J Mol Evol (1993) 36:144-152 Journal of Molecular Evolution © Springer-Verlag NewYork Inc. 1993 Molecular Evolution of the Intergenic Spacer in the Nuclear Ribosomal RNA Genes of Cucurbitaceae Klaus King, 2 Ramon A. Torres, 1 Ulrike Zentgraf, 2 and Vera Hemleben 2 Institut f~ir Mikrobiologie und Genetik der Universitfit Mtinchen, Maria Ward Strage la, D-8000 Mtinchen 19, Federal Republic of Germany 2 Biologisches Institut der Universit~it Tfibingen, Lehrstuhl fiir Allgemeine Genetik, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany Summary. The intergenic spacer (IGS) of a 10- kbp repeat (clone pRZ7D) of the nuclear 18S, 5.8S, and 25S ribosomal RNA genes of Cucurbita pepo (zucchini) was sequenced and compared to the IGS sequences of two other Cucurbitaceae, Cucurbita maxima (squash), and Cucumis sativus (cucumber). The nucleotide sequence and the structural organi- zation of the IGS of C. pepo and C. maxima are rather similar (between 75 and 100% sequence sim- ilaritydepending on the region compared), The IGS are mainly composed of three different repeated el- ements interspersed into unique sequences: GC- rich clusters, a 422-bp AT-rich element including the transcription initiation site (TIS) for RNA poly- merase I, and 260-bp repeats in the 5' external tran- scribed spacer (D repeats). The TIS is duplicated in the 10-kbp repeat class of C. pepo, as it is also described for the 11.5-kbp rDNA repeat of C. max- ima. The IGS of Cucumis sativus is also composed of different repeated elements; however, obvious sequence identity to the Cucurbita species only oc- curs around the TIS and the preceding AT-rich re- gion. GC-rich clusters with different primary se- quences are present in the IGS of all three plants. Remarkably, the repeated elements in the 5'ETS accumulate TpG and TpNpG motifs, whereas CpG and CpNpG motifs less frequently occur. This ac- Abbreviations: IGS (intergenic spacer), ITS (internal transcribed spacer), ETS (external transcribed spacer), NTS (nontranscribed spacer), rDNA (ribosomal DNA), rRNA (ribosomal RNA), TIS (transcription initiation site), TTS (transcription termination site) Offprint requests to: V. Hemleben cumulation might be caused by the transition of methylated cytosines (in mCpG or ~CpNpG mo- tifs) into thymidine via deamination in a previously GC-rich ancestor. The following singular region ex- hibits 50% G + C in C. pepo, 53% G + C in C. maxima, and 63% G + C in C. sativus. A model for a common ancestor of the 3'IGS for these Cucur- bitaceae is proposed. Key words: "Ancestor" IGS -- Cucum& -- Cu- curbita -- C to T transition -- Intergenic spacer DNA methylation -- Ribosomal DNA The nuclear ribosomal RNA genes (rDNA) of higher eukaryotic organisms are organized in tran- scription units containing the 18S, 5.8S, and 25S/28S rRNA coding regions which are arranged in tandem arrays located in the nucleolus organizer region (NOR), whereas the 5S rRNA genes are lo- calized at other parts of the genome. The 18S, 5.8S, and 25S rRNA coding regions are separated by the large intergenic spacer (IGS); it includes the tran- scription initiation site (TIS) and the transcription termination site (TTS) of RNA polymerase I, the 5' and 3' external transcribed spacers (ETS), and a nontranscribed spacer (NTS) region. All IGSs of higher plants investigated so far are composed of different repeated elements interspersed into unique sequences. Whereas the coding regions are strongly conserved among different organisms, the spacer regions show an enormous variability in se- quence and often in length (Rogers and Bendich