Supplemental Figure S1
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Fig. S1. Transcripts for dally are not provided maternally, but are present in 4-6 hour embryos. (A) RT-PCR analysis for dally transcripts using three independent primer pairs generates no product from an RNA template isolated from unfertilized wild-type eggs (lanes 1-3). By contrast, the same template generates a robust RT-PCR product of the expected size (410 nucleotides) using sotv-specific primers (lane 4), indicating the RNA is intact. The dally-specific primer pairs generate RT-PCR products of the predicted sizes using RNA isolated from 4-6 hour embryos (lanes 5-7), as does the sotv primer pair control (lane 8). (B) The embryonic RT-PCR products are specific and do not result from the amplification of contaminating DNA. The schematic represents the dally transcript (roughly to scale), with triangles indicating the location and sizes of the excised introns (FlyBase, http://flybase.bio.indiana.edu/). Primer pairs are represented with colored arrowheads, and are connected by lines that indicate the predicted lengths of the RT-PCR products and locations of template sequences along the transcript. Dally primer-pair F4-B4, yields a 411-bp product from an embryonic RNA template. A PCR product resulting from contaminating DNA would span 3 introns and total over 57 kb. Likewise, Dally primer-pair F1-R1 generates an RT-PCR product of the expected size of 321 bp. The corresponding product from a DNA template would include over 9 kb of additional intronic sequences. Finally, primer pair F2-R2 would generate a PCR product from contaminating DNA that would span 3 introns and yield a 2 kb product instead of the predicted size of 437 bp. In each case, only sizes predicted for products generated from dally RNA templates were observed.
