Figure 4. An evolutionarily conserved site in
egl-1 is required for programmed cell death of specific cells in the P11 and P12 lineages. (A) Light-gray boxes in the
egl-1 genes of C. briggsae and C. elegans indicate regions with evolutionarily conserved sequences. The dark-gray boxes indicate the
egl-1 open reading frame. Numbered asterisks indicate the locations of four evolutionarily conserved matches to the TGATNNAT Hox/Hox co-factor consensus. F23B12.1 encodes a predicted phosphatase that is not present in the C. briggsae (or C. remanei)
egl-1 region. (B) C. elegans genomic DNA sequence is shown flanked by nucleotide positions relative to the
egl-1 ATG. The positions of candidate binding sites are indicated. Nucleotides conserved in C. briggsae are indicated by black boxes. (C) Site 1 at position +5995 from C. elegans is shown. Mutated nucleotides are underlined. (D) The percentage of transgenic animals with the indicated number of corpses among the descendants of specific P cells. The diameters of the spots are proportional to the percentage of animals with the indicated number of corpses. Transgenic animals were constructed by biolistic transformation (Praitis et al., 2001) of
ced-1(
e1735);
unc-119(
ed3);
egl-1(
n1084n3082) mutants with the 7.6 kb genomic DNA of C. elegans
egl-1 (see Materials and methods). Wild-type (WT) indicates introduction of wild-type genomic DNA. In general, 15 animals were scored for each independently derived transgenic line and the data were pooled (11 independent transgenic lines for the wild-type construct; 13 transgenic lines for the Site 1 NcoI construct; and six transgenic lines for the others). The F23B12.1 phosphatase was not required for the effects on programmed cell deaths of transgenic animals, and mutations affecting sites 2, 3 and 4 did not alter the pattern of programmed cell deaths in the ventral nerve cord (see Fig. S1 in the supplementary material). Deletion of the 470 nucleotide evolutionarily conserved region, including Site 1 (sequences 3' of an XhoI site), resulted in a phenotype like that of mutations in Site 1. (E) DIC (a,c) and epifluorescence (b,d) images of some of the P11.a descendants of transgenic
egl-1(
n1084n3082) mutants carrying either a (a,b) wild-type Pegl-1histone:gfp reporter or a (c,d) mutant reporter in which Site 1 was changed to an NcoI site. Thirty out of 30 transgenic animals with a wild-type reporter expressed gfp in P11.aaap, and 29 of 30 expressed gfp in P11.aap. By contrast, of 90 descendants of three independent transgenic lines with a Site1 NcoI mutant reporter, only 11 expressed gfp in P11.aaap, while 83 out of 90 expressed gfp in P11.aap. P11.aap undergoes programmed cell death in wild-type animals and in
mab-5 and
ceh-20 mutants.