Figure 1.
One of the parental X chromosomes (y wam v2 f 66) has functional distorter(s), whereas the other (w) does not. Single-male matings were set up for all X chromosome recombinants in G3. All recombinants, represented by X in G4 and G5, were tested for sex-ratio in an isogenic background nt; nmy in G5 (I) as well as in a control background of nt; +/nmy (II). Two classes of recombinants (F and H) with the same visible phenotype were distinguished by progeny from additional crosses with y wam v2 f 66 females in G5. However, class B (+ w ? +) may be a mixture of + w + + and + w v2 +, which were not distinguished.
Figure 2.
Sex Ratios (k) Scored for the Preliminary Mapping
Each recombinant X chromosome in classes A–H (Figure 1) was tested against nmy (filled circle) and its sex ratio was rank-ordered within each class. Also shown are the corresponding sex ratios tested against nmy/+ (open circles) as control and the number of recombinants tested for each class (in parenthesis). Three exceptional recombinants are indicated with arrows.
Figure 3.
Fine Mapping and Positional Cloning of dox
(A) The crossover intervals and the sex-ratio phenotype of the definitive 43 recombinants between lz and v. dox: normal sex ratio; Dox: sex ratio distorted. The number of similar recombinants is included in parenthesis. The seven most informative recombinant events fall within the interval 5dox_III and C14/17 (green region). Some of the annotated genes in D. melanogaster in this region are also shown (yellow region) (http://www.flybase.org/cgi-bin/gbrowse/dmel/). The two regions marked as 18.7k and 31k in the D. simulans simB strain have been sequenced from PCR products and phage clones. * represents Dox at left and E(Dox) at right.
(B) Annotations of the 18.7k and 31k regions in D. simulans. Genes orthologous to D. melanogster are shown (arrow: orientation of transcripts; line: introns; open box: untranslated region of mRNA; filled box: coding sequence). A transposition of 3,833 bp (double lines, Tp3833) in the Dox region can be recognized as originating from the downstream region of CG32702 (Tp3549). The positions of the 105-bp deletion (Δ105) and the exons of CG32702 within Tp3833 (CG32702d in gray) are shown. A repetitive sequence with a 360-bp consensus has also been recognized in both regions. Primers DoxF4 and DoxR4 were used to amplify this region for cross-species comparisons. In the Tp3549 region, a sequence of 1,458 bp (red line) has no homolog in D. melanogaster CG32702.
(C) Comparison of the Dox region among several species of the D. melanogaster subgroup. Sequences from eight strains of D. simulans, and one strain each from D. sechellia (3588), D. mauritiana (w12), and D. melanogaster (y; cn bw sp) were compared. Tp3833 is present in seven strains of D. simulans (represented by simB) but is absent in SR6. It is also absent in all other species examined.
Figure 4.
Molecular Structure of Dox and MDox
Both Dox and MDox have transcripts in the opposite direction of CG32702d or CG32702, respectively. The genomic sequences of Tp3833 (Dox) and Tp3549 (MDox) are largely homologous except for one indel of 135 bp (*135) and another short sequence (*61 in MDox versus *163 in Dox). The 360-bp repetitive sequence may have been involved in the transposition event of Tp3833. Five homologous segments of sequences are recognized. The nucleotide divergence is shown for each of them (#1–#5) expressed as nucleotide difference over segment length. Segment #1 corresponds to the last exons of CG32702 and it has only one nucleotide substitution (A versus C in Ex 13) between these two paralogs. R26 and F26 are the two PCR primers used to amplify genomic DNA and cDNA in this region (see Figure S4). The transcripts of Dox and MDox were determined by RT-PCR, 5′- and 3′-RACE with primers that can distinguish between these two genes. The intron–exon boundaries of these two genes are largely the same between Dox and Mdox, so they are annotated together. However, the 91-bp intron I of Dox has never been found from MDox. Several alternative splicing forms including earlier termination have been found. In some RT-PCR products, introns III and IV of MDox are not spliced out. Other splice forms may yet be uncovered. The 105-bp deletion (dashed line) in dox causes the loss of exon III in the otherwise identical transcripts as compared to Dox. Identical tandem repeats of a 42-bp element (green and red rectangles) are present in both Dox and MDox transcripts. It is unclear which ORF, if any, is actually translated in each transcript. All potential ORFs larger than 100 bp are shown for all three reading frames (thick black lines on thin red lines). Two ORFs of 157 and 107 aa across the exon III region in the Dox and MDox transcripts, respectively, are marked with brackets and are the strongest candidate for translation (see Text S2). The best ORF predicted by GENSCAN are marked with an asterisk (http://genes.mit.edu/GENSCAN.html).
Figure 5.
Homology Among Dox, Mdox, and Nmy
(A) Schematic of the Winters sex-ratio system. An autosomal suppressor, Nmy, is apparently a new gene created by a 2,041-bp insertion (red) in the gene CG14370. The insert contains a pair of almost perfect IRs of 345 bp (red arrows, IR' and IR'') and they are required for the suppression of Dox. Nmy originated through a retrotransposition event from Dox, because all of the 2,041 bp consists of paralogous sequence from cDNA of Dox (red line) and the 5′ region upstream of Dox transcription (dashed red line) [24].
