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When the temperature is above 250 °C, thermogravimetric analysis (TGA) shows a weight loss of 30.8 wt%, similar to the theoretical loss of 31.5 wt% according to the formula when the ligand is removed completely (Supplementary Figure 43).
Multiple copies of tub-AOX also rescued this lethality, though less completely (Supplementary Material, Fig. S4C).
In total, 51 (Exon, 26; Intron, 8; Intergenic, 17) of 54 short indels were validated completely (supplementary table S9, Supplementary Material online).
Oocytes failed to enter the spermatheca (8/11), were fragmented upon entry because the valve closed prematurely (2/11), or fell back into the gonad arm (1/11) because the valve failed to close completely (supplementary material Fig. S2A C).
Different crosses were implemented, so as to study females heterozygous for the driver, plus males hemizygous for the driver, or control males lacking the driver completely (Supplementary Material, Fig. S4E, Supplementary Material, Table S1), as well as any rescue produced by UAS-AOX (or UAS-Ndi1).
From these data, it is evident that this locus has not evolved strictly in association with MLST lineages and that, for example, in ST-45 several different alleles are found and a large portion of the isolates (39%) lack this locus completely (supplementary table S3, Supplementary Material online).
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Strains AltDE1 two cassettes and UM7 single cassette are completely different (supplementary table S2, Supplementary Material online), illustrating the highly dynamic nature of this genomic region.
According to our data, the tyrosine residue just upstream of the CxGG motif in these organisms is also completely conserved (supplementary fig. S2, Supplementary Material online).
After the treatment of resultant RNA samples with TURBO DNase free (Ambion) for 1 h at 37 °C to degrade possibly remaining DNA fractions completely (see supplementary fig. 2, Supplementary Material online), reverse transcription reaction was carried out using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the random hexamers primer, following the manufacturer's protocol.
Upon trap expression, the PC-3 derived xenografts significantly decreased in size, some completely regressed (Supplementary Fig S5A) with only small foci remaining (Supplementary Fig S5B).
We next turned to Opa1flx/flx MAFs where 48 h after transfection with CRE recombinase OPA1 was almost completely absent (Supplementary Fig. 2).
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