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. 2020 Nov 9;9:e52416. doi: 10.7554/eLife.52416

Figure 1. Single-copy rDNA transgene allows study of rDNA expression.

Figure 1.

(A) Scheme of endogenous rDNA units and the rDNA transgene. Native rDNA units are composed of non-transcribed spacer (NTS) and a transcribed portion that produces the 47S pre-rRNA. The pre-rRNA contains a 5' external transcribed sequence (ETS) and internal transcribed spacers (ITSs), which are removed from the pre-rRNA to generate mature 18S, 5.8S, 2S and 28S rRNAs. Positions of R1 and R2 transposon integration sites in the 28S rRNA are indicated. The 9 Kb transgene contains one complete rDNA unit together with a 28 bp of the 28S rDNA from the upstream unit. The transgene is marked by insertion of a 21 bp unique identification sequence (UID, red bar) into the ETS and inserted using ΦC31-mediated recombination into a common att site on the second chromosome (chr 2L: 1,582,820). (B) rDNA transgene expression is detected by RT-PCR in fly ovaries. RT-PCR amplicon of the UID ETS region is only detected in transgenic but not in wild-type flies or in the absence of reverse transcriptase (-RT). rDNA transgene expression in ovaries was measured by RT-qPCR and normalized to rp49 mRNA. Error bars indicate standard deviation of three biological replicas. Statistical significance is estimated by two-tailed Student’s t-test; ***p<0.001. (C) rDNA transgene expression is detected by HCR-FISH in fly ovaries. Nascent transcripts of the pre-rRNA transgene (arrowhead) were detected in nurse cell nuclei using a probe against the UID sequence (red). Control wild-type flies lack the UID sequence. Scale bar: 5 µm.