Recognition of ribosomal RNA sites in DNA. II. The HeLa cell system.

Abstract

As reported in a preceding paper,' the analysis of the sedimentation behavior and base composition of the RNA recovered from hybrids between E. coli 16S and 238 RNA and homologous DNA, after RNase digestion of non-base-paired segments, has given results which suggest a regular and complete hydrogen bonding of the hybridized RNA with specific sites in DNA. A similar analysis has now been applied to the investigation of ribosomal RNA sites in HeLa and other human DNA's and has made it possible to resolve the ambiguities in specificity pattern and base composition presented by the raw hybridization data, and to identify among the RNA-DNA complexes a fraction which appears to involve specific sites in DNA. Materials and Methods.-Methods of growth in suspension of HeLa cells have been described previously.2 Ribosomal RNA labeled to a high specific activity with p32 was prepared by growing the cells for 48 hr in a modified Eagle's medium3 with 4.5 X 10-5 M phosphate and 5% dialyzed horse serum, in the presence of 20-25 ,.C/ml p32 carrier-free orthophosphate. In order to reduce the specific activity of the unstable RNA fraction,4 the cells were subjected to a chase for 16-20 hr in the presence of 10-2 M unlabeled phosphate. Ribosomal RNA was extracted from the ribosomal fraction2 with redistilled, water-saturated phenol containing 0.1% hydroxyquinoline,5 in the presence of 0.5% sodium dodecylsulfate, 5 mg/ml naphthalene disulfonate,2 and 1.3 mg/ml bentonite.6 Following 3 ethanol precipitations, the final product was dissolved in acetate buffer 0.01 AT, pH 5.0, NaCl 0.1 M, and resolved into the 28S and 18S components by two cycles of sucrose gradient (linear, 5-20%) centrifugation. Several P32-labeled preparations made in this way had at the time of isolation specific activities varying between 1.0 and 1.5 X 106 dpm/igg RNA. While the 288 preparations were always found to be free of any detectable P32 contaminant (less than 10-6), determined as RNase-resistant counts excluded by Sephadex G-100 or alkali stable counts, the 18S preparations usually showed between 0.1 and 1% RNase-resistant radioactivity associated with macromolecular material, which could be reduced 'to less than 10-4 by two or more passages of the 18S preparation through nitrocellulose membranes.' Reference is made to the preceding paper' for the procedures followed for 1)NA preparation and denaturation, RNA-DNA hybridization (incubation here was carried out at 70'C for 2'/2-8 hr), isolation of hybrids, and analysis of sedimentation properties and base composition of hybridized RNA after RNase digestion.

The Genes for Cytoplasmic Ribosomal Ribonucleic Acid in Higher Plants

“…Much less is known about rRNA genes in plants, and determination of the absolute redundancy of these genes is complicated by the apparent cross-hybridization between the two high molecular weight rRNAs (1.3 and 0.7 X 106) and their complementary sequences in the DNA, which confuses the determination of saturation levels (15). Similar cross-hybridization has been noted with the rRNAs from Escherichia coli (1,4,21), yeast (25), rabbit (22), and HeLa cells (2), which contrasts with the situation observed with Bacillus megaterium (31), Bacillus subtilis (3,21), Xenopus (7,29), Drosophila (24), and rat (27). The cross-hybridization initially reported with HeLa cells (2) was, however, virtually eliminated by the use of better preparations of rRNA and hybridization under conditions of very low RNA/DNA ratios, whereby measurements were limited to the initial stages of the hybridization reactions (17) MATERIALS AND METHODS DNA Preparation and Fractionation.…”

