Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Jul 25;11(14):4753–4774. doi: 10.1093/nar/11.14.4753

High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A D D'Andrea, U Tantravahi, M Lalande, M A Perle, S A Latt
PMCID: PMC326084  PMID: 6192392

Abstract

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.

Full text

PDF
4753

Images in this article

4759Image
on p.4759
4760Image
on p.4760
4761Image
on p.4761
4763Image
on p.4763
4766Image
on p.4766
4768Image
on p.4768

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amaldi F., Giacomoni D., Zito-Bignami R. On the duplication of ribosomal RNA cistrons in Chinese hamster cells. Eur J Biochem. 1969 Dec;11(3):419–423. doi: 10.1111/j.1432-1033.1969.tb00790.x. [DOI] [PubMed] [Google Scholar]
  2. Balazs I., Brown E. H., Schildkraut C. L. The temporal order of replication of some DNA cistrons. Cold Spring Harb Symp Quant Biol. 1974;38:239–245. doi: 10.1101/sqb.1974.038.01.027. [DOI] [PubMed] [Google Scholar]
  3. Balazs I., Schildkraut C. L. DNA replication in synchronized cultured mammalian cells. VI. The temporal replication of ribosomal cistrons in synchronized cell lines. Exp Cell Res. 1976 Sep;101(2):307–314. doi: 10.1016/0014-4827(76)90382-7. [DOI] [PubMed] [Google Scholar]
  4. Balazs L., Schildkraut C. L. DNA replication in synchronized cultured mammalian cells. II. Replication of ribosomal cistrons in thymidine-synchronized HeLa cells. J Mol Biol. 1971 Apr 14;57(1):153–158. doi: 10.1016/0022-2836(71)90125-2. [DOI] [PubMed] [Google Scholar]
  5. Berkner K. L., Folk W. R. The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases. J Biol Chem. 1979 Apr 10;254(7):2551–2560. [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Braunstein J. D., Schulze D., DelGiudice T., Furst A., Schildkraut C. L. The temporal order of replication of murine immunoglobulin heavy chain constant region sequences corresponds to their linear order in the genome. Nucleic Acids Res. 1982 Nov 11;10(21):6887–6902. doi: 10.1093/nar/10.21.6887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  9. Crouse G. F., Simonsen C. C., McEwan R. N., Schimke R. T. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed] [Google Scholar]
  10. Epner E., Rifkind R. A., Marks P. A. Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells. Proc Natl Acad Sci U S A. 1981 May;78(5):3058–3062. doi: 10.1073/pnas.78.5.3058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Furst A., Brown E. H., Braunstein J. D., Schildkraut C. L. alpha-Globulin sequences are located in a region of early-replicating DNA in murine erythroleukemia cells. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1023–1027. doi: 10.1073/pnas.78.2.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giacomoni D., Finkel D. Time of duplication of ribosomal RNA cistrons in a cell line of Potorous tridactylis (rat kangaroo). J Mol Biol. 1972 Oct 14;70(3):725–728. doi: 10.1016/0022-2836(72)90570-0. [DOI] [PubMed] [Google Scholar]
  13. Gimmler G. M., Schweizer E. rDNA replication in a synchronized culture of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1972 Jan 14;46(1):143–149. doi: 10.1016/0006-291x(72)90642-0. [DOI] [PubMed] [Google Scholar]
  14. Goodpasture C., Bloom S. E. Visualization of nucleolar organizer regions im mammalian chromosomes using silver staining. Chromosoma. 1975 Nov 20;53(1):37–50. doi: 10.1007/BF00329389. [DOI] [PubMed] [Google Scholar]
  15. Goto K., Akematsu T., Shimazu H., Sugiyama T. Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining. Chromosoma. 1975 Dec 10;53(3):223–230. doi: 10.1007/BF00329173. [DOI] [PubMed] [Google Scholar]
  16. Gray J. W., Pallavicini M. G., George Y. S., Groppi V., Look M., Dean P. N. Rates of incorporation of radioactive molecules during the cell cycle. J Cell Physiol. 1981 Aug;108(2):135–144. doi: 10.1002/jcp.1041080204. [DOI] [PubMed] [Google Scholar]
  17. Hamlin J. L., Biedler J. L. Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines. J Cell Physiol. 1981 Apr;107(1):101–114. doi: 10.1002/jcp.1041070112. [DOI] [PubMed] [Google Scholar]
  18. Hamlin J. L., Pardee A. B. S phase synchrony in monolayer CHO cultures. Exp Cell Res. 1976 Jul;100(2):265–275. doi: 10.1016/0014-4827(76)90147-6. [DOI] [PubMed] [Google Scholar]
  19. Holmquist G., Gray M., Porter T., Jordan J. Characterization of Giemsa dark- and light-band DNA. Cell. 1982 Nov;31(1):121–129. doi: 10.1016/0092-8674(82)90411-1. [DOI] [PubMed] [Google Scholar]
  20. Housman D., Huberman J. A. Changes in the rate of DNA replication fork movement during S phase in mammalian cells. J Mol Biol. 1975 May 15;94(2):173–181. doi: 10.1016/0022-2836(75)90076-5. [DOI] [PubMed] [Google Scholar]
