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We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in 25 the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 26 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism.27 Using single strand conformation polymorphism (SSCP) analysis, we demonstrate the existence of two classes 28 of 5S rRNA, one which is embryo-specific and encoded by the smallest (700 bp) cluster and the other which is 29 expressed at every stage and encoded by longer clusters (900 and 950 bp). We also demonstrate that the 30 embryo-specific class of 5S rRNA is expressed in oocytes and embryonic stages and is silenced in adult tissue 31 and that this phenomenon appears to be due exclusively to DNA methylation, as indicated by sensitivity to 5-32 azacytidine, unlike Xenopus where this mechanism is necessary but not sufficient to maintain the silenced status Q3 .33