Figure 5. Cloning of
dpy-19. (A) Schematic diagram showing intron and exon boundaries in
dpy-19. Black arrows mark sites of point mutations found in four different
dpy-19 alleles. Open arrows mark sites of mutations found in intragenic revertants of
dpy-19(
e1259). Exact mutation sites:
mu78(L218->F),
e1259(G185->D),
e1314(splice acceptor mutation disrupts protein after R517),
mu79(W653->STOP),
e1259mu298(G185->N),
e1259mu300(P127->S, G185->D). asterisk, revised first exon that was not predicted by Genefinder analysis of genomic sequence (C. elegans Sequencing Consortium). (B) Northern blot of total RNA probed with sequences from the
dpy-19 gene. N2 lane contains wild-type RNA and
n1347n1348 lane contains RNA from the deletion allele,
dpy-19(
n1347n1348). (White bands reflect a decrease in background from excess rRNA.) (C) Alignment of DPY-19/F22B7.10 and the human full-length cDNA KIAA0877 (Genbank Accession: AB020684, Unigene cluster Hs. 11217.). DPY-19 and KIAA0887 are 41% identical and 61% similar over 547 amino acids. Gray lines above F22B7.10 sequence indicate position of 13 potential membrane spanning domains predicted by TMPred (Hofmann and Stoffel, 1993). The position of the loss of function mutations described in (A). (D) Expression of GFP-tagged DPY-19 in QL and surrounding cells. Rings of GFP expression are coincident with cell nuclei outlines. This construct fully rescues mutations in
dpy-19 (see Materials and Methods). (E,F) Expression of nuclear-localized GFP under control of the
dpy-19 promoter. (E) shows GFP expression faintly in QL and strongly in surrounding epidermal cells. (F) shows a Nomarski image of the same worm. In animals containing the array, 5/15 QL cells and 9/18 QR cells expressed GFP faintly during the time of the Q migrations. In D-F, arrows mark dorsal (
hyp7) and ventral (P) epidermal nuclei and arrowheads mark lateral (V) epidermal nuclei. The lack of expression in V6 in E is presumed to be the result of sporadic loss of the extrachromosomal array carrying the GFP reporter since other animals showed strong expression in V6. Scale bar: 5 µm.