“…Similar cross-hybridization has been noted with the rRNAs from Escherichia coli (1,4,21), yeast (25), rabbit (22), and HeLa cells (2), which contrasts with the situation observed with Bacillus megaterium (31), Bacillus subtilis (3,21), Xenopus (7,29), Drosophila (24), and rat (27). The cross-hybridization initially reported with HeLa cells (2) was, however, virtually eliminated by the use of better preparations of rRNA and hybridization under conditions of very low RNA/DNA ratios, whereby measurements were limited to the initial stages of the hybridization reactions (17) MATERIALS AND METHODS DNA Preparation and Fractionation. Nuclear preparations were made from embryo-axes of 3-day-old pea seedlings and young shoots from swisschard and artichoke plants by a short homogenization of the tissue at top speed in a VirTis 45 in a medium containing 250 mm sucrose, 2.5% Ficoll, 5% dextran, 25 mm tris-HCl, pH 7.8, 2 mm CaCl2, and 10 mm magnesium acetate (5 volumes/g of tissue).…”

The Amount of Ribosomal RNA Genes in Tetrahymena pyriformis in Different Physiological States

“…1 7 3 rRNA strands occupy 25-S rRNA gene sites preventing 25-8 rRNA from entering these sites, but not forming stable hybrids). Apparent competition between the two rRNAs has been observed in many competition studies where the two rRNAs are present in saturating amounts [32,33]. I n an attempt to investigate the effect of high concentrations of unlabeled competitor RNA on the hybrid formation the experiments depicted in Fig.…”

Localization of highly repeated HindIII fragments of novikoff rat DNA to the nucleolus

“…Novlkoff rat ascltes hepatoma cells have large nucleoh compared to normal rat cells and nucleoli from these hepatoma cells have been isolated m high yield and purity [ 1 ] The nucleolus IS known to be the site of nbosomal RNA synthesis and the rlbosomal RNA genes are repeated lOO-SOO-tlmes/dlplold genome [ 1,2] It hds been estimated that the DNA content/nucleolus 1s 1 3 pg and that the DNA content/cell 1s 8 pg for Novlkoff rat cells [ 1 ] Rlbosomai DNA comprised 0 008% of the total DNA [3] and -0 05% of nucleoldr DNA The remaining 99 95% of nucleolar DNA has not yet been chdracterlzed Restriction endonuclease dIgestIon followed by gel electrophoresls has been used to detect repeated sequences m DNA from several mammahan species [4-61 Novlkoff rat hepatoma DNA was analyzed by gel electrophoresls after EcoRI restrIction endonuclease digestIon m this laboratory SrnallEcoRI fragments of Novlkoff rat DNA were exammed [7] by acrylamide gel electrophoresls and large EcuRI fragments of Novlkoff rat DNA [8] by agarose gel electrophorests The largest EcoRI fragment, fragment A, was reported [8] localized to the nucleolus Here, HlndIII digestIon patterns of total nuclear DNA, nucleolar DNA, and extranucleolar nucleoplasmlc DNA have been studied by agarose and acrylamlde gel electrophoresls From quantltatlve analysis of the amount of the smallest HzndIII DNA fragment m various fractions of the nucleus, this fragment has been found to be localized to the nucleolus 2 Materials and methods Novlkoff hepatoma ascltes cells were mamtamed m male Holtzman rats for 6 days and the cells were labeled with 32P for '74 h as m [9] Nucleoh and extranucleolar nucleoplasm, 1 e , the nucleus minus the nucleolus, were prepared by the sucrose--Ca'+ procedure [ 1,101 After sedunentatlon of nucleoli by centrlfugatlon at 1100 X g for JO mm from somcated nuclei, the supernatant contained the extranucleolar nucleoplasm The extranucleolar chromatm was collected by centrlfugatlon at 20 000 X g for 30 mm after 2 vol ethanol were added Nucleolar contammatlon in the extranucleolar chromatm was muumlzed by momtormg the somcatlon process by phase microscopy to -80% nuclear dlsruptlon DNA was prepared generally as m Ill] To ehmlnate small DNA fragments m the extranucleolar nucleoplasm preparation, 6 cycles of DNA spoohng were repeated by adding 2 vol ethdnol and dlssolvmg the spooled DNA m SSC (0 15 M NaCI, 0 0 15 M sodnun citrate)…”