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Fig. 5

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ZDB-IMAGE-200331-11
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Figures for Bhargava et al., 2019
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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.

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Reprinted from Developmental Cell, 52(1), Bhargava, V., Goldstein, C.D., Russell, L., Xu, L., Ahmed, M., Li, W., Casey, A., Servage, K., Kollipara, R., Picciarelli, Z., Kittler, R., Yatsenko, A., Carmell, M., Orth, K., Amatruda, J.F., Yanowitz, J.L., Buszczak, M., GCNA Preserves Genome Integrity and Fertility Across Species, 38-52.e10, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell