1
Characterization of the gpa-16(pk481) phenotype
Viability of the embryos and larvae (n>200) produced by stably balanced (see Materials and
methods) + gpa-16(pk481)/bli-3(e767) + hermaphrodites (subsequently abbreviated as gpa16/bli) was similar to that seen with wild-type hermaphrodites at 16°C and 20°C, i.e. almost all
(>97%) survive. Therefore, at these temperatures 33% of the resulting nonBli progeny adults
should be of genotype pk481/pk481. However, when these nonBli progeny were plated singly, at
either 16°C (n=200), 20°C (n=300) or 25°C (n=200), about 40% were completely sterile or
nearly so (laying 1-5 eggs and producing 1-2 viable larvae). The remaining 60% produced larger
broods of 20-100 progeny, which in all cases included Bli animals, indicating a parental
genotype of pk481/bli-3. None of the surviving progeny from the nearly sterile animals were Bli
(n=25). These results indicate that the phenotype of pk481 homozygotes is sterility, but that
about 10% of heterozygous pk481/bli-3 animals are also sterile. Examination of a few sterile
hermaphrodites by Nomarski microscopy revealed variable defects in gonad morphology that
were not further analyzed.
gpa-16(pk481) appears to be a complex rearrangement
When tested in single worm PCR experiments using the primers shown in Fig. 4B, sterile,
presumed homozygous pk481 animals derived as described above (n>50) appeared to contain a
copy of the wild-type sequence including the first three exons, in addition to the deletion gene
lacking exon 2 (Fig. S2). Genetic experiments (not shown) using this PCR assay to analyze
progeny showed that the wild-type sequence was closely linked to the deletion sequence (as
opposed to being present in an unlinked duplication that could have been selected for during the
backcrossing as necessary for viability). RT-PCR using primers flanking the deleted region
showed that this region was present in transcripts from progeny of pk481/bli-3 animals,
suggesting that the mutant protein is expressed.