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Fig. 3. Molecular analysis of the csul locus. (A) Restriction map of the genomic DNA covering the csul locus with the location of the P element insertion in csul^P. Below the map are shown three transcription units in this locus and their orientation, as well as the three genomic fragments used for constructing transgenes. Transcription orientation of the fidipidine (), csul (ß), and Kinesin heavy chain (Khc) genes is indicated by arrows. The analysis of complementation of the csul^P mutation by transgenes shown on the right demonstrates that the ß gene corresponds to csul. (B) Restriction map of the csul gene, with alignment of the csul transcript. Exons are drawn as boxes and the putative open reading frame is indicated in black. (B) BamHI; (P) PstI; (R) EcoRI; (Xb) XbaI; (Xh) XhoI. (C) Northern blot showing a single ovarian csul transcript of 2 kb. RNA marker sizes (in kb) are indicated on the left. (D) Western blot detection of Csul in wild-type and csul^P ovarian protein extracts using anti-Csul antibodies. A band of 65 kDa (arrow) is detected in wild-type but not in csul^P. Protein load in each well was normalized by using anti-actin antibodies (data not shown). The anti-Csul antibodies recognize another protein of 38 kDa unrelated to Csul. (E) Alignment of the amino acid sequences of Csul and homologous proteins performed using the Pileup program of the Wisconsin Package (Genetics Computer Group) reveals that csul encodes a protein-arginine methyltransferase similar to human PMRT5. Gaps in the amino acid sequence, indicated by dots, were introduced for optimal alignment. At each position of the alignment, residues identical in all sequences are background-shaded blue, and functionally conserved (i.e. more than half of the amino acids of a column) residues with strong or weak similarities are background-shaded red and orange, respectively. The asterisks over the positions 243 and 244 indicate the substitutions made in the csul^G343A;R344L transgene. GenBank accession number sequences are as follows: CP4855_AG: EAA14767; PRMT5_HS: AF167572; zC14A17.2_DR: CAD60861; F6E21.40_AT: T10666; Skb1_SP: U59684; YLB5_CE: U10402; B208.200_NC: T49572; Hsl7p_SC: U65920.

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