster ovary cells

1987 ◽  
Vol 65 (3) ◽  
pp. 219-229 ◽  
Author(s):  
J. Tim Westwood ◽  
Robert B. Church ◽  
Emile B. Wagenaar
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Specific Cell ◽  
Ovary Cells ◽  
Temporal Properties ◽  
Gradient Gel Electrophoresis

The protein synthesis patterns at various stages of the cell cycle of Chinese hamster ovary cells were examined by labelling cells with [35S]methionine and then separating the proteins by isoelectric focussing and two-dimensional, nonequilibrium pH gradient gel electrophoresis. We have observed a number of proteins which display quantitative differences in synthesis at specific cell cycle stages and of these the α- and β-tubulins have been identified. A few proteins appear to be uniquely synthesized at specific times during the cell cycle. These include the histones and a modified version of them, which are synthesized only in S phase, and a pair of 21 kilodalton (kDa), pI 5.5 proteins, which appear only in late G2 and mitosis. We have also identified a 58-kDa, pI 7.5 protein which is present at all cell cycle stages except during late G2. This protein appears to have the same temporal properties as a 57-kDa protein called "cyclin" originally described in sea urchin embryos.

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth

1981 ◽  
Vol 1 (7) ◽  
pp. 594-599
Author(s):  
J J Harada ◽  
D R Morris
Keyword(s):  
Cell Cycle ◽  
Chinese Hamster Ovary ◽  
Doubling Time ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Limited Growth ◽  
Ovary Cells ◽  
Index Analysis ◽  
Thymidine Autoradiography

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.

scholarly journals Relationship between nuclear DNA-content of sperm cells and the timing of events in the cell cycle of Brassica campestris L. (Brassicaceae)

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Hua Deng
Keyword(s):  
Cell Cycle ◽  
Quantitative Analysis ◽  
Dna Content ◽  
Nuclear Dna ◽  
Brassica Campestris ◽  
Nuclear Dna Content ◽  
Pollen Grain ◽  
S Phase ◽  
Sperm Cells ◽  
Generative Cells

In this work we conducted a quantitative analysis of the nuclear DNA-content of developing sperm cells of the plant <em>Brassica campestris</em> L. The sperm cells were in young pollen grain, mature pollen grain and pollen tubes. When generative cells, at the pre-anthesis stage, split into two sperm cells, we have established that the newly-formed sperm cells begin to synthesize nuclear DNA in developing pollen grain of <em>B. campestris</em>. We measured this DNA-content during the development of sperm cells. The results indicate that during development, sperm cells of <em>B. campestris</em> have passed the G<sub>1</sub> phase of the cell cycle and entered the S phase, presumably then fusing with egg cells at a level of 2C, as is characteristic of G<sub>2</sub> type fertilization in angiosperms.

scholarly journals Pattern of DNA increase in macronuclear anlagen of Tetrahymena

1986 ◽  
Vol 83 (1) ◽  
pp. 155-164
Author(s):  
J. Roth ◽  
G. Cleffmann
Keyword(s):  
Cell Cycle ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
S Phase ◽  
The Mean

By combining cytophotometry with autoradiography, five stages of macronuclear anlagen can be discriminated by their DNA content until the end of the first cell cycle after conjugation in Tetrahymena. DNA increases from 2C to about 32C. Each S-phase is followed by a non-synthetic period. Comparing the mean nuclear DNA content after and before each S-phase revealed that 16C anlagen contain significantly less DNA than twice the amount of 8C anlagen. This is unlike the situation in other S-phases during which the amount of DNA is precisely doubled. In the second cell cycle after conjugation some of the cells increase their macronuclear G2 DNA content beyond the 64C stage, while the majority show a mean G2 content of about 64C.

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