Fig. 7. X-gal staining of C. elegans hermaphrodites transgenic for unc-
ll9::
f3gal fusions. Left column, unc-
ll9 (
ed4); edEx42 (C. briggsae fusion; edEx42 contains the fusion plasmid pDP#MM082 and the rescuing clone pDP#MM016); right column [except (h)], unc-
ll9(ed3); edEx43 (C. elegans fusion; edEx43 contains the fusion plasmid pDP#MM081 and pDP#MM016); (h) wild-type animal transgenic for pRF4 (rol-6Dmarker) and pDP#MM082. Despite the nuclear localizing signal (NLS) present in the reporter gene constructs, staining is still visible outside of nuclei (as verified by DAPI staining; data not shown). Panels a and b, comma-stage embryos showing anteriorly localized staining in presumptive neuronal cell precursors; (c) adult and (d) fourth larval stage animals, showing staining in the nerve ring (arrows), pre-anal ganglia (open arrowheads) and ventral nerve cord (filled arrowheads); (e) and (f), adults showing staining in the nerve ring (arrows) and ventral nerve cord (filled arrowheads); (g) and (h), ventral view of hermaphrodite vulva (V) showing the ventral nerve cord and a VC neuron (*). A diffusely staining early embryo (E) is visible in each panel. Methods: Construction of pDP#MM081, predicted to encode the first 101 aa of C. elegans UNC-119, has been described (Maduro and Pilgrim, 1995). For construction of the C. briggsae fusion, two intermediate plasmids were used to generate suit- able restriction sites. The plasmid UR#91 (a gift from W. Wadsworth) contains an EcoRI-HindIII fragment cloned into pBluescript SK+; this fragment was removed from the polylinker of pUC19 which con- tained a 120 bp insertion at the BglII site. UR#91 was digested with EcoRI and BglII (both enzymes BRL) to liberate the 120 bp insertion and part of the pUC polylinker, and allow the insertion of a BamHI- EcoRl fragment from C. briggsae. This intermediate plasmid, pDP#MM070, was digested with HindIII (Pharmacia) and PstI (BRL) to allow its subcloning into similarly digested pPD22.04 (Fire et al., 1990), which is predicted to join the first 130 aa of C. briggsae UNC-119 with bgal.