mpH-1 is a gained splice site mutation in TTHERM_00564170. (A) The diagram shows the locations of predicted introns and exons in TTHERM_00564170. Below is the sequence of intron 8 and the location of the mpH-1 G/A substitution, 2-bp upstream of the predicted 3′ splicing junction. (B) Sequences of fragments of RT-PCR products amplified using TTHERM_00564170-specific primers positioned outside of exon 8. Arrows mark the positions at which exon 8 sequence transitions into exon 9 sequence following splicing of intron 8. Note the presence of two extra bases in the mutant sequence. (C) Sequencing of a portion of TTHERM_00564170 gene in the WT (top), mpH-1 homozygote (IA486 strain, middle), and a rescued clone after transformation with the targeting fragment. (D and E) SR-SIM images of cells from transformants clones with GFP-mNeon-neo5 sequence integrated at the end of the coding region that have the either variant G base and the mutant phenotype (D) or the WT A base at the variant site and the WT (rescued) phenotype (E). Note a strong signal of MPH-GFP-mNeonGreen in the phenotypically rescued transformant.