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Nucleic Acids Research, 1985, Vol. 13, No. 10 3551-3560
© 1985

Articles

Isolated HeLa cell nuclei synthesize meaningful DNA

Tom Kristensen and Hans Prydz

Research Institute for Internal Medicine, University of Oslo Rikshospitalet, Oslo 1, Norway

Received January 28, 1985. Revised March 27, 1985. Accepted April 30, 1985.

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.


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