Hybridization between rat liver DNA and complementary RNA

Abstract

The hybridization of rat liver DNA with complementary RNA has been studied using the membrane technique of Gillespie & Spiegelman (1965). Complementary RNA was synthesized in vitro using Micrococcus RNA polymerase and rat liver DNA. Hybridization was rather rapid, but only about 5% of the total DNA became involved in hybrid formation. The hybrid melted sharply with a mean temperature of 72 °C, only 3 deg. C lower than the mean melting temperature of enzymic DNA-RNA hybrid. RNA complementary to at least half of the total DNA was isolated from a 2:1 enzymic DNA-RNA hybrid. Only about 5% of the DNA hybridized with this complementary RNA under standard annealing conditions, showing that the limited hybridization of rat liver DNA is caused by factors other than limited complementarity of this RNA. It is suggested that the factor responsible is the extreme complexity of mammalian DNA. Mammalian DNA is known to contain a minor fraction which, following denaturation, renatures rapidly compared with the bulk of the DNA. Renaturing rat liver DNA reduced its capacity to hybridize with complementary RNA by two-thirds, showing that it is the rapidly renaturing DNA fraction which hybridizes with the RNA. In confirmation of this, when the rapidly renaturing and slowly renaturing DNA fractions were separated by differential adsorption to hydroxylapatite at 70 °C, preferential hybridization of complementary RNA to the rapidly renaturing DNA fraction was observed. Liver nuclear RNA competed completely against the hybridization of complementary RNA with rat liver DNA. DNA-like RNA from liver, kidney, spleen and brain all competed strongly against complementary RNA. It is concluded that the hybridization of DNA and RNA from higher organisms under the usual conditions permits mainly or only the hybridization of the minor DNA fraction which renatures rapidly. RNA complementary to most of this DMA fraction is found in several different tissues.