Analysis of the human genome by complementary RNA/DNA gradient hybridization and relaxed cesium sulfate-silver ion density centrifugation

Abstract

3H-Labeled complementary RNA (cRNA) transcribed from total nuclear human DNA was hybridized to homologous DNA and the hybrids formed localized in CsCl density gradients. Radioactive peaks indicative of cRNA/DNA hybrids identified ten density components. The experimental conditions used indicated that these components correspond to DNA rich in repeated sequences. In addition, cRNAs transcribed from five C o t fractions (31% of total DNA; C o t < 100) were hybridized to total DNA in CsCl gradients. The hybrids sedimented at similar densities to those seen in total cRNA but showed a differential distribution along the five fractions. To isolate native double-stranded DNA components containing the various families of repeated sequences observed, nuclear DNA was fractionated using relaxed Cs 2 SO 4 Ag + density gradient centrifugation. These fractions revealed the presence of seven major components at densities of 1.715, 1.711, 1.708, 1.705, 1.702, 1.700 and 1.698 g/cm 3 and three minor components at densities of 1.696, 1.693 and 1.687 g/cm 3. The density components corresponded to those observed by cRNA/DNA hybridization, thus suggesting that the bulk of the human genome is made up of ten density-biased components containing sequences of varying degrees of repetitiveness.