(A–F) Expression of Grk in okr, spnB, and spnD mutant ovaries. Ovarioles (A,C,E) and stage 10 egg chambers (B,D,F) with Grk in green and cortical actin detected with Phalloidin in red. In wild-type ovaries, Grk is detected in the oocyte throughout oogenesis (A), and becomes localized to the presumptive dorsal–anterior corner in stages 9 and 10 (B). In okr mutant ovaries, Grk expression is reduced or undetectable in many egg chambers, and these are interspersed among egg chambers that have apparently normal Grk expression (C,D). In spnB and spnD mutant ovaries, Grk expression is apparently normal in the early stages, but less and less protein is detectable in the oocyte as oogenesis proceeds (E). The majority of stage 10 spnB and spnD mutant egg chambers have no detectable Grk (F). (G,H) Expression of fs(1)K10 in okr, spnB, and spnD mutant ovaries. Triple stainings of egg chambers with K10 shown in red and cortical actin and DNA shown in green. In wild type (G), K10 protein is observed in the oocyte nucleus, and is particularly concentrated around the karyosome. In the mutant egg chambers (H) K10 protein is reduced or absent. (I–K) Defects in oocyte nuclear morphology in okr, spnB, and spnD. Stage 8 egg chambers stained for cortical actin (red) and DNA (green). In wild type (H), the DNA in the oocyte nucleus is condensed into a tight sphere. In ovaries mutant for okr, spnB, spnD, or a number of other genes (including aub, del, squ, vas, zuc, spnA, spnC, and spnE), the DNA is more diffuse (J) or threadlike and fragmented (K).

Molecular characterization of spnB. (A) Physical map of the 88B region on 3R. (Shaded boxes) Regions that are deleted in Df(3R)redP93 and Df(3R)redP52, (light stippling) areas of uncertainty. Molecular coordinates are numbered with respect to the published cosmid walk in the region (see Materials and Methods). The restriction map, indicating all EcoRI sites, was devised from existing maps and fine mapping of subclones of cos144. Two previously characterized genes, Supressor of Hairy wing [Su(Hw)] and the 15-kilodalton subunit of RNA polymerase II (RpII15) (Harrison et al. 1992) are shown as open boxes with arrows indicating the direction of transcription. (Hatched boxes) Unmapped transcripts defined by Northern analysis of ovarian poly(A)⁺ RNA using genomic fragments spanning the region, with sizes indicated beneath each box (in kilobases, except c550, in basepairs). Four rescue constructs are shown, pR144Xb14w+, pR144NS9.5w+, pR144E5.9w+, and pRE5.9BE3.5w+, that cover RpII15 and c3.5/c4.4, c5.5, and c500 and c1.3, and exclusively c1.3, respectively. (B) Fine map of the p144E5.9w+ construct that rescues the spnB mutant phenotype. Restriction sites: (E) EcoRI; (H) HindIII; (B) BamHI; (S) SalI; and (St) StuI. The smallest genomic rescue construct, pRE5.9BE3.5w+ extends from the BamHI site in c550 to the EcoRI site at the right end of the map. Gene structures were determined from a comparison of cDNA and corresponding genomic sequences. (Dark shading) Coding region of each transcript. c5.5, c1.0/c1.2 are ovarian transcripts defined by Northern analysis using small subclones of the complete 5.9-kb fragment. The unidentified ORF was deduced from the genomic sequence. (C) Protein structure of SpnB as compared with the related protein Dmc1. (Light shading) Region of homology between the two proteins, (dark shading) A- and B-type nucleotide binding consensus sequences (Walker et al. 1982). Black bars in the SpnB diagram indicate the positions of the lesions associated with spnB alleles (cf. Table 2). An alignment of the A-type nucleotide binding domains of SpnB, Dmc1, yeast Rad57, Drosophila Rad51, yeast Rad51, and human Rad51, is shown, indicating the location of the spnB¹⁵³, spnB^BU, spnB^CN, and spnB⁰⁵⁶ mutations. The P-loop motif corresponds to the GXXXXGKT/S at the right end of the alignment.

Dorsal–ventral patterning defects in okr, spnB, and spnD. (A–D) Ventralized eggshells representing the four phenotypic classes described in the text. Anterior is up. (A) Class 1 (B) class v2 (C) class v3 (D) class v4. (E–G) Ventralization of the embryos in okr, spnB, and spnD: Cellular blastoderm embryos stained for the mesodermal marker Twist, anterior is to the left, dorsal is up, except for the embryo in G, which is shown from a ventral view. Mutant embryos show ventralization of the embryonic dorsal–ventral axis as evidenced by the expansion of the mesoderm. (E) Mutant embryo with a fairly normal mesodermal domain that is only slightly expanded at the posterior. This differential ventralization along the anterior–posterior axis is characteristic of the phenotype. (F) A more severe example than that in E with a posterior expansion of the mesoderm extending to almost 50% egg length (in some cases the entire embryo expresses Twist). (G) An example of an embryo in which the posterior expansion has resulted in a split mesodermal domain that runs up the lateral sides of the embryo and meets at the dorsal midline. Splitting of the mesodermal domain has been observed in strong combinations of grk and Egfr (Roth and Schüpbach 1994), but the process generating the duplication is not understood. (H) Anterior–posterior patterning defects in okr: Chorion of okr^AB/Df(2L)JS17. In up to 42% of the eggs laid by okr^AB/Df(2L)JS17 females, a second micropyle is observed at the posterior pole of the egg (micropyles are indicated by arrows) indicating that the follicle cells have adopted an anterior fate.

Molecular characterization of okr. (A) Physical map of the 23C region of 2L. (Shaded boxes) Regions deleted in Df(2L)C144, Df(2L)JS17, and Df(2L)JS7. Df(2L)JS17 extends off the map both proximally and distally. (Stippled boxes) Regions of uncertainty. Simplified restriction map showing only XbaI (Xb) sites, was derived from the maps of genomic phage that are indicated as bars below the map. A more detailed restriction map of λ7-4 is shown with all HindIII (H), EcoRI (E), SalI (S), and XbaI sites indicated. Three previously characterized genes, Rbp9, Rrp1, and γTub23C are shown as open boxes with arrows indicating the direction of transcription. (Hatched boxes) Unmapped ovarian transcripts defined by Northern analysis of poly(A)⁺ RNA using corresponding genomic fragments. Approximate sizes are show below each box. The rescue construct, pRRa54E4.7w+, which rescues the okr mutant phenotype, is shown beneath the 2.7-kb transcription unit which it includes. (B) Restriction map of the 4.7-kb EcoRI fragment included in the rescue construct shown in A. The structure of the okr gene was derived from the existing sequences for DmRad54 (Kooistra et al. 1997), and our genomic and cDNA sequences (see Materials and Methods). The 4.3-kb cDNA was identified by Northern analysis of ovarian poly(A)⁺ RNA with a fragment extending from the left EcoRI site to just before the transcriptional start of okr. The 1.6-kb transcript that overlaps with okr was identified by Northern analysis by use of either the entire rescue fragment or the okr cDNA, and the overlap was verified by sequencing the 3′ ends of the EST corresponding to this gene. (C) Protein structure of Okr. (Light shading) Six domains of homology shared with other members of the Snf2 family of DNA helicases (Bork and Koonin 1993). (Black bars) Positions of mutation in the alleles indicated (Table 2).

MMS sensitivity of okr, spnB, and spnD mutants. Graph of fraction of percent expected (+MMS) to percent expected (control) for okr, spnB, and spnD mutants.