Nodeotide Seqnence of a Complete Ribasomal Spacer of D. Melanogaster

We determined the nucleotide sequence of a D. melanogaster ribosomal DNA spacer. Sequences of various portions of different cloned ribosomal spacers have been previously reported. We extend the analysis to cover the entire nontranscribed and external transcribed regions. Comparison to other cloned ribosomal DNA gene units of this species confirms a conserved general organization of the ribosomal spacer through different size classes. JX melanogaster ribosomal gene units interrupted by insertions are known to be transcribed at a much lower level than the continuous gene units. Nonetheless previous sequence analysis of a region around the trasenption initiation site did not reveal significant differences in rDNA genes with and without insertions. We extend such analysis to cover the last two promoter duplications in the spacer and the entire external transcribed spacer up to the 5' cleavage site of the 18S rRNA. INTRODUCTION Ribosomal genes (rDNA) of every higher organism studied are tandemly repeated and clustered with contiguous transcribed regions separated by nontranscribed spacers (NTS) (1). The 28S, 18S and 5.8S rRNA molecules are transcribed as parts of a large precursor molecule which is subsequently processed in several steps to yield the mature rRNA species (2, 3, 4). While the mature rRNA coding regions are largely conserved among eukaryotes, spacer regions evolved much more rapidly and diverge in all but the most closely related species (5, 6). Spacer regions comprise the NTS region and the portion of the transcribed region upstream to the 18S rRNA coding region, termed external transcribed spacer (ETS). In X. laevis and D. melanogaster, among others, the NTS region is highly variable in length within the same locus, and the size distribution of fragments may be quite different in individual animals (7, 8). This length © IR L Press Limited, Oxford, England. 1 ° 8 9 Nucleic Acids Research heterogeneity appears to be originated by different numbers of internally repetitious portions of the spacers (7, 9, 10). In D. melanogaster there are 150-250 ribosomal gene units in each nudeolus organizer present on the X chromosome and the Y chromosome. A large fraction of gene units are interrupted in the 28S rRNA coding region by noncodinq DNA segments which have been called ribosomal insertions. These insertions occur in two distinct sequence types, each represented by several size classes (11, 12). rDNA repeating units containing an insertion are generally not transcribed (13, 14, 15, 16, 17, 18). Nucleotide sequences around the transcription initiation site do not differ in cloned genes with and without insertions (19). Various portions of different cloned NTS regions have been sequenced (19, 20, 21, 22, 23, 24). We report here on the nucleotide sequences of the complete spacer, i.e. NTS and ETS regions, of a cloned D. melanogaster gene and extend the sequence comparison of cloned genes with and without insertions. MATERIALS AND METHODS rDNA clones pDmr a56 and pDmr Y22 were obtained from P. K. Wellauer (25). Recombinant phage ADmr275 contains a ribosomal spacer upstream to a continuous gene unit, whereas /lDmr231, ADmr241 and ADmr214 contain spacers upstream to gene units with type 1 insertions (19). These phage clones were kindly provided by I. B. Dawid. P. melanogaster stocks Stock 1 males are X~Y, y* su(w') w* bb/Ybb . Stock 2 males are R( 1)2, cv v f bb/Ybb . Wild-type Oregon-R is from the University of Naples. Genomic DNA analysis Genomic DNA was digested, electrophoresed on agarose gels, transferred onto nitrocellulose filters according to Southern (26) and hybridized to a P-nick-translated probe (27). Terminal labelling and fine mapping The fragments to be analyzed were end-labelled at the 5 end using T4