Fig. 2. Mapping, cloning and expression of twin. (A) Genomic region around the ry3 and ry5 P-element insertions. The insertions (black arrow) fall in a 15 kb intron of ccr4. Distal probe: 1.5 kb of genomic DNA isolated from inverse PCR generated from the 5' end of the ry3 or the 3' end of the ry5 strains. Proximal probe: 1.5 kb of genomic DNA isolated from inverse PCR generated from the 3' end of the ry3 or the 5' end of the ry5 strains. The three exons of ccr4 included in the distal probe are shown. The proximal probe lies in the 15 kb intron. (B) Different splice forms of Drosophila ccr4 represented by ESTs isolated by the Drosophila Genome Project. The three exons preceding the 15 kb intron containing the P insertions are shown in gray. M, putative start methionine; (M), start methionine less certain because there is no in-frame stop codon upstream in the EST isolated; ^*, stop codon. (C) Northern blot of poly(A)-selected RNA from wild-type and twin^ry3/twin^ry3 ovaries. The distal and proximal probes are as described in A. RP49 was used as a loading control. The proximal probe seems to be upregulated in twin^ry3 homozygotes, but this is unlikely to contribute to the twin phenotype. The alleles are all recessive and phenotypically similar, and the point mutants have missense mutations in ccr4 and not in the predicted ORFs of the intron. (D-K) mRNA in situ hybridization. Anterior is leftwards in all panels. (D-G,I-J) ccr4 antisense probe. (H,K) ccr4 sense negative control. (D) Maternal ccr4 is present at very high levels in early embryos. (E) Expression is quite reduced in the blastoderm. (F,G) Expression is general, with strong staining in the central nervous system. (H) No staining observed in sense control. (I,J) Expression is present throughout the ovary, with stronger expression in germ cells than in soma. Expression is particularly strong in late (stage 10 and later) nurse cell. Staining in germarium region 1 was not strong, but this tissue is refractory to in situ hybridization. (K) No staining observed in sense control.