Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells

Publisher Website
Google Scholar
Abstract
The DNA of Novikoff hepatoma ascites cells contains a 3.6 Mdalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker, et al., 1979). C0t [C0 = initial concentration of DNA; t = time of hybridization] analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in .lambda. gt WES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from 1 terminus which was used to generate mono-5''-32P-labeled fragments. The larger PstI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence island, TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver.