Isolated HeLa cell nuclei synthesize meaningful DNA.

Nucleic Acids Res. 1985 May 24; 13(10): 3551–3560.

PMCID: PMC341258

T Kristensen and H Prydz

This article has been cited by other articles in PMC.

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

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (907K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Image
on p.3556

Click on the image to see a larger version.

Selected References

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

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. [PubMed]
Friedman, DL; Mueller, GC. A nuclear system for DNA replication from synchronized HeLa cells. Biochim Biophys Acta. 1968 Jul 23;161(2):455–468. [PubMed]
Bernard, O; Brent, TP. DNA replication in isolated HeLa cell nuclei. Biochem Biophys Res Commun. 1973 Aug 21;53(4):1213–1219. [PubMed]
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. [PubMed]
Kumar, KV; Friedman, DL. Initiation of DNA synthesis in HeLa cell-free system. Nat New Biol. 1972 Sep 20;239(90):74–76. [PubMed]
Nagata, K; Enomoto, T; Yamada, MA. A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis. Biochim Biophys Acta. 1981 May 29;653(3):316–330. [PubMed]
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. [PubMed]
Muller, MT; Kajiwara, K; Mueller, GC. 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. [PubMed]
Berger, SL; Birkenmeier, CS. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. [PubMed]
Thomas, PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. [PubMed]
Thomas, PS. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. [PubMed]
Lehrach, H; Diamond, D; Wozney, JM; Boedtker, H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. [PubMed]
Winnacker, EL; Magnusson, G; Reichard, P. Synthesis of polyoma DNA by isolated nuclei. Biochem Biophys Res Commun. 1971 Aug 20;44(4):952–957. [PubMed]
Qasba, PK. Synthesis of simian virus 40 DNA in isolated nuclei. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1045–1049. [PubMed]
Henderson, AS; Warburton, D; Atwood, KC. Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3394–3398. [PubMed]
Schmickel, RD. Quantitation of human ribosomal DNA: hybridization of human DNA with ribosomal RNA for quantitation and fractionation. Pediatr Res. 1973 Jan;7(1):5–12. [PubMed]