Temporal Order of Gene Replication in Chinese Hamster Ovary Cells

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1989 Jul 1

PMID 2476659

Citations 18

Authors

J Taljanidisz

J Popowski

N Sarkar

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Insights into the Link between the Organization of DNA Replication and the Mutational Landscape.

Gaboriaud J, Wu P Genes (Basel). 2019; 10(4).

PMID: 30934791 PMC: 6523204. DOI: 10.3390/genes10040252.

Developmental control of the DNA replication and transcription programs.

Nordman J, Li S, Eng T, MacAlpine D, Orr-Weaver T Genome Res. 2010; 21(2):175-81.

PMID: 21177957 PMC: 3032921. DOI: 10.1101/gr.114611.110.

DNA replication timing: random thoughts about origin firing.

Rhind N Nat Cell Biol. 2006; 8(12):1313-6.

PMID: 17139278 PMC: 2861037. DOI: 10.1038/ncb1206-1313.

Specific signals at the 3' end of the DHFR gene define one boundary of the downstream origin of replication.

Mesner L, Hamlin J Genes Dev. 2005; 19(9):1053-66.

PMID: 15879555 PMC: 1091740. DOI: 10.1101/gad.1307105.

Coordination of replication and transcription along a Drosophila chromosome.

MacAlpine D, Rodriguez H, Bell S Genes Dev. 2004; 18(24):3094-105.

PMID: 15601823 PMC: 535919. DOI: 10.1101/gad.1246404.

References

Holmquist G, Caston L . Replication time of interspersed repetitive DNA sequences in hamsters. Biochim Biophys Acta. 1986; 868(2-3):164-77. DOI: 10.1016/0167-4781(86)90019-9. View

Land H, Chen A, Morgenstern J, Parada L, Weinberg R . Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts. Mol Cell Biol. 1986; 6(6):1917-25. PMC: 367729. DOI: 10.1128/mcb.6.6.1917-1925.1986. View

DAndrea A, Tantravahi U, Lalande M, Perle M, LATT S . High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells. Nucleic Acids Res. 1983; 11(14):4753-74. PMC: 326084. DOI: 10.1093/nar/11.14.4753. View

Brennand J, Konecki D, Caskey C . Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts. J Biol Chem. 1983; 258(16):9593-6. View

Huberman J, Riggs A . On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol. 1968; 32(2):327-41. DOI: 10.1016/0022-2836(68)90013-2. View

Amaldi F, GIACOMONI D . On the duplication of ribosomal RNA cistrons in Chinese hamster cells. Eur J Biochem. 1969; 11(3):419-23. DOI: 10.1111/j.1432-1033.1969.tb00790.x. View

Balazs L, SCHILDKRAUT C . DNA replication in synchronized cultured mammalian cells. II. Replication of ribosomal cistrons in thymidine-synchronized HeLa cells. J Mol Biol. 1971; 57(1):153-8. DOI: 10.1016/0022-2836(71)90125-2. View

Gimmler G, Schweizer E . rDNA replication in a synchronized culture of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1972; 46(1):143-9. DOI: 10.1016/0006-291x(72)90642-0. View

GIACOMONI D, Finkel D . Time of duplication of ribosomal RNA cistrons in a cell line of Potorous tridactylis (rat kangaroo). J Mol Biol. 1972; 70(3):725-8. DOI: 10.1016/0022-2836(72)90570-0. View

10.

Stambrook P . The temporal replication of ribosomal genes in synchronized Chinese hamster cells. J Mol Biol. 1974; 82(3):303-13. DOI: 10.1016/0022-2836(74)90592-0. View

11.

Housman D, Huberman J . Changes in the rate of DNA replication fork movement during S phase in mammalian cells. J Mol Biol. 1975; 94(2):173-81. DOI: 10.1016/0022-2836(75)90076-5. View

12.

Lewis J . Structure and expression of the Chinese hamster thymidine kinase gene. Mol Cell Biol. 1986; 6(6):1998-2010. PMC: 367739. DOI: 10.1128/mcb.6.6.1998-2010.1986. View

13.

Iqbal M, Chinsky J, Didamo V, SCHILDKRAUT C . Replication of proto-oncogenes early during the S phase in mammalian cell lines. Nucleic Acids Res. 1987; 15(1):87-103. PMC: 340399. DOI: 10.1093/nar/15.1.87. View

14.

Ariga H, Itani T . Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro. Mol Cell Biol. 1987; 7(1):1-6. PMC: 365034. DOI: 10.1128/mcb.7.1.1-6.1987. View

15.

Brown E, Iqbal M, Stuart S, Hatton K, Valinsky J, SCHILDKRAUT C . Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon. Mol Cell Biol. 1987; 7(1):450-7. PMC: 365088. DOI: 10.1128/mcb.7.1.450-457.1987. View

16.

Holmquist G . Role of replication time in the control of tissue-specific gene expression. Am J Hum Genet. 1987; 40(2):151-73. PMC: 1684080. View

17.

Taljanidisz J, Karnik P, Sarkar N . Messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane is polyadenylated. J Mol Biol. 1987; 193(3):507-15. DOI: 10.1016/0022-2836(87)90263-4. View

18.

Itani T, Yamaguchi M, Ariga H . c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication. Nucleic Acids Res. 1987; 15(12):4889-99. PMC: 305925. DOI: 10.1093/nar/15.12.4889. View

19.

Gannon J, Lane D . p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987; 329(6138):456-8. DOI: 10.1038/329456a0. View

20.

Sarkar N, List J, Banfalvi G . Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells. Eur J Biochem. 1987; 168(2):263-8. DOI: 10.1111/j.1432-1033.1987.tb13415.x. View