Evidence for the resumption of DNA replication prior to histone synthesis in HeLa cells after release from treatment with hydroxyurea

Shephard, Elizabeth A.; Phillips, Ian; Davis, Jeudi; Stein, Janet L.

doi:10.1016/0014-5793(82)80891-0

Cited by 11 publications

(7 citation statements)

References 11 publications

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“…DNA synthesis rates were monitored by measuring the incorporation of radioactivity into 5% trichloroacetic acid precipitable material after pulse labeling of duplicate samples for 30 min with [3H]thymidine (Shephard et al, 1982).…”

Section: Methodsmentioning

confidence: 99%

Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle

Moreno¹,

Chrysogelos²,

Stein³

et al. 1986

Biochemistry

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The organization of nucleosomes associated with a cell cycle regulated human H4 histone gene was examined in synchronized HeLa S3 cells. At various times during the cell cycle, nuclei were digested with micrococcal nuclease, and the nucleosomal pattern of the gene was obtained by Southern blot analysis using radiolabeled human histone H4 gene probes. We have detected reversible changes during the cell cycle in the chromatin structure of this gene, as reflected by the shortening of the nucleosomal spacing after replication and the peak of transcription. This variation is also observed when DNA and protein syntheses are inhibited. By using a probe that comprises 250 base pairs (bp) of the coding region and 240 bp of the 5' end of the gene, containing the promoter and DNase I sensitive sequences, we also have observed a general disruption of the nucleosomal organization, which is reflected by a degeneration of the characteristic nucleosomal ladder produced by micrococcal nuclease digestion. This modification coincides with the replication and active transcription of the gene (early S phase), which recovers its regular nucleosomal appearance when both processes have been completed, although the nucleosome linker length is shortened. When the probe utilized comprises the distal 3' end of the gene, there is no disruption of the nucleosomal pattern, but the linker region also exhibits a shortened length. A non-cell cycle regulated gene (beta-globin) does not exhibit such modifications in any of the situations analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle

Moreno¹,

Chrysogelos²,

Stein³

et al. 1986

Biochemistry

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show abstract

“…Relative rates of DNA synthesis were monitored by measuring the incorporation of 14C-thymidine (0.1 uiCi/ml, 5 x 105 cells/ml) into acid-precipitable material in a 20 min pulse (10,26 (10), and 50 jig resolved electrophoretically in 1.5% (w/v) agarose, 6% (w/v) formaldehyde gels (10), using 6% (w/v) formaldehyde, 20 nM MOPS (pH 7.0), 5 ntl sodium acetate, 1 mnM EDTA, as an electrolyte (27). RNA was transferred to nitrocellulose filters, which were hybridized to [32P]-labelled cloned human histone DNA, and washed as described (10).…”

Section: Materials and Methods Materials Smentioning

confidence: 99%

“…RNA was transferred to nitrocellulose filters, which were hybridized to [32P]-labelled cloned human histone DNA, and washed as described (10). Filters were exposed to preflashed XAR5 or Cronex X-ray film at -70°C, and hybridization quantitated by liquid scintillation spectrometry (10,26). Quenching was corrected by external standardization.…”

Section: Materials and Methods Materials Smentioning

confidence: 99%

Influence of DNA synthesis inhibition on the coordinate expression of core human hlstone genes during S phase

Plumb¹,

Stein

Stein³

1983

Nucl Acids Res

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Core histone mRNA metabolism has been examined in S phase HeLa cells recovering from DNA synthesis inhibition by 1 mM hydroxyurea. Using cloned human histone genes as probes for histone mRNA quantitation, the response to and recovery from DNA synthesis inhibition is shown to depend on the position of the cell with respect to the initiation of DNA replication. The incorporation of 3H-uridine into multiple histone mRNAs in recovering cells does not exceed preinhibition levels, and as this incorporation is maximal in early S phase, the synthesis of core histone mRNA is apparently related to the ordered replication of the genome. The total histone mRNA present in interrupted S phase cells after recovery is not significantly different from that present in control cells, and a temporal and functional coupling between histone mRNA levels and the relative rate of DNA synthesis is maintained in perturbed cells.

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“…Rates of DNA synthesis in synchronized cells were monitored by pulse labelling 106 cells with 0.2pCi of 14C-thymidine for 20 minutes and measuring the incorporation of radioactivity into 10% (w/v) trichloroacetic acidinsoluble material (35).…”

Section: Materials Smentioning

confidence: 99%

Coordinate regulation of multiple histone mRNAs during the cell cycle in HeLa cells

Plumb

Stein

Stein³

1983

Nucl Acids Res

266

195

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Core histone gene expression in HeLa S3 cells has been examined as a function of the cell cycle using cloned human histone gene probes. Total cellular histone mRNAs were analyzed by Northern blot analysis, and their relative abundance shown to be temporally coupled to DNA synthesis rates in S phase. The in vivo incorporation of 3H-uridine into at least fifteen heterologous histone mRNAs (in one hour pulse intervals at various times in the cell cycle), was monitored by hybrid selection. Hybridized RNAs were eluted and resolved electrophoretically to give both a quantitative and qualitative assay for multiple mRNA species. Maximal incorporation of 3H-uridine into histone mRNAs precedes their maximal accumulation, indicating that transcriptional regulation is predominant in early S phase. The turnover of histone mRNAs in late S occurs in the presence of a reduced apparent transcription rate, indicating that post-transcriptional regulation is predominant in late S. All the detected multiple histone mRNAs are coordinately regulated during the HeLa cell cycle.

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Evidence for the resumption of DNA replication prior to histone synthesis in HeLa cells after release from treatment with hydroxyurea

Abstract: FEBS Letters 140 (1982) 189-192. doi:10.1016/0014-5793(82)80891-0Received by publisher: 1981-12-30Harvest Date: 2016-01-04 12:19:43DOI: 10.1016/0014-5793(82)80891-0Page Range: 189-19

Cited by 11 publications

References 11 publications

Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle

Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle

Influence of DNA synthesis inhibition on the coordinate expression of core human hlstone genes during S phase

Coordinate regulation of multiple histone mRNAs during the cell cycle in HeLa cells

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