Figure 1. Characterization of the long isoforms (UNC-53L) of
unc-53. (A) Structure of the
unc-53 gene. The start of the various UNC-53L and UNC-53S isoforms are indicated by arrows. The promoter for UNC-53SA is between exons 5 and 8, and the promoter for UNC-53SB is located between exons 8 and 13 (Choi and Newman, 2006; Stringham et al., 2002). 2.9 kb of DNA upstream of the transcriptional start site of UNC-53LA was used to construct
punc-53L::gfp. Alternatively spliced exons are shown in pink.
unc-53(
n152) is a 319-bp deletion removing parts of exons 18 and 19, producing a stop codon in exon 20 (Stringham et al., 2002), and
n166 is a single nucleotide C to T transition in exon 19 that introduces a premature stop codon. (B) The longest polypeptide, UNC-53LA, is 1654 amino acids and contains a calponin homology domain (CH, red; amino acids 11-109), two LKK motifs (LKK, purple; 114-133 and 1097-1116), two proline-rich SH3-binding motifs (SH3b, green; 487-495 and 537-545), two coiled-coil regions (CC, blue; 890-923 and 1078-1113) and an AAA domain (yellow; 1292-1425).
n166 introduces a premature stop codon at amino acid 949. Both
n152 and
n166 remove the coiled-coil, LKK and AAA domains from all isoforms. The first five exons of UNC-53 (UNC-53N; amino acids 1-139) were used for the production of PAB-UNC-53N antisera and the GAL4 DNA-binding domain (GAL4DBD) in pVA200 for yeast two-hybrid studies. (C,D) Expression pattern using
punc-53L::gfp. (C) Adult hermaphrodite (anterior is left), showing GFP expression in head (HNeu) and tail (TNeu) neurons, the excretory cell (EXc), and the ventral nerve cord (VC). (D) Midbody, showing expression in the sex myoblasts (SM) and the ventral cord (VC). (E,F) Expression pattern using PAb-UNC-53N antisera. (E) Expression of UNC-53L throughout the excretory cell and canals (EXc). (F) UNC-53L expression in a pair of coelomocytes (CC). Scale bars: 100μ m.