Fig 1: Intragenic complementation analysis within a multigene family.(A) Cartoon of chromosomal loci used in (B) and in subsequent experiments. For complete genotypes, see figs. S1, S2, and S4. The H3.3B gene (chr. X) is either WT, K36R, or ? (null). The H3.3A gene (chr. 2L) is either WT or ? (null). The endogenous replication-dependent histone gene cluster HisC (chr. 2L) is either intact (WT) or ? (null). The transgenic insertion site VK33 (chr. 3L, band 65B2) was used for histone gene replacement analysis. 12xH3.2 transgenes contain 12 copies of the histone repeat unit, each of which contains all five replication-dependent histone genes. Transgenes used in this study carry the following alleles of H3.2: HWT, K9R, K27R, or K36R. Panel was created using BioRender.com. (B) Developmental viability assay for complementation analysis of 12xH3.2 transgenes. All genotypes are HisC? and carry two 12x histone transgenes in trans (24x total). Pairs of transgenes are represented on the x axis for each set of bars. For each genotype, % pupation and % eclosion of four to six biological replicates (50 larvae/replicate vial) were calculated, and means and SD of these percentages were plotted. Statistical significance for % pupation was calculated with GraphPad Prism software using a mixed-effects analysis (can accommodate missing values) on the four genotype pairs indicated by brackets, followed by Šidák’s multiple comparisons test. **P < 0.01; ***P < 0.001. ns, not significant.