Planta 146, 467-473 (1979) Planta by Springer-Verlag 1979 Purification and Restriction Endonuclease Mapping of Soybean 18 S and 25 S Ribosomal RNA Genes Harald Friedrich 1, Vera Hemleben 1, Richard B. Meagher 2, and Joe L. Key 2 1 Institut f/ir Biologic II der UniversitM, Lehrstuhl ffir Genetik, Auf der Morgenstelle 28, D-7400 Tfibingen, Federal Republic of Germany, and 2 Department of Botany, University of Georgia, Athens, GA 30601, USA Abstract. The restriction endonuclease map of the 25S and 18S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifuga- tion in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm- 3 by analytical centrifugation in CsC1. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Barn H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9.106 or ap- proximately 9,000kb. The 25S and 18S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [125I]25 S or [125I]18 S rRNA. It was dem- onstrated that there is no heterogeneity even in the spacer region of the soybean rDNA. Key words: Gene mapping - Glycine - Restriction enzymes Ribosomal RNA genes. sequentially to the final molecules. However, the size of the precursor and the processing intermediates are generally smaller in size (Rogers etal., 1970; Leaver and Key, 1970; Grierson and Loening, 1974; Hadjio- lov and Nikolaev, 1976). There was some uncertainty whether the genes coding for 5 S rRNA are inter- spersed with the high-molecular-weight ribosomal RNA genes as reported for bacteria (Nomura, 1976), chloroplasts (Bedbrook et al., 1977), and some lower eucaryotes (Maizels, 1976; Bell et al., 1977), or are arranged separately on different sites of the chromo- somes as indicated by in situ hybridization studies (Wimber et al., 1974; Durante et al., 1977). But there is now evidence that the 5 S rRNA genes are not linked to the 25 S and 18 S rRNA genes in higher plants (Hemleben and Grierson, 1978). We used the method of preparative centrifugation in CsCl-actinomycin D gradients for purification and enrichment of the repetitive ribosomal RNA genes from soybeans (Hemleben et al., 1977). The enriched rDNA sequences were characterized by mapping with restriction endonucleases and specific hybridization of the fragments with [125I]25 S and 18 S rRNA. Introduction The organization of the repetitive genes coding for ribosomal 28 S and 18 S RNA has been analyzed in detail in animals such as Xenopus or Drosophila (Wellauer and Dawid, 1977 a, b). Studies on this group of genes have been enhanced by cloning in Escherichia coli cells. For higher plants the internal organization of the ribosomal RNA genes is not ex- actly known. The processing scheme of the ribosomal RNA synthesis is similar to that described for animal cells. The 25 S and 18 S rRNA m~olecules are transcribed in a common precursor which is processed Materials and Methods Isolation and Characterisation of Soybean DNA 50 g of soybean embryonic axes were imbibed in 200 ml of Tris- EDTA-buffer (I00 mM Tris, 5 mM EDTA/pH 7.5) for 2 h. The DNA was isolated and purified as described by Hemleben et al. (1975). The neutral CsC1 gradients and the CsCl-actinomycin D gradients were prepared as described by Hemleben et al. (1975, 1977). Saturation hybridization with soybean DNA on filters and [3H]25S and 18S rRNA and gradient hybridization were performed as described by Grierson and Hemleben (1977). For determination of the buoyant density, 3 gg of rDNA-enriched soy- bean DNA were dissolved in Tris-EDTA-buffer (10mM Tris, 1 mM EDTA/pH 7.5), 1 gg SPO l DNA was added and the solu- tion was adjusted with saturated CsC1 solution to a final refractive 0032-0935/79/0146/0467/$ 01.40