Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 May 1;104(5):1125–1132. doi: 10.1083/jcb.104.5.1125

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus

PMCID: PMC2114460  PMID: 3553204

Abstract

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti- rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Allen R. L., Kennel S. J., Cacheiro L., Olins A. L., Olins D. E. Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies. J Cell Biol. 1986 Jan;102(1):131–136. doi: 10.1083/jcb.102.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen R. L., Olins A. L., Harp J. M., Olins D. E. Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus. Eur J Cell Biol. 1985 Nov;39(1):217–223. [PubMed] [Google Scholar]
  3. Allen R. L., Olins D. E. Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide. Chromosoma. 1984;91(1):82–86. doi: 10.1007/BF00286488. [DOI] [PubMed] [Google Scholar]
  4. Allison L., Arndt-Jovin D. J., Gratzner H., Ternynck T., Robert-Nicoud M. Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies. Cytometry. 1985 Nov;6(6):584–590. doi: 10.1002/cyto.990060613. [DOI] [PubMed] [Google Scholar]
  5. Cadilla C. L., Harp J., Flanagan J. M., Olins A. L., Olins D. E. Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus. Nucleic Acids Res. 1986 Jan 24;14(2):823–841. doi: 10.1093/nar/14.2.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cadilla C. L., Roberson A. E., Harp J., Olins A. L., Olins D. E. Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus. Nucleic Acids Res. 1986 Nov 11;14(21):8501–8512. doi: 10.1093/nar/14.21.8501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  8. Enomoto T., Tanuma S., Yamada M. Characterization of deoxyribonucleic acid synthesis in reconstituted nuclear systems. Biochemistry. 1983 Mar 1;22(5):1128–1133. doi: 10.1021/bi00274a021. [DOI] [PubMed] [Google Scholar]
  9. Evenson D. P., Prescott D. M. Disruption of DNA synthesis in Euplotes by heat shock. Exp Cell Res. 1970 Dec;63(2):245–252. doi: 10.1016/0014-4827(70)90210-7. [DOI] [PubMed] [Google Scholar]
  10. Evenson D. P., Prescott D. M. RNA metabolism in the macronucleus of Euplotes eurystomus during the cell cycle. Exp Cell Res. 1970 Jul;61(1):71–78. doi: 10.1016/0014-4827(70)90259-4. [DOI] [PubMed] [Google Scholar]
  11. GALL J. G. Macronuclear duplication in the ciliated protozoan Euplotes. J Biophys Biochem Cytol. 1959 Mar 25;5(2):295–308. doi: 10.1083/jcb.5.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gratzner H. G. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science. 1982 Oct 29;218(4571):474–475. doi: 10.1126/science.7123245. [DOI] [PubMed] [Google Scholar]
  13. Haraguchi T., Oguro M., Nagano H., Ichihara A., Sakamura S. Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Nucleic Acids Res. 1983 Feb 25;11(4):1197–1209. doi: 10.1093/nar/11.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harms G., van Goor H., Koudstaal J., de Ley L., Hardonk M. J. Immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections. Histochemistry. 1986;85(2):139–143. doi: 10.1007/BF00491761. [DOI] [PubMed] [Google Scholar]
  15. Herman T. M., Lefever E., Shimkus M. Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs. Anal Biochem. 1986 Jul;156(1):48–55. doi: 10.1016/0003-2697(86)90152-1. [DOI] [PubMed] [Google Scholar]
  16. Kaufman S. J., Robert-Nicoud M. DNA replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5-bromodeoxyuridine, actin, and alpha 2-macroglobulin. Cytometry. 1985 Nov;6(6):570–577. doi: 10.1002/cyto.990060611. [DOI] [PubMed] [Google Scholar]
  17. Kraut H., Lipps H. J., Prescott D. M. The genome of hypotrichous ciliates. Int Rev Cytol. 1986;99:1–28. doi: 10.1016/s0074-7696(08)61422-9. [DOI] [PubMed] [Google Scholar]
  18. Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lin M., Prescott D. M. Electron microscope autoradiography of DNA synthesis in the replication band of two hypotrichous ciliates. J Protozool. 1985 Feb;32(1):144–149. doi: 10.1111/j.1550-7408.1985.tb03028.x. [DOI] [PubMed] [Google Scholar]
  20. Madsen P., Celis J. E. S-phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis. A role for cyclin in DNA replication? FEBS Lett. 1985 Nov 25;193(1):5–11. doi: 10.1016/0014-5793(85)80068-5. [DOI] [PubMed] [Google Scholar]
  21. Mathews C. K., Slabaugh M. B. Eukaryotic DNA metabolism. Are deoxyribonucleotides channeled to replication sites? Exp Cell Res. 1986 Feb;162(2):285–295. doi: 10.1016/0014-4827(86)90335-6. [DOI] [PubMed] [Google Scholar]
  22. Miller M. R., Ulrich R. G., Wang T. S., Korn D. Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells. J Biol Chem. 1985 Jan 10;260(1):134–138. [PubMed] [Google Scholar]
  23. Murti K. G., Prescott D. M. Replication forms of the gene-sized DNA molecules of hypotrichous ciliates. Mol Cell Biol. 1983 Sep;3(9):1562–1566. doi: 10.1128/mcb.3.9.1562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Olins A. L., Olins D. E., Franke W. W., Lipps H. J., Prescott D. M. Stereo-electron microscopy of nuclear structure and replication in ciliated protozoa (Hypotricha). Eur J Cell Biol. 1981 Aug;25(1):120–130. [PubMed] [Google Scholar]
  25. PRESCOTT D. M., KIMBALL R. F. Relation between RNA, DNA, and protein syntheses in the replicating nucleus of Euplotes. Proc Natl Acad Sci U S A. 1961 May 15;47:686–693. doi: 10.1073/pnas.47.5.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shimkus M. L., Guaglianone P., Herman T. M. Synthesis and characterization of biotin-labeled nucleotide analogs. DNA. 1986 Jun;5(3):247–255. doi: 10.1089/dna.1986.5.247. [DOI] [PubMed] [Google Scholar]
  27. Van der Velden H. M., Poot M., Wanka F. In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells. Biochim Biophys Acta. 1984 Sep 10;782(4):429–436. doi: 10.1016/0167-4781(84)90050-2. [DOI] [PubMed] [Google Scholar]
  28. Wickremasinghe R. G., Yaxley J. C., Hoffbrand A. V. Solubilization and partial characterization of a multienzyme complex of DNA synthesis from human lymphoblastoid cells. Eur J Biochem. 1982 Sep 1;126(3):589–596. doi: 10.1111/j.1432-1033.1982.tb06821.x. [DOI] [PubMed] [Google Scholar]
  29. Yumura S., Mori H., Fukui Y. Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence. J Cell Biol. 1984 Sep;99(3):894–899. doi: 10.1083/jcb.99.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES