Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 Aug 15;238(1):13–21. doi: 10.1042/bj2380013

Incorporation of nucleotide tracers into nucleic acids in permeabilized cells and cellular homogenates.

K Pawlak, C Lawi-Berger, W Sadée
PMCID: PMC1147091  PMID: 2432880

Abstract

The validity of permeabilized cells as a model of DNA and RNA synthesis was studied with the use of mouse S-49 lymphoblastoma cells rendered permeable by exposure to L-alpha-lysophosphatidylcholine. The permeabilized cells readily incorporated exogenously supplied cytosine and uracil nucleotides into HClO4-insoluble macromolecular material. However, the incorporation of these tracers did not require the three other complementary nucleotides, and adenine, guanine or thymine nucleotide tracers were incorporated at much lower rates. These results, which were also obtained with permeabilized Abelsohn-leukaemia-virus-transformed mouse macrophages, mouse neuroblastoma cells and S-49 lymphoblastoma homogenates, are inconsistent with semi-conservative DNA replication or RNA transcription; rather, they suggest the involvement of terminal nucleotidyltransferase(s) that mediate the incorporation of uracil and cytosine nucleotides. DNA synthesis was restored when permeabilized cells or cellular homogenates were supplemented with denatured salmon testes DNA. These results suggest that endogenous cellular DNA is impaired in its function as a template for DNA replication and transcription in vitro. Metabolic channelling or compartmentation of nucleic-acid-precursor pathways could not be demonstrated in the permeabilized cells.

Full text

PDF
13

Selected References

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

  1. Berger N. A., Johnson E. S. DNA synthesis in permeabilized mouse L cells. Biochim Biophys Acta. 1976 Feb 18;425(1):1–17. doi: 10.1016/0005-2787(76)90211-2. [DOI] [PubMed] [Google Scholar]
  2. Bestwick R. K., Mathews C. K. Unusual compartmentation of precursors for nuclear and mitochondrial DNA in mouse L cells. J Biol Chem. 1982 Aug 25;257(16):9305–9308. [PubMed] [Google Scholar]
  3. Biedler J. L., Helson L., Spengler B. A. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 1973 Nov;33(11):2643–2652. [PubMed] [Google Scholar]
  4. Cohen M. B., Maybaum J., Sadee W. Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells. J Biol Chem. 1981 Aug 25;256(16):8713–8717. [PubMed] [Google Scholar]
  5. Gottesman M. E., Canellakis E. S. The terminal nucleotidyltransferases of calf thymus nuclei. J Biol Chem. 1966 Oct 10;241(19):4339–4352. [PubMed] [Google Scholar]
  6. Kato K. I., Gonçalves J. M., Houts G. E., Bollum F. J. Deoxynucleotide-polymerizing enzymes of calf thymus gland. II. Properties of the terminal deoxynucleotidyltransferase. J Biol Chem. 1967 Jun 10;242(11):2780–2789. [PubMed] [Google Scholar]
  7. Klee W. A., Nirenberg M. A neuroblastoma times glioma hybrid cell line with morphine receptors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3474–3477. doi: 10.1073/pnas.71.9.3474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lee H. J., Pawlak K., Nguyen B. T., Robins R. K., Sadée W. Biochemical differences among four inosinate dehydrogenase inhibitors, mycophenolic acid, ribavirin, tiazofurin, and selenazofurin, studied in mouse lymphoma cell culture. Cancer Res. 1985 Nov;45(11 Pt 1):5512–5520. [PubMed] [Google Scholar]
  9. Miller M. R., Castellot J. J., Jr, Pardee A. B. A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry. 1978 Mar 21;17(6):1073–1080. doi: 10.1021/bi00599a021. [DOI] [PubMed] [Google Scholar]
  10. Nguyen B. T., Sadée W. Compartmentation of guanine nucleotide precursors for DNA synthesis. Biochem J. 1986 Mar 1;234(2):263–269. doi: 10.1042/bj2340263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nicander B., Reichard P. Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1347–1351. doi: 10.1073/pnas.80.5.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Noguchi H., Prem veer Reddy G., Pardee A. B. Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell. 1983 Feb;32(2):443–451. doi: 10.1016/0092-8674(83)90464-6. [DOI] [PubMed] [Google Scholar]
  13. Oda T., Watanabe S., Hanakawa S., Hosogi N. Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells. Acta Med Okayama. 1981 Apr;35(2):149–154. doi: 10.18926/AMO/31271. [DOI] [PubMed] [Google Scholar]
  14. Raschke W. C., Baird S., Ralph P., Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell. 1978 Sep;15(1):261–267. doi: 10.1016/0092-8674(78)90101-0. [DOI] [PubMed] [Google Scholar]
  15. Schlabach A., Fridlender B., Bolden A., Weissbach A. DNA-dependent DNA polymerases from HeLa cell nuclei. II. Template and substrate utilization. Biochem Biophys Res Commun. 1971 Aug 20;44(4):879–885. doi: 10.1016/0006-291x(71)90793-5. [DOI] [PubMed] [Google Scholar]
  16. Spyrou G., Reichard P. Ribonucleotides are not channeled into DNA in permeabilized mammalian cells. Biochem Biophys Res Commun. 1983 Sep 30;115(3):1022–1026. doi: 10.1016/s0006-291x(83)80037-0. [DOI] [PubMed] [Google Scholar]
  17. Taheri M. R., Wickremasinghe R. G., Hoffbrand A. V. Metabolism of thymine nucleotides synthesized via the 'de novo' mechanism in normal, megaloblastic and methotrexate-treated human cells and in a lymphoblastoid cell line. Biochem J. 1981 Apr 15;196(1):225–235. doi: 10.1042/bj1960225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. veer Reddy G. P., Pardee A. B. Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells. J Biol Chem. 1982 Nov 10;257(21):12526–12531. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES