—Localization of each gene probe by in situ hybridization to polytene chromosomes. (a) exu1 probe hybridized to XL of D. miranda. (b) exu2 probe hybridized to XL of D. miranda. In some cells, cross hybridization with exu1 is detectable, as indicated by a red arrow. The strain of D. miranda shown is MA32. (c) exu1 probe hybridized to D. pseudoobscura chromosome 3. (d) exu2 probe hybridized to D. pseudoobscura XL. (e) exu1 probe hybridized to D. affinis chromosome 3. (f) exu1 probe hybridized to D. subobscura chromosome E.
—(A) Genomic Southern blotting results for determining the copy numbers of exu-related loci in D. pseudoobscura and D. miranda. Hybridization was done overnight at 55°. The exu2 locus of D. miranda was amplified by PCR and then gel extracted. To better detect the homologous copies in the genome, a 400-bp fragment of a region that is highly conserved between exu1 and exu2 was generated by restriction digestion using HaeIII and then labeled as described in materials and methods. Lanes 1-3 correspond to the digests of D. pseudoobscura genomic DNA. The enzymes used are BamHI (lane1), BamHI and SalI (lane 2), and SalI and XbaI (lane 3). Lanes 4-6, digests of D. miranda female genomic DNA using the same set of restriction enzymes as in the previous lanes, in the same order; lanes 7-9, digests of D. miranda male genomic DNA prepared in the same way. BamHI digests in females (lane 4) show the presence of two bands with strong homology to the probe. The bands with stronger intensity in males are indicated by arrows (compare lanes 6 and 9; the band with the highest molecular weight is stronger in the male lane, while the other bands are relatively weaker in the male lane). This difference also exists between lanes 4 and 7 and between lanes 5 and 8, which are blurred in the male lanes due to the close proximity of the high molecular weight bands. This pattern appeared consistently using other enzymes with overnight restriction digestions, suggesting that it is not an artifact of partial digestion. (B) Differences between female and male genomic DNA Southern blots of D. miranda shown against a 1-kb ladder. The probe used for these blots is the exu1 locus from D. pseudoobscura amplified by PCR and prepared in the same way as above. Hybridization was done overnight at 60°. The restriction enzymes used are AvaI (lanes 1 and 4), BamHI and HindIII (lanes 2 and 5), and EcoRI (lanes 3 and 6). Multiple bands appear due to internal digestion of target material. Lanes 1-3, D. miranda female DNA; lanes 4-6, male genomic DNA of D. miranda. In lanes 1 and 4, the same high molecular weight band is stronger in males, while all the other bands are generally weaker in male digests, as was seen in A. This suggests that the high molecular weight band (indicated by arrows) corresponds to the neo-sex-linked alleles, with the neo-Y allele possessing higher homology to the probe. In the remaining lanes, male-specific bands with relatively high intensity were detected (arrows in lane 5 and 6). These bands must correspond to the neo-Y-linked exu-like locus.
—Polymorphic sites from exu1 and exu2 loci in D. miranda. The exon/intron structures of the two loci are very similar, as is shown for exu1. Sequenced regions are shown as starting from number 1, ending at nucleotides 1423 and 1021, respectively. The endpoints of each exon/intron block are numbered. For exu2, the structure of the gene is shown only for the sequenced regions. The orientation of exu2 relative to exu1 is not known. Each site has a letter R, S, or I, standing for replacement, synonymous, or intron substitution, respectively, and a number corresponding to the position within the sequenced region.
m, length of sequence surveyed (in base pairs); n, number of alleles sampled; S, number of segregating sites; θ and π, Watterson’s (1975) estimator of 4Nμ and the average nucleotide pairwise difference (Nei 1987), respectively. The subscripts t, r, and s mean total sites, replacement sites, and silent sites (synonymous + noncoding sites), respectively.