Figure 2.

Lam inactivation determines the silencing of gypsy sequences when gypsy is active. (A–C) Representative egg chambers subjected to β-galactosidase staining as readout for gypsy-lacZ reporter activity. (A) gypsy-lacZ reporter activity is derepressed when the X chromosome from wild type Canton S flies is combined with the Df(1)l11 deficiency encompassing the flamenco locus. (B) Derepression of the gypsy-lacZ reporter activity in flies with the X chromosome carrying the permissive flam^A allele in combination with the Df(1)l11 deficiency (Df(1)l11/flam^A). (C) gypsy-lacZ reporter activity of Df(1)l11/flam^A gonads is repressed in the Lam^4643/K2 genetic background. (D–H) Gene expression analysis of gypsy and flamenco sequences in RNAs isolated from female head tissues. wt: X chromosomes from the wild type Canton S strain. flam^A: X chromosomes carrying the flamenco permissive allele flam^A. ct^A: presence of a gypsy-induced mutation in the cut locus. Flies can be homozygous for the wild type Lam allele (+/+) or transheterozygous (4643/K2). Data are mean values from three independent experiments, and error bars indicate SD (* P < 0.05; *** P < 0.005) (D) gypsy expression levels in wild type Canton S (wt) and flam^A female head tissues. (E) gypsy expression levels in control and Lam^4643/K2 female head tissues carrying X chromosomes from the wild type Canton S (wt) strain. (F) gypsy expression levels in control and Lam^4643/K2 female head tissues carrying X chromosomes from the flam^A strain. (G) Upper part: schematic representation of the genomic region containing the 5′ upper region of the cut gene (grey) where the gypsy element (cyan) is inserted. The two strand-specific primers used for RT experiments are represented by grey arrows, while the two qPCR couples of primers are represented by black arrows (see Table S1). Lower part: strand-specific qRT-PCR analysis of the transcription levels of the boundaries of the gypsy elements and the surrounding genomic regions of the cut locus (ct^A allele) in ct^A flam^A/flam^A; Lam^4643/K2 female head tissues compared to the ct^A flam^A/flam^A; Lam⁺ control. ct-gyp up is the upstream boundary, and ct-gyp dw is the downstream boundary. (H) qRT-PCR analysis of three flamenco fragments in head tissues of flam^A females with or without a copy of the ct^A allele and carrying wild type or Lam transheterozygous combination compared to the flam^A; Lam⁺ control. flam1 sequence was selected inside the flamenco locus in a region without homology with gypsy, while flam5 and flam6 were selected inside gypsy fragments [31].