Figure 2. Characterization of the
nhr-8 spatial expression pattern and
nhr-8(
ok186) deletion allele. (a,b) The
nhr-8::GFP was constructed by a two-step PCR cloning strategy and contains ~6.8 kb of upstream genomic sequence, the
nhr-8 transcription and translation start sites, and 49
nhr-8 codons fused in frame to GFP-coding sequences. A total of five independently isolated
nhr-8::GFP fusion plasmids were used to generate transgenic nematode strains. All transgenic strains exhibited identical GFP activity. (a) DIC micrograph of a transgenic larva (arrows mark the anterior/posterior extent of the intestine of the transgenic larva) and a wild-type larva (indicated by an arrowhead). (b) Epifluoresence micrograph of the animals in (a), revealing fusion protein expression only in the transgenic animal. (c) PCR amplicons from
nhr-8(
ok186) genomic DNA were sequenced to reveal a large deletion removing 1.3 kb including four exons and the 3' splice junction of the last intron. The DBD is designated by the stipled box. The
nhr-8(
ok186) sequence is indicated with the exon sequence in capital letters, the intron sequence in lower case letters, and the lesion represented by a tilde. (d) Northern blot. Each lane contains 30 μg total RNA purified from mixed stage cultures of wild-type (lane 1) and
nhr-8(
ok186) (lane 2) animals. An
nhr-8 probe detects a wild-type transcript of the expected size (~1.6 kb). A smaller ~0.8 kb transcript is detected in RNA from
nhr-8(
ok186) animals, consistent with the size predicted for a spliced mRNA produced from the remaining 5' end of the gene. A
pgp-3 probe detects
pgp-3 RNA in both the wild-type and
nhr-8(
ok186) lanes.
ama-1 mRNA was detected on the same blot to control for RNA loading and integrity (
ama-1 encodes the large subunit of RNA polymerase II,).