Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 May 24;13(10):3551–3560. doi: 10.1093/nar/13.10.3551

Isolated HeLa cell nuclei synthesize meaningful DNA.

T Kristensen, H Prydz
PMCID: PMC341258  PMID: 4011435

Abstract

DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The results obtained indicate that the isolated nuclei make "meaningful" DNA, as judged by the melting characteristics of the hybrids between rRNA and the in vitro replicated DNA. Hybridization of the labelled DNA against rRNA fractionated by electrophoresis and blotted onto nitrocellulose verified the presence of sequences complementary to 18 S and 28 S rRNA.

Full text

PDF
3553

Images in this article

3556Image
on p.3556

Selected References

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

  1. Berger S. L., Birkenmeier C. S. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. doi: 10.1021/bi00590a018. [DOI] [PubMed] [Google Scholar]
  2. Bernard O., Brent T. P. DNA replication in isolated HeLa cell nuclei. Biochem Biophys Res Commun. 1973 Aug 21;53(4):1213–1219. doi: 10.1016/0006-291x(73)90594-9. [DOI] [PubMed] [Google Scholar]
  3. Friedman D. L., Mueller G. C. A nuclear system for DNA replication from synchronized HeLa cells. Biochim Biophys Acta. 1968 Jul 23;161(2):455–468. doi: 10.1016/0005-2787(68)90122-6. [DOI] [PubMed] [Google Scholar]
  4. Henderson A. S., Warburton D., Atwood K. C. Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3394–3398. doi: 10.1073/pnas.69.11.3394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Krokan H., Bjorklid E., Prydz H. DNA synthesis in isolated HeLa cell nuclei. Optimalization of the system and characterization of the product. Biochemistry. 1975 Sep 23;14(19):4227–4232. doi: 10.1021/bi00690a012. [DOI] [PubMed] [Google Scholar]
  6. Krokan H., Cooke L., Prydz H. DNA synthesis in isolated HeLa cell nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and their subsequent ligation. Biochemistry. 1975 Sep 23;14(19):4233–4237. doi: 10.1021/bi00690a013. [DOI] [PubMed] [Google Scholar]
  7. Krokan H., Wist E., Prydz H. Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei. Biochem Biophys Res Commun. 1977 Mar 21;75(2):414–419. doi: 10.1016/0006-291x(77)91058-0. [DOI] [PubMed] [Google Scholar]
  8. Kumar K. V., Friedman D. L. Initiation of DNA synthesis in HeLa cell-free system. Nat New Biol. 1972 Sep 20;239(90):74–76. doi: 10.1038/newbio239074a0. [DOI] [PubMed] [Google Scholar]
  9. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  10. Muller M. T., Kajiwara K., Mueller G. C. Role of cytosol proteins in DNA chain growth and chromatin replication in Friend erythroleukemia cell nuclei. Biochim Biophys Acta. 1981 May 29;653(3):391–407. doi: 10.1016/0005-2787(81)90195-7. [DOI] [PubMed] [Google Scholar]
  11. Nagata K., Enomoto T., Yamada M. A. A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis. Biochim Biophys Acta. 1981 May 29;653(3):316–330. doi: 10.1016/0005-2787(81)90188-x. [DOI] [PubMed] [Google Scholar]
  12. Qasba P. K. Synthesis of simian virus 40 DNA in isolated nuclei. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1045–1049. doi: 10.1073/pnas.71.4.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schmickel R. D. Quantitation of human ribosomal DNA: hybridization of human DNA with ribosomal RNA for quantitation and fractionation. Pediatr Res. 1973 Jan;7(1):5–12. doi: 10.1203/00006450-197301000-00002. [DOI] [PubMed] [Google Scholar]
  14. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  16. Winnacker E. L., Magnusson G., Reichard P. Synthesis of polyoma DNA by isolated nuclei. Biochem Biophys Res Commun. 1971 Aug 20;44(4):952–957. doi: 10.1016/0006-291x(71)90804-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES