Nucleic Acids Research, 2007, Vol. 35, No. 16 5435 Figure 5. Structure and expression of the reporter construct copiaLTR-lacZ. (A) copia LTR comprises the known upstream regulatory region, including 137 bp of the transcribed fragment. (B) b-gal activity in ovaries and carcasses. Bars indicate the ratio of b-gal activity in spn-E1/hls3987 trans- heterozygous and piwi2, armi1 homozygous to heterozygous ﬂies. The strong increase in b-gal activity due to the mutations was revealed in ovarian extracts (dark bars), whereas no signiﬁcant changes were detected in extracts of carcasses (gray bars). Measurements of b-gal activity were normalized to total level of protein in extracts. (C) Expression of copiaLTR-lacZ located on the X-chromosome in spn-E1, piwi2, armi1 homozygous and trans- heterozygous spn-E1/hls3987 ovaries. In heterozygous spn-E1/+, spn-Ehls3987/+, piwi2/+ and armi1/+ ovaries lacZ expression occurs at a low level. In ovaries of homozygous and trans-heterozygous (spnE1/hls3987) females expression is increased in germinal nurse cells (arrow 2) and developing oocyte (arrow 1), but not in somatic follicle cells (arrow 3). (D) lacZ expression level is not increased in somatic organs of spn-E1 and piwi2 larvae. lacZ staining of larval brain (ﬁrst row), haltere imaginal discs (second row) and salivary glands (third row) is shown. homozygous and spn-E1/hls3987 trans-heterozygous ovaries (Figure 5C). lacZ expression level remained unchanged in brain, imaginal discs and salivary glands of spn-E1, piwi2 and armi1 larvae as compared with heterozygous or wild- type controls (Figure 5D; data not shown). Thus, the chromatin-based regulation of tested retrotransposons mediated by rasiRNAs is realized in the germline. DISCUSSION We demonstrated that short rasiRNA species, known to be associated with Piwi subfamily proteins (3–6), have a germline-speciﬁc function in the maintenance of chroma- tin modiﬁcations of retrotransposons. The spn-E, piwi and armi genes are predominantly expressed in germ cells and their mutant states lead to abnormalities in germ-line development and sterility (41–44). Moreover, evidence of germ-line speciﬁcity of the rasiRNA-mediated silencing pathway is supported by the observation, that rasiRNAs are signiﬁcantly more abundant in the germline than in somatic tissues. Germline-speciﬁc silencing of mobile elements is considered an important defense mechanism against mutations caused by mobile element transposi- tions, because selﬁsh transposable elements are thought to be expressed mainly in germinal cells to ensure their ampliﬁcation and transmission to the progeny. A distinct function of rasiRNA-mediated silencing concerns the maintenance of Drosophila telomeric state. Extension