Perturbation of growth and differentiation of friend murine erythroleukemia cells by 5‐bromodeoxyuridine incorporation in early S phase

Brown, Elissa J.; Schildkraut, Carl L.

doi:10.1002/jcp.1040990213

Cited by 44 publications

(18 citation statements)

References 35 publications

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“…In the case of several genes induced by serum, an increase was the consequence of serum induction of quiescent cells and not of cell cycle-specific events (15,30). Proliferating cells can be separated by centrifugal-flow elutriation into size populations that are in different stages of the cell cycle (6). To study the cell cycle fluctuation of p49 mRNA in growing cells, mouse lymphoma L1210 cells were grown and separated into 10 cell-size fractions by centrifugal elutriation.…”

Section: Resultsmentioning

confidence: 99%

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Tseng¹,

Prussak²,

Almazan³

1989

Mol. Cell. Biol.

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Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased -10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased-the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated Gl cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

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Section: Resultsmentioning

confidence: 99%

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Tseng¹,

Prussak²,

Almazan³

1989

Mol. Cell. Biol.

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“…We have previously shown that as cells progress through the S phase of the cell cycle, cell size increases proportionately along with nuclear DNA content (5). Therefore, we used centrifugal elutriation to sort a random population of growing cells on the basis of size into progressive intervals of the S phase, as described previously (4,6,21). Briefly, exponentially growing hybrid cells (10') or fibroblast cells (2 x 108) were pulsed with bromodeoxyuridine (BUdR; 40 and 20 ,ug/ml, respectively) for 2 to 2.5 h, after which cells were injected into the centrifugal elutriator chamber at a constant rotor (Beckman JE-1OX) speed.…”

Section: Methodsmentioning

confidence: 99%

Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

1989

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To investigate whether a switch in the transcriptional activity of a gene is associated with a change in the timing of replication during the S phase, we examined the replication timing of the I-globin genes in two different types of somatic cell hybrids. In mouse hepatoma (Hepa la) x mouse erythroleukemia (MEL) hybrid cells, the j8-globin gene from the MEL parent is transcriptionaily inactivated and is later replicating than in the parental MEL cell line. In human fibroblast (GM3552) x MEL hybrid cells, the human ,3-globin gene is transcriptionally activated, and all of the sequences within the human 0-globin domain (200 kilobases) we have examined appear to be earlier replicating than those in the parental fibroblast cell line. The chromatin configuration of the activated human 0-globin domain in the hybrids is relatively more sensitive to nucleases than that in the fibroblasts. Furthermore, major nuclease-hypersensitive sites that were absent in the chromatin flanking the distal 5' region of the human ,(-globin gene cluster in the parental fibroblast cell line are present in the transcriptionally activated domain in the hybrid cell line. These results suggest that timing of replication of globin genes has been altered in these hybrid cells and thus is not fixed during the process of differentiation.Genes for specialized functions usually replicate very early during the S phase in mammalian cell lines in which they are expressed (e.g., see reference 28). When they are not expressed, some of these tissue-specific genes replicate late during the S phase while others can replicate at any time during the S phase. The time at which these genes replicate in cell lines in which they are not expressed appears to depend in part on their chromosomal location (7). In erythroid cell lines, P-globin genes are expressed and replicate early in the S phase, but in lymphoid cell lines, these genes replicate late in the S phase and are inactive. Transcriptionally active immunoglobulin heavy-chain variable-region genes (such as V1 of the T15 family) and T-cell receptor p-chain constant-region genes (CTP1 and CTP2) replicate early in the S phase in B-and T-cell lines, respectively, but late in the S phase in erythroid cell lines (28). Since these represent functionally and morphologically distinct blood cell lines which arise from the same progenitor or pluripotent stem cells, our findings suggest that some tissue-specific genes undergo a change in their replication timing concomitant with the turning on of their transcription during development and differentiation.We performed studies to determine whether the time at which a gene replicates in a differentiated cell is a permanent feature or whether it can be changed when the expression of the gene is altered. Ideally, we would like to be able to turn on the expression of a gene from a completely silent level and compare the replication times of the gene in these two states. There are few genes, however, whose activities can be modulated in this manner in cultured cells. The glob...

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“…Growth in the presence of BUdR, centrifugal elutriation, and flow microfluorimetric (FMF) analysis of propidium iodidestained MEL and MPC11 cells were as described previously (4)(5)(6)9). BUdR labeling and centrifugal elutnation conditions for Hepa 1.6, 22D6, 300-19P, and RL 11 were as described elsewhere (Hatton et al, in preparation; S. Stuart, F. W. Alt, and C. L. Schildkraut, manuscript in preparation).…”

Section: Methodsmentioning

confidence: 99%

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

Brown¹,

Iqbal²,

Stuart³

et al. 1987

Mol. Cell. Biol.

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We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) contained mitochondrial origin sequences. Two independently isolated clones contained the same middle repetitive sequences. Thus, as expected of replication origins, these clones represent specific sets of sequences rather than a random sampling of genomic DNA.Studies by Heintz et al. (12) on methotrexate-resistant Chinese hamster ovary cells suggested that a 135-kb amplified region (amplicon) containing the dihydrofolate reductase gene may in fact be a replicon. Furthermore, the initiation of DNA synthesis in each amplicon was shown to be confined to a small subset of restriction segments during the first 40 min of the S phase. These studies suggested that the early replicating fragments contain or flank origins of replication.While specific origins appear to exist in the genome, the available evidence suggests that only a fraction of them are active in a single cell type (36). For example, more origins are active in early Drosophila melanogaster embryonic cells than at later stages in development (3). The arrest of cells in the S phase with fluorodeoxyuridine results in the initiation of DNA synthesis from additional sites after release from the block, suggesting the existence of potential origins that are not normally active (37). These authors (37) found that such potential origins are spaced about 12 kb apart. In a particular cell type, only a fraction of these origins may be used.The transcriptional activity of DNA also appears to influence the activation of origins. For example, some genes replicate early in the S phase in cell types in which they are expressed and at later times in cell types in which they are not expressed (6, 9, 10; Hatton et al., manuscript in preparation). It has also been suggested that the initiation of replication from an upstream origin is required for transcriptional activation of a gene (31,34). A recent study (13) 450 on May 11, 2018 by guest

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Perturbation of growth and differentiation of friend murine erythroleukemia cells by 5‐bromodeoxyuridine incorporation in early S phase

Cited by 44 publications

References 35 publications

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

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