Figure 3

unc-7 transformation rescue. (A) Exon/intron structure of unc-7. Cosmid F56B12 includes presumptive upstream promoter sequences and ends within intron 3. ATG indicates UNC-7L (exon 2), UNC-7S (exon 1S, blue shading), and UNC-7SR (exon 5) start sites. Recombination in vivo between F56B12 and plasmid constructs reconstituted the unc-7 locus. Rescue of forward (F) or reverse (R) locomotion by plasmid constructs, with or without co-injection of F56B12, was determined (isoforms predicted to be expressed are listed in red). Rescue was determined from at least three lines, except (G) (single line generated), and (F) (no rescued lines obtained). (B) Fully rescuing unc-7 min plasmid expressing UNC-7S and UNC-7SR. Red slashes indicate frameshift mutation eliminating UNC-7L expression. (C) unc-7S::gfp construct lacks UNC-7L translational start site and shares 1.4-kb with F56B12. (D) unc-7SΔ1S lacks exon 1S. (E) unc-7-2cDNA6 with exons 2–6 replaced by corresponding cDNA sequence, shares 1 kb of intron 1 overlap with F56B12. (F) unc-7SΔ1S-M121L cannot initiate translation of UNC-7S or UNC-7SR. (G) unc-7S-M121L intiates translation of UNC-7L and UNC-7S with the M121L mutation. (H) Western blot using anti-GFP antibodies detecting UNC-7::GFP isoforms. Predicted sizes of UNC-7L::GFP, -S::GFP, and -SR::GFP are 85, 78, and 72 kDa, respectively. Lane 1, wild type (N2); lanes 2 and 3, unc-7(e5) rescued with unc-7S::gfp + cosmid F56B12, expected to express all isoforms; lane 4, unc-7(e5) rescued with unc-7SΔ1S + F56B12, predicted to express UNC-7L:: and SR::GFP. ND = not determined.