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. 2017 May 12;8:15231. doi: 10.1038/ncomms15231

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Copyright © 2017, The Author(s)

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(a) CTD amino acid sequence alignment from 12 species of Drosophila. (b) Experimental design of the human CTD rescue experiment. UAS-Rpb1i is a GAL4 inducible transgene that produces an RNAi against endogenous Rpb1. UAS-Rpb1 is a GAL4 inducible transgene that encodes a FLAG-tagged, RNAi-resistant derivative of Rpb1. Fly images were created with Genotype Builder⁵⁴, (c) Results of crosses to test if ectopically expressed derivatives of Rpb1 rescue the lethality caused by ubiquitously expressing Rpb1 RNAi. Each bar on the histogram indicates the percentage of progeny with straight wings. yw: ActGAL4/CyO × yw (n=137 progeny); Rpb1i: ActGAL4/CyO × UAS-Rpb1i (n=86 progeny); Rpb1^wt[sens],Rpb1i: ActGAL4/CyO × UAS-Rpb1i,UAS-Rpb1_sen (n=102 progeny); Rpb1^wt[res],Rpb1i: ActGAL4/CyO × UAS-Rpb1i,UAS-Rpb1_res (n=91 progeny); Rpb1^hu,Rpb1i: ActGAL4/CyO × UAS-Rpb1i, UAS-Rpb1^hu (n=78 progeny). (d) Immunofluorescence of polytene chromosomes from salivary glands of third instar larvae. Larvae were derived from mating ActGAL4/CyO and UAS-Rpb1i, UAS-Rpb1 parents, so half of the larvae ectopically expressed Drosophila or humanized Rpb1 (Left and Middle panels). FLAG Rpb1 detected on chromosomes co-localizes with the Rpb3 subunit of Pol II (Right panel). Arrows highlight chromosomes from individuals not expressing FLAG Rpb1 variants (CyO/+; UAS-Rpb1i,UAS-Rpb1/+). (e) Genetic complementation assay mating scheme and results. Complementation was scored in males, which have a single copy of the X chromosome on which the endogenous Rpb1 gene resides. Female flies carrying the early embryonic lethal Rpb1 mutant allele, G0040 are crossed to male flies expressing Rpb1^wt or Rpb1^hu under the control of da-GAL4, or to yw control flies. Crosses were carried out in triplicate with 8 males and 8 female parents per cross and flies were grown at 24C and 70% humidity. Shown in e are the total counts of male progeny; counts from individual vials are shown in Supplementary Table 1. ‘B' males indicate that the ectopically expressed Rpb1 complements of the lethal allele. We observe no ‘B' flies from the yw control crosses.