Figure 2.

Correlation between Brat recruitment in yeast and hb regulation in embryos. (A) Activity of Pum mutants in yeast. Genes encoding either the wild-type (wt) Pum RNA-binding domain (RBD) or various mutant derivatives (as indicated) or the empty vector were introduced into yeast to monitor recruitment of Nos into ternary complexes and Brat into quaternary complexes. Transformants were streaked on medium lacking histidine and containing the concentration of the His3 competitor 3-aminotriazole (3AT) indicated on the right. (B) Activity of Pum mutants in embryos. Transgenes encoding each of the indicated RBD derivatives were introduced into flies to monitor complementation of the pum⁻ embryonic phenotype. The Western blot of embryonic extracts prepared from representative transgenic lines shows that each Pum RBD fragment accumulates to essentially the same level in vivo (endogenous full-length Pum serving as a loading control). Below each lane of the blot is shown the typical number of abdominal segments in embryos from pum⁻ females that also bear a single copy of the indicated pum–RBD transgene. Below this list are shown four representative dark-field photographs of embryonic cuticle in which the extent of abdominal segmentation can be assessed. In the absence of pum function (none), hb mRNA is translated in the posterior of the embryo and no abdominal segments form. In this mutant background, expression of the wild-type RBD (wt) results in repression of hb translation sufficient to allow formation of six abdominal segments, on average (Wharton et al. 1998). In this assay, the T1366D derivative is completely inactive, whereas activity of the C1365R derivative typically yields embryos with a single (arrow) abdominal segment. (C) Activity of the Nos^M379K mutant in yeast and embryos. Above are shown the results of yeast interaction experiments (as in A) that monitor the ability of Nos and Nos^M379K to enter a ternary complex with Pum and the NRE (Sonoda and Wharton 1999), or to recruit Brat into a quaternary complex, as described in Fig. 1. Transgenes encoding either wild-type or M379K mutant protein were then introduced into flies, and the distribution of encoded protein was examined by whole mount in situ histochemical methods (center) by crossing each transgene into a nos^BN background (which otherwise contains no detectable Nos). Dark-field photographs at the bottom show that expression of Nos⁺ supports development of a normal complement of eight abdominal segments, whereas Nos^M379K is completely inactive in this assay (although it does rescue the oogenesis defects of nos^RC/Df flies and the lethality of nos⁷¹¹⁷/Df or nos^j3B6/Df animals; not shown). The Nos^M379K embryo (and the embryos in B) is surrounded by the vitelline membrane.