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A 3.6-Megadalton, Highly Repetitive Eco R_l Fragment in the Genome of Novikoff Hepatoma¹

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

The DNA of Novikoff hepatoma cells was compared to that of normal rat liver by Eco R_l restriction endonuclease digestion, followed by agarose gel electrophoresis. Nuclear and nucleolar DNA from both sources was utilized. Three Eco R_l fragments were found in the Novikoff hepatoma DNA: Fragment A, 3.6 megadaltons; Fragment B, 1.3 megadaltons; and Fragment C, 0.91 megadaltons. Fragments B and C appear in equal amounts in both nuclear and nucleolar DNA of normal rat liver and Novikoff hepatoma cells. In hybridization experiments, radioiodinated Fragment A hybridized to excess Novikoff and normal liver DNA's with C₀t_1/2 values of 1.3 x 10^-1 and 3.0 x 10¹, respectively. These data demonstrated a 220-fold enrichment of Fragment A in the genome of Novikoff hepatoma cells relative to the normal rat liver. By comparing these data with C₀t_1/2's of single-copy rat DNA, reiteration frequencies of 6600 and 30 were calculated for Fragment A in Novikoff hepatoma and normal rat liver, respectively. In preliminary studies, the relative amount of Fragment A present in digests of Novikoff nuclear and nucleolar DNA was compared; the results provided evidence that this fragment is localized to the nucleolus.

As Eco R_l Fragment A is present in only 30 copies in the genome of the normal rat liver, it is probably not a part of the ribosomal cistrons, which are present in 200 to 400 copies. Fragment A did not hybridize to either 18S or 28S ribosomal RNA. However, the fragment may be one of a set of heterogeneous spacer sequences and, thus, part of the ribosomal cistron. This DNA fragment may be important to structural aspects of the nucleolus or to nucleolar hyperfunction in the Novikoff hepatoma.

¹ This investigation was supported by Grant CA-10893, P. 1, awarded by the National Cancer Institute, Department of Health, Education and Welfare; Bristol-Myers Fund; the Pauline Sterne Wolff Memorial Foundation; and a generous gift from Mrs. Jack Hutchins.

Received 8/ 7/78. Accepted 1/ 3/79.