The c(3)G (CG17604) locus. (A) The genomic structure of c(3)G (CG17604), based on the GM04379 and LD07655 cDNAs (Rubin et al. 2000); surrounding genes are shown as boxes representing exons. Genomic DNA coordinates in base pairs are from the Gadfly Drosophila genome annotation database (FlyBase 1999). The c(3)G (CG17604) open reading frame is shown in gray. c(3)G mutations are represented below the intron/exon structure of CG17604. CG18505 is encoded on the opposite strand as c(3)G, largely within the c(3)G 5′ untranslated region. (B) Rescue constructs P{X203} and P{X204} include genomic DNA from between the XbaI and XhoI sites indicated in A. P{X204} bears a deletion between the HindIII sites (shown in A) within the CG17604 gene.

C(3)G protein localizes between paired homologous chromosomes. (A) Deconvolved optical section through a pro-oocyte nucleus showing C(3)G localization (red). (B) DAPI image showing DNA (cyan) of the nucleus shown in A. Much of the chromatin is visible as paired linear tracks of staining (arrows). (C) Merged image of A and B, showing the association of the thread-like localization of C(3)G (red) to the paired linear tracks of DNA (cyan). In parts of this section, C(3)G can be observed between the tracks of DNA (arrows). Twisting of the chromosomes and/or position of the chromosomes within the section prevents the visualization of this arrangement of C(3)G and chromatin throughout the nucleus. A–C, bar, 5 μm. (D) Deconvolved optical section showing fluorescence in situ hybridization (FISH) of an X-chromosome probe (red) to an immunostained germarium. Pairs of FISH signals, representing paired homologous chromosomes, are visible in two cells (arrows). (E) Anti-C(3)G immunofluorescence (cyan) superimposed with the FISH signals (red) from D. The paired FISH signals are arranged on either side of the thread-like C(3)G immunofluorescence signal (arrows). D–E, bar, 10 μm.

Comparison of C(3)G and Orb localization in wild-type and egl germaria. (A) Maximum intensity projection of a deconvolved image stack containing an entire w¹¹¹⁸ germarium. Anti-C(3)G immunofluorescence (red) is visible in a subset of nuclei (visualized by DAPI staining, blue). (B) Comparison of C(3)G (red) and Orb localization (green) in the germarium shown in A. C(3)G is expressed in germ-line cells beginning at the time when Orb is first detected in the cytoplasm of region 2a cysts. In early cysts, C(3)G is detected in ∼4 cells per cyst. By region 2b, only two cells retain C(3)G, one of which is the oocyte, as determined by the accumulation of Orb protein. C(3)G is then lost from the other pro-oocyte by the time cysts leave the germarium. (C) Germarium from A, showing C(3)G (red), Orb (green), and DNA (blue). Regions 1, 2a, 2b, and 3 of the germarium are indicated at left. (D) Maximum intensity projection of a deconvolved image stack containing an entire germarium from a female homozygous for egl¹. Anti-C(3)G immunofluorescence (red) is visible in numerous nuclei (visualized by DAPI staining, blue) in region 2a and early region 2b, in contrast to wild type. (E) Comparison of C(3)G (red) and Orb localization (green) in the germarium shown in D. Orb protein is expressed and is present in the cytoplasm of germ-line cells starting in region 2a, but Orb fails to accumulate in the oocyte cytoplasm, because no oocyte is determined in egl mutant ovaries. C(3)G is present in the nuclei of all 16 cells of the germ-line cyst starting in region 2a, at the same time that Orb becomes detectable. By late region 2b, all 16 cells lose anti-C(3)G staining. (F) Germarium from D, showing C(3)G (red), Orb (green), and DNA (blue).

Localization of C(3)G in recombination-defective mutants. (A–C) Maximum intensity projections of anti-C(3)G immunofluorescence in three pro-oocyte nuclei from w¹¹¹⁸ females. (D–F) Maximum intensity projections of anti-C(3)G immunofluorescence in three pro-oocyte nuclei from P{X204}/+; c(3)G⁶⁸/c(3)G⁶⁸ females. The mutant form of C(3)G encoded by the P{X204} construct localizes to chromosomes (data not shown), but forms an increased number of stained segments suggestive of incomplete synapsis. (G–I) Maximum intensity projections of anti-C(3)G immunofluorescence in three pro-oocyte nuclei from mei-P26¹ homozygous females. Pro-oocytes in mei-P26¹ germaria were identified by cytoplasmic accumulation of Orb protein (data not shown). C(3)G forms spots and short segments of linear localization, indicating an extensive lack of normal SC.

C(3)G protein colocalizes in a thread-like pattern associated with nuclear DNA. (A) Anti-C(3)G immunofluorescence (red) in an early germ-line cyst (germarium region 2a), showing a thread-like pattern visible in four nuclei. The lower two adjacent nuclei are more intensely stained and are probably the two pro-oocytes. (B) Nuclear DNA in the cyst shown in A visualized by DAPI staining (cyan). (C) Merged image of A and B, showing colocalization of C(3)G (red) with the DNA (cyan). (D) C(3)G–eGFP (pseudocolored red) localizes in a thread-like pattern similar to the pattern visualized by anti-C(3)G immunofluorescence. Two brightly fluorescing nuclei are visible in the center of the frame, and two dimly fluorescing nuclei are positioned at the bottom and upper right edges of the frame. (E) Nuclear DNA in the cyst shown in D visualized by DAPI staining (cyan). (F) Merged image of D and E, showing colocalization of C(3)G–eGFP (red) with the DNA (cyan). Bars, 10 μm.

Computer model of C(3)G localization. (A) Maximum intensity projection of all anti-C(3)G immunofluorescence signals from one nucleus (lower left nucleus from Fig. 2A). (B) Wire frame model of the anti-C(3)G immunofluorescence signals shown in A. The contiguous thread-like immunofluorescence signals were modeled in three dimensions using the deconvolved image stack used to create Figure 5A. Each contiguous segment of the model is shown in a different color. Bar, 5 μm.

Localization of C(3)G in vitellarial cysts from w¹¹¹⁸ females. (A) Deconvolved optical section showing C(3)G (red) in a stage 3 oocyte nucleus. The Orb protein (green) is accumulated in the oocyte cytoplasm. C(3)G remains localized in thread-like patterns associated with DNA (blue) at this stage. (B) Deconvolved optical section showing C(3)G (red) in a stage 4 oocyte nucleus. Orb protein and DNA are stained as in A. Thread-like C(3)G colocalization with DNA is visible, but is beginning to dissipate. (C) Deconvolved optical section showing C(3)G (red) in a stage 6 oocyte nucleus. Orb protein and DNA are stained as in A. C(3)G localization to chromatin is decreased, and hazy extrachromosomal anti-C(3)G immunofluorescence is starting to become visible in the nucleus. In A–C, the posterior of each egg chamber is to the right. Bar, 10 μm.