Fig. 5

Functional Analysis of GCNA Domain Structure (A and B) Expression of a UAS-GcnaWT transgene tagged at the C terminus driven by nanos (nos)-gal4 shows prominent cytoplasmic staining. Germaria co-labeled for the Gcna-GFP transgene and (A) the nuage component, Aubergine (red) or (B) DAPI (red) to visualize DNA. Yellow arrow points to a mitotic germ cell. Scale bars represent 20 μm. (C) Structure of the Drosophila GcnaWT and catalytic dead, GcnaHE>AA, mutant transgenes. (D) Comparison the GcnaWT and GcnaHE>AA proteins tagged at their N termini. Note the abundant GcnaHE>AA punctae in the germarium and germ cells. DNA (red), Gcna (anti-HA, green). Scale bars represent 20 μm. (E–G) The catalytic dead transgene confers a separation-of-function phenotype and (E and F) cannot suppress the accumulation of γH2Av foci in (E) meiotic and (F) non-meiotic nuclei from control, GcnaKO, GcnaKO; nos>GcnaWT, and GcnaKO; nos>GcnaHE>AA ovaries, but (G) can partially rescue the maternal-effect lethality conferred by loss of Gcna. Plotted are the percentages of eggs derived from females of the indicated genotypes that hatch into larvae. (E) Control (n = 16), GcnaKO (n = 18), GcnaKO; nos > GcnaWT(n = 18), GcnaKO; nos > GcnaHE>AA (n = 15). (F) n = 8 for all four genotypes. (G) Control (n = 1035), GcnaKO (n = 749), GcnaKO; nos > GcnaWT(n = 1059), GcnaKO; nos > GcnaHE>AA (n = 615). (E–G) Graphs depict mean values with standard deviation. (H) Expression of C. elegans OLLAS::GCNA-1. Germ cells are labeled for DNA (blue), GCNA-1 (anti-OLLAS, yellow), and the nuclear pore (antibody mAb414, magenta). Scale bar represents 5 μm. (I) Structure of the C. elegans “IDR-only”, C-terminal truncation allele, gcna-1(ea76). (J) Comparison of brood sizes from N2 control, and homozygous, F3 and F8 gcna-1(ea43) null, and gcna-1(ea76) truncation mutants. The IDR-only mutation does not exhibit the rapid transgenerational sterility seen in the null.