Genetic tests confirm that TTHERM_007700760/IMP9 is MPD. (A–F) Cortical organization in cells stained with the anti-centrin antibody (red) and DAPI (blue) imaged using confocal microscopy. The genotypes are as indicated. White arrows point to OAs with a 4 M organization. The cells shown in C are mpD-1 mutant homozygotes transformed with a targeting fragment that tags the variant allele of IMP9 with GFP-neo5 sequence. In D, the mpD-1 homozygotes were transformed with a targeting fragment that replaced the variant base with the WT base and added the GFP-neo5 sequences. Note fewer OAs with 4 M rows. (E and F) A knockdown of TTHERM_007700760/IMP9 partially phenocopies mpD-1/39°C. (E) Cells (grown at 30°C) that were subjected to a mock knockdown using an empty “co-deletion vector” that does not mediate production of small RNAs targeting IMP9-coding region for elimination. (F) Cells (grown at 30°C) in which a portion of the coding region of IMP9 was eliminated using the co-deletion method that produces a knockdown. (G and H′) SR-SIM images of cells expressing IMP9-GFP that where the IMP9 portion is either WT (G939) (G and G′) or has the mpD-1–linked mutation G939R (H and H′), grown at 39°C. Note that the mutant protein is depleted from M1, suggesting that the mutation reduced the stability or cortical targeting of MpD. (I) Quantification of the extra M row phenotype in various genetic backgrounds. The following genotypes were scored: WT; mpD-1, mutant homozygous (strain IA305); mpD-1+IMP9-G939R, an mpD-1 homozygous strain carrying the mpD-1–linked variant (G939R codon substitution) and GFP-neo5 integrated at the end of the coding region of IMP9; mpD-1+IMP9-G939, an mpD-1 homozygote strain in which the mutant codon (R939) was replaced by the WT codon (G939) and GFP-neo5 was integrated at the end of the coding region of IMP9; mock knockdown; cells transformed with a negative control empty pMCodel (co-deletion) vector; and IMP9 knockdown, cells transformed with a co-deletion vector pMCodel expressing an RNA that targets a portion of IMP9 gene for deletion during macronuclear development. There are 90 copies of IMP9 in the macronucleus, and likely not all copies were eliminated, resulting in a knockdown of IMP9 expression. Mean ± SD. N = 3 experiments indicated by different symbol colors (100 cells scored per experiment and genotype). The P values were obtained using an unpaired t test with Welch’s correction. (J) A gel image showing PCR products of amplification of a portion of IMP9 after digestion with MnII restriction endonuclease that distinguishes between the WT and mpD-1–linked alleles. Genomic DNAs were purified from a strain IA305 (mpD-1 homozygous) subjected to gene replacement using a fragment of IMP9 that contained either the mpD-1–linked variant codon (G939R) (lanes 2–4) or the reference codon G939 (lanes 5–10). Lane 11, WT genomic DNA and lane 12, mpD-1 mutant genomic DNA (strain IA305). MnII distinguishes between the mpD-1–linked variant and reference IMP9 allele. Note a partial (lanes 5, 6, 8, and 9) replacement of the variant base with the reference base (see Fig. 6 I). (J′) The diagram shows the strategy that used homologous DNA recombination to tag the 3′ end of the coding region of IMP9 in the mutant homozygous strain IA305, using fragments that encode either the reference (G939) or mpD-1–linked variant codon R939.