DNA sequences in Neurospora crassa that code for ribosomal RNA have been isolated in a highly purified state by DNA-RNA hybridization. About 100 separate ribosomal RNA genes exist in each nucleus. These genes are composed of repeated sequences that are present about 100 times per nucleus. The individual members of this group of DNA sequences are essentially identical.The amount of DNA that codes for ribosomal RNA is different in different organisms (1)(2)(3)(4)(5). The amount of Neurospora crassa DNA homologous to ribosomal RNA (rRNA) is almost 1% (4). Neurospora contains very little (unpublished data), if any, repeated DNA, besides multiple mitochondrial and ribosomal gene sequences. Thus, DNA sequences coding for rRNA are readily isolated by techniques used for the isolation of highly purified rRNA cistrons from bacteria (1).
METHODSNeurospora crassa strain 74A was used in these studies. Conventional methods for isolation of DNA do not work well with Neurospora; Neurospora DNA was isolated by a modification of the urea-phosphate method (6). Lyophilized mycelial powder was suspended in a solution of 8 M urea, 0.24 M phosphate buffer (made of equimolar quantities of Na2HPO4 and NaH2PO4; molarity refers to the phosphate; pH 6.8), 1% sodiuni dodecyl sulfate, and 0.1 mM ethylenediamine tetraacetic acid (EDTA). The mixture was homogenized in a closed container (filled with liquid to suppress foaming) with a Waring blendor for 2 min to reduce the size of DNA pieces.The solution was passed through a hydroxyapatite (Bio-Gel HTP, BioRad Labs, Richmond, Calif.) column equilibrated with 0.24 M phosphate buffer-8 M urea at room temperature (230); the column was washed with the same solution until the A260 of the effluent was zero. It was then washed with 14 mM phosphate buffer (pH 6.8) for removal of urea from the column.DNA was removed from the column by elution with 0.4 M phosphate buffer and concentrated by centrifugation overnight at 100,000 X g. The pellet was suspended in a small volume of 1 mM EDTA. The resulting DNA was very pure and ready to use. 90% or more of the diphenylamine-reacting material was recovered from the Neurospora powder by this method.[32P]DNAs (4) were checked for alkaline lability and acid solubility.[82P]DNAs used in these experiments routinely had less than 1% acid-soluble radioactivity, virtually no alkali-labile radioactivity (<0.1%o), and 95% or better DNase lability. All DNAs were sheared before use by passing them through an orifice with a pressure drop of 50,000 lb/in.2. The resulting pieces were about 300-500 nucleotides in length.t Present address: Biology Department, University of California, San Diego, La Jolla, Calif., 92037. 3256As a final purification step, a dilute solution of sheared Neurospora [82P]DNA (20-100 ,g/ml) was heat-denatured in 0.14 M phosphate buffer-0.4% sodium dodecyl sulfate and passed immediately through hydroxyapatite equilibrated to 0.14 M phosphate buffer-0.4% sodium dodecyl sulfate at 600. Single-stranded DNA passes through the column under these con...