Structure of GLS variants.

<p>The wild-type GLS is shown at the left for comparison. The GLS mutant (referred to as <i>grkGLS^mut</i> in Text) contains 12 point mutations (shown in red), which are predicted to disrupt the predicted base pairing pattern of the GLS at five sites (circled). None of the 12 mutations affect the protein coding sequence as shown at the bottom portion of the figure.</p

Conservation and predicted secondary structure of the GLS.

<p>(A) Sequence alignment of the <i>gurken</i> transcription unit displayed using the Vista Browser at <a href="http://pipeline.lbl.gov/cgi-bin/gateway2" target="_blank">http://pipeline.lbl.gov/cgi-bin/gateway2</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015448#pone.0015448-Nielsen1" target="_blank">[49]</a>. The estimated years in millions (MYA) of evolution between <i>D</i>. <i>melanogaster</i> and each of the other five species is from Heger and Ponting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015448#pone.0015448-Heger1" target="_blank">[24]</a>. The most highly conserved region is circled and includes the first 39 nt of the GLS. The last 25 nt of the GLS map to the 3′ side of the abutting intron. The arrow indicates the direction of transcription. The red shaded region corresponds to a putative transposable element. The numbers at the bottom of the graph indicate nucleotide position along the chromosome. (B) The 5′ end of the <i>gurken</i> mRNA, where the green dot denotes the translation start site, the red arrows the boundaries of the GLS, and the asterisk the position of the intron. The nucleotides beneath the aligned sequence blocks highlight differences between the <i>D. Willistoni</i> and <i>D. melanogaster</i> sequences. (C) Predicted secondary structure of the GLS, with non-conserved residues shown in red.</p

The GLS is required for <i>gurken</i> RNA localization and gene function.

<p>(A–B) Wild-type expression patterns of endogenous <i>gurken</i> RNA (A) and protein (B) as revealed by whole mount <i>in situ</i> hybridization and immunofluorescence, respectively. Anterodorsal localization of transcripts and protein is only apparent in the rightmost egg chambers, which are stage 8 and 9, respectively. (C–E) The <i>gurken</i> RNA and protein distribution patterns of <i>gurken</i> null mutants (<i>grk^ΔFRT</i>) carrying the wild-type <i>gurken</i> transgene, <i>grk^wt</i> (C–D) or no transgene (E). (F–H) <i>grk^ΔFRT</i> eggs and egg chambers (from <i>gurken</i> null mothers) carrying the <i>grkGLS^mut</i> transgene. (F) Left panel: representative <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> egg exhibiting a completely ventralized phenotype, i.e., complete loss of dorsal appendage material. Right panel; anterior end of a <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> egg exhibiting a strong, but not complete, ventralized phenotype. Note, for example the short, fused dorsal appendage. (G) <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> ovariole following <i>in situ</i> hybridization with <i>gurken</i> probe. Transcripts are dispersed throughout the germ-line cysts with only slight enrichment in the oocyte and no subcellular localization. (H) <i>grk^ΔFRT</i>; <i>grkGLS^mut</i> ovariole following immunofluorescence using an anti-Grk antibody. The protein is generally dispersed throughout the germ-line cysts, although slight enrichment around the oocyte nucleus is seen in rare stage 10 and 11 egg chambers.</p