Open AccessThe Involvement of RNA in the Initiation of DNA Synthesis in Mammalian Cells
Author(s) -
PEARSON Colin K.,
DAVIS Peter B.,
TAYLOR Alison,
AMOS Norma A.
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10178.x
Subject(s) - nucleic acid , dna , rna , centrifugation , buoyant density , caesium , chemistry , covalent bond , differential centrifugation , uridine , thymidine , biochemistry , sodium , microbiology and biotechnology , biology , organic chemistry , gene
Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesised DNA is covalently attached to RNA. Newly synthesised DNA (in baby hamster kidney cells, BHK‐21/C13), pulse‐labelled with [ 3 H]thymidine for 10 min at temperatures below 25 °C, banded at a greater buoyant density than mature [ 14 C]DNA (heatdenatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesised RNA, labelled with [ 3 H]uridine, banded at a buoyant density slightly greater than the DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse‐labelled at 37 °C, nor when samples were heat‐denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA. Such artifacts can also be produced when centrifuging mixtures of mature (non‐replicating) nucleic acids by appropriate manipulation of experimental conditions. Mature [ 3 H]RNA was shown to aggregrate with mature [ 14 C]DNA when the DNA concentration was greater than 100 μg/ml. Sonicated mature [ 3 H]DNA banded with a mean buoyant density in caesium chloride gradients greater than non‐sonicated mature [ 14 C]DNA. This density difference was prevented by heat denaturation in the presence of formaldehyde.