(B) A detailed comparison between transcripts of Dox and Nmy. Paralogous sequences are in red. The 5′ and 3′ ends of Nmy (black line) are from CG14370. IR' might have duplicated from IR” after the retroposition [24], while some sequences (black between the two small arrow heads) in Dox no longer have paralogs in Nmy. The 42-bp elements are shown in red and green color, as in Figure 4. The dox allele has lost one of the 42-bp elements in the transcript. A dsRNA stem is presumably formed between the two IR's. The critical region marked as “C” is detailed in (C).
(C) The critical region “C” with a base-by-base comparison among Dox, Nmy, and MDox. Identical bases or amino acids are represented by a period, and deletion by a dash, and divergent bases are in red. This region starts at position 844 (778) of the Dox (MDox) transcript and the beginning of IR', respectively. IR' and IR'' are identical except for a 6-bp (TAGGGA) deletion in IR' (cyan). The two tandem 42-bp elements are also shown in red and green, respectively on the Dox sequence. The amino acids encoded by the ORF in this region are shown in the top (Dox) and bottom lines (MDox), respectively. These two ORFs have an amino acid identity of 94/107 (88%) and similarity of 100/107 (93%) (underlined).
Figure 6.
Spermatogenesis of Double-Mutant dox; nmy Is Normal
(A–C) TEM images of sperm maturation.
(A) Early post elongation stage of a spermatid head (upper) and tail (lower). Normal nuclear condensation can be seen apposed to rows of microtubules (white arrowheads), while nucleoplasm is eliminated (v).
(B) Late post elongation stage of spermatid heads with homogeneously condensed nuclei (upper left and right). Also note the normal head-tail alignment (middle) and the tail (lower right).
(C) Post individualization stage of a sperm bundle with 61 normal tails and one degenerated tail in the waste bag (WB).
(D) DAPI images of 64 spermatid heads in a bundle at the stage of post elongation.
Abbreviations used in annotation: AX (axoneme); BB (basal body); mM (minor mitochondrial derivative); MM (major mitochondrial derivative); mS (minor strip); MS (major strip); MT (microtubule); WB (waste bag). Scale Bars: 500 nm (A–C); 20 μm (D).
Figure 7.
The Y Chromosome from D. sechellia Is Sensitive to Dox but Distorts SR6, Resulting in a Male-Biased Sex Ratio
(A) Fingerprinting the Y chromosomes from D. melanogaster sibling species. Genomic DNA was cut with Hind III and probed sequentially with the probes Y5g and RpL32. The probe Y5g is composed of sequences from five known Y-specific genes (Figure S6). RpL32 is an autosomal gene at 99D3. Stocks used: D. mel: D. melanogaster Canton-S; D. mau: D. mauritiana w12; D. sim: D. simulans simB; D. sech: D. sechellia 3588; D. sim Y[sech]: isogenic to simB (w; nt; Nmy) except that the Y is from D. sechellia 3588, as confirmed here.
(B) Test of the sensitivity of Y[sech] to Dox. Dox/Y[sim] or Dox/Y[sech] males in the background of nmy or nmy/Nmy were constructed by the following scheme: simB or D. sim Y[sech] males were crossed to P40L12 (w; nt; P40L12 nmy/Nmy] or P40B13 (w; nt; P38B13 Nmy/Nmy) females. Ten sublines of each combination were set up by mating single F1 male (P40/Nmy) to SR1227 (w; nt; nmy) females. The following four genotypes of males were obtained in the F2: (1) Dox/Y[sim]; nmy (w Dox/Y[sim]; nt; P40L12 nmy/nmy). (2) Dox/Y[sech]; nmy (w Dox/Y[sech]; nt; P40L12 nmy/nmy). (3) Dox/Y[sim]; Nmy/nmy (w Dox/Y[sim]; nt; P40B13 Nmy/nmy). (4) Dox/Y[sech]; Nmy/nmy (w Dox/Y[sech]; nt; P40B13 Nmy/nmy). Three males of each subline were tested for sex-ratio at room temperature. Dox; nmy males express sex-ratio regardless of the origin of the Y chromosome. Y[sech] and Y[sim] are similar in their sensitivity to the sex ratio distortion effect of Dox (t-test, p = 0.341).
(C) Test the sensitivity of Y[sech] to SR6. The X chromosome SR6 carries the Paris sex-ratio distorters, while ST8 is the standard nondriving chromosome. Four genotypes of SR6/Y[sech] and its controls were constructed by crossing F1 females obtained from the crosses simB or D. sim Y[sech] x C(1)RM yw; nt; Nmy to SR6 or ST8 males: (1) SR6/Y[sech] (SR6/Y[sech], nt/+; Nmy); (2) SR6/Y[sim] (SR6/Y[sim], nt/+; Nmy); (3) ST8/Y[sech] (ST8/Y[sech], nt/+; Nmy); and (4) ST8/Y[sim] (ST8/Y[sim], nt/+; Nmy). Individual males of the above four genotypes were tested for sex-ratio by crossing to three w; e females (column ± SEM, with sample size). SR6 distorts Y[sim] as reported before [81], and Nmy does not suppress SR6. Unexpectedly, Y[sech] actually distorts SR6. F%: F3 males obtained in the above sex-ratio test matings were crossed singly again to three w; e females.