  21. Huberman J. A., Riggs A. D. On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol. 1968 Mar 14;32(2):327–341. doi: 10.1016/0022-2836(68)90013-2. [DOI] [PubMed] [Google Scholar]
  22. Hutchinson F. The lesions produced by ultraviolet light in DNA containing 5-bromouracil. Q Rev Biophys. 1973 May;6(2):201–246. doi: 10.1017/s0033583500001141. [DOI] [PubMed] [Google Scholar]
  23. Kapuściński J., Skoczylas B. Simple and rapid fluorimetric method for DNA microassay. Anal Biochem. 1977 Nov;83(1):252–257. doi: 10.1016/0003-2697(77)90533-4. [DOI] [PubMed] [Google Scholar]
  24. Kunkel L. M., Tantravahi U., Eisenhard M., Latt S. A. Regional localization on the human X of DNA segments cloned from flow sorted chromosomes. Nucleic Acids Res. 1982 Mar 11;10(5):1557–1578. doi: 10.1093/nar/10.5.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Latt S. A. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3395–3399. doi: 10.1073/pnas.70.12.3395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Latt S. A., Sahar E., Eisenhard M. E. Pairs of fluorescent dyes as probes of DNA and chromosomes. J Histochem Cytochem. 1979 Jan;27(1):65–71. doi: 10.1177/27.1.86582. [DOI] [PubMed] [Google Scholar]
  27. Lehmann A. R., Ormerod M. G. The replication of DNA in murine lymphoma cells (L5178Y). I. Rate of replication. Biochim Biophys Acta. 1970 Mar 19;204(1):128–143. doi: 10.1016/0005-2787(70)90496-x. [DOI] [PubMed] [Google Scholar]
  28. Lin M. S., Comings D. E., Alfi O. S. Optical Studies of the interaction of 4'-6'-diamidino-2-phenylindole with DNA and metaphase chromosomes. Chromosoma. 1977 Mar 7;60(1):15–25. doi: 10.1007/BF00330407. [DOI] [PubMed] [Google Scholar]
  29. Loveday K. S., Latt S. A. A high buoyant density fraction in mammalian DNA. Characterization and impact on the detection of heteroduplex DNA. Exp Cell Res. 1981 Nov;136(1):177–187. doi: 10.1016/0014-4827(81)90049-5. [DOI] [PubMed] [Google Scholar]
  30. Marchionni M. A., Roufa R. J. Digestion of 5-bromodeoxyuridine-substituted lambda-DNA by restriction endonucleases. J Biol Chem. 1978 Dec 25;253(24):9075–9081. [PubMed] [Google Scholar]
  31. Mariani B. D., Slate D. L., Schimke R. T. S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4985–4989. doi: 10.1073/pnas.78.8.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mueller G. C., Kajiwara K. Early- and late-replicating deoxyribonucleic acid complexes in HeLa nuclei. Biochim Biophys Acta. 1966 Jan 18;114(1):108–115. doi: 10.1016/0005-2787(66)90258-9. [DOI] [PubMed] [Google Scholar]
  33. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  34. Rosenstein B. S., Setlow R. B., Ahmed F. E. Use of the dye Hoechst 33258 in a modification of the bromodeoxyuridine photolysis technique for the analysis of DNA repair. Photochem Photobiol. 1980 Mar;31(3):215–222. doi: 10.1111/j.1751-1097.1980.tb03710.x. [DOI] [PubMed] [Google Scholar]
  35. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  36. Stambrook P. J. The temporal replication of ribosomal genes in synchronized Chinese hamster cells. J Mol Biol. 1974 Jan 25;82(3):303–313. doi: 10.1016/0022-2836(74)90592-0. [DOI] [PubMed] [Google Scholar]
  37. TAYLOR J. H. Asynchronous duplication of chromosomes in cultured cells of Chinese hamster. J Biophys Biochem Cytol. 1960 Jun;7:455–464. doi: 10.1083/jcb.7.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taichman L. B. The use of ultraviolet light in the fractionation of chromatin containing unsubstituted and bromodeoxyuridine-substituted DNA. Nucleic Acids Res. 1979;6(5):2029–2038. doi: 10.1093/nar/6.5.2029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taylor J. H. Increase in DNA replication sites in cells held at the beginning of S phase. Chromosoma. 1977 Jul 18;62(4):291–300. doi: 10.1007/BF00327029. [DOI] [PubMed] [Google Scholar]
  40. Taylor J. H. Rates of chain growth and units of replication in DNA of mammalian chromosomes. J Mol Biol. 1968 Feb 14;31(3):579–594. doi: 10.1016/0022-2836(68)90429-4. [DOI] [PubMed] [Google Scholar]
  41. Tiemeier D. C., Tilghman S. M., Leder P. Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda. Gene. 1977;2(3-4):173–191. doi: 10.1016/0378-1119(77)90016-6. [DOI] [PubMed] [Google Scholar]
  42. Tobey R. A., Crissman H. A. Preparation of large quantities of synchronized mammalian cells in late G1 in the pre-DNA replicative phase of the cell cycle. Exp Cell Res. 1972 Dec;75(2):460–464. doi: 10.1016/0014-4827(72)90453-3. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES