Purification and Some Characteristics of 5S DNA from Xenopus laevis

Abstract

DNA containing the multiple genes for 5S RNA The small RNA component of ribosomes, termed 5S RNA, comprises about 1-2% of total cellular RNA in prokaryotes and eukaryotes. In the South African toad, Xenopus laevis, 5S RNA genes are known to be in greater abundance than the genes for 18S and 28S ribosomal RNA and are not intermingled with them (1). This report documents the purification of the DNA containing the genes for 5S RNA from the genome of X. laevis and describes some characteristics of 5S DNA. MATERIALS AND METHODSCrude Cs2SO4 was purchased from Gallard Schlessinger (more than 99% pure, catalogue no. 27573) and was found to be as good for the experiments described here as the much more expensive optical-grade product. CsCl was purchased from Harshaw, and actinomycin D was a gift of Merck. The antibiotic was dissolved in a stock aqueous solution (1 mg/ml) and stored at 4°C in the dark.Purification of DNA. DNA was purified from isolated nuclei of X. laevis erythrocytes (1). To maintain the DNA at the highest possible molecular weight, it was never precipitated during purification. The purified nuclei, suspended in SSC (0.15 M NaCl-0.015 M sodium citrate), were lysed with 0.5% sodium dodecyl sulfate and digested with 1 mg/ml of Pronase overnight at 370C. The DNA was extracted gently with phenol three or four times. Fig. la).For saturation-hybridization experiments, about 1.5 mg of 5S RNA isolated from ovaries of X. laevis was methylated in vitro with 5 mCi of [3H]dimethyl sulfate (6). The 5S [3H]RNA was purified by four ethanol precipitations that removed all acid-soluble radioactivity; the specific activity of the RNA was 10,000 cpm/ug.Radioactive RNA was hybridized with DNA immobilized on Millipore HA filters. Aliquots of DNA complexed with silver were mixed with 10 volumes of 20 X SSC to complex the silver before the DNA was denatured and trapped on filters.The hydribization medium contained 50% formamide, 4 X SSC, and 0.05 M Tris (pH 7.8); incubation took place at 40'C overnight (7). The filters were washed, treated with RNase, and counted in a scintillation counter.Denaturation Map of 5S DNA. The 5S DNA was partially denatured with alkali for electron microscopy (8). The exact procedure that we used for 5S DNA has been described in detail for ribosomal DNA (9). RESULTSPurification of 5S DNA. Two characteristics of 5S DNA from X. laevis made its isolation possible. First, hybridization experiments have shown that thousands of genes for 5S RNA are present in a haploid complement of X. laevis DNA, so that 5S DNA is abundant in the genome (ref. 1, and see Table 2). Second, 5S DNA has a lower buoyant density in CsCl than does the bulk of X. laevis DNA (1) [however, this difference (about 7 mg/cm8) is not enough to allow its separa-

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

“…These genes are tandemly arranged in 720-base pair (bp) repeating units (9) which include the 5S RNA gene, a pseudogene (25), and an untranscribed spacer region which varies in length (7,11) (Fig. 1).…”

Gene pioneers: Donald Brown and Thomas Maniatis win the 2012 Lasker~Koshland Special Achievement Award in Medical Science

“…A few years later, in 1971, Brown and his group were able to isolate the 5s ribosomal genes (5). Just the second gene sequences to be isolated, the 5s ribosomal genes were the basis for many of the studies that illuminated the nature of eukaryotic gene structure and regulation.…”

Chromatin directed transcription of 5S and tRNA genes.

“…RNA-DNA hybridization to purified Xenopus laevis 5S DNA prepared as described by Brown et al (16) was carried out as described previously (10).…”