Nucleic Acids Research

The organization of differently sized genes coding for rRNA in Xenopus laevis has been investigated by partial EcoRI or Hindlll digestion. The electrophoretic patterns obtained revealed that most adjacent repeating units are equally sized. This conclusion is in agreement with a previous suggestion that the nucleolar organizer is made up of internally homogeneous blocks of rRNA genes. INTRODUCTION The length variability of the multiple copies of rRNA genes in Xenopus laevis (l) has been shown to be due to variations of the number of short subrepeats within the non transcribed spacer region (2). Recently we have studied this variability in a population of Xenopus laevis (3); while a high degree of length polymorphism has been found in the population, each individual exhibited a few discrete classes of genes. As for the organization of differently sized genes along the nucleolar organizer, we found that equally sized genes are tandemly arranged. The method used was the examination at the electron microscope of reassociated high molecular weight rDNA. This conclusion was different from the one reached by other authors (4); they used a cloned spacer containing fragment as a probe for the analysis of the length of contiguous genes along the chromosomal rDNA and found them very often different in length. © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 1 3 3 5 Nucleic Acids Research Because of the relevance of this point for the understanding of gene rectification, that is of the mechanism(s) responsible for the parallel evolution of reiterated genes, this discrepancy needed further investigation by different approaches. The method used here is the electrophoretic analysis of fragments resulting from partial EcoRl or Hindlll digestion of purified rDNA from Xenopus laevis individuals. This allows a direct investigation of adjacent genes with no involvement of intermediate steps such as cloning and reannealing procedure. The results obtained confirm that equally sized rDNA repeat units are mainly clusterd in blocks internally homogeneous. MATERIALS AND METHODS Extraction and purification of rDNA: Adult Xenopus laevis individuals were chosen with restriction patterns suitable for the analysis of partial digests (number, intensity and distribution of bands) (3)rDNA was extracted from erythrocytes and purified by two cycles of CsCl gradient centrifugation, as described previously (3)Care was taken to keep the molecular weight of the DNA as high as possible; in the various experiments it ranged from 40 to 70x10 daltons, as determined by analytical ultracentrifugation. Partial digestion with restriction enzymes: 2 to 3 pg of purified rDNA in 0.5 ml of 0.05 M NaCl, 0.01 M MgCl2, 0.1 M Tris-HCl (pH 7-5) were added with EcoRI and incubated at 37°C. The amount of the enzyme and the time of incubation were such to give a proper partial digestion. Hindlll partial digestion was carried out in 0.05 M NaCl, 0.006 M MgCl,, 0.006 M Tris-HCl (pH 7.S), at 37°C. Gel electrophoresis: Partially digested rDNA was ethanol precipitated and redissolved in 100 pi of electrophoresis buffer (0.036 M Tris base, 0.001 M EDTA, O.O3O M NaH.PO , pH 7-7) conz 4 taining 8% (x/v) sucrose and 0.025% (w/v) bromophenol blue. The