Worm Breeder's Gazette 12(5): 54 (February 1, 1993)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
unc-101 is a negative regulator of the vulval induction pathway, and encodes the homolog of AP47 ,the medium chain of mammalian Golgi-associated clathrin adaptor protein complex.
I. Physical basis of unc-101 mutations. We have determined the locations of the mutations of eight alleles of unc-101 .The mutations were determined by directly sequencing PCR-amplified DNA from genomic DNA's or single worms of each mutant. Sequence analysis of the mutant alleles showed that all but one allele are deletions or nonsense mutations, encoding truncated, and probably non-functional proteins. sy108 is a deletion of 115 nucleotides in exon 3 and intron 3. It also has an insertion of 8 nucleotides at the deletion point. sy168 , sy169 ,and sy241 ,recoveredin a non-complementation screen against sy108 ,are the same mutation as sy108 ;these mutations are likely the results of gene conversion events. sy237 , sy242 , m1 and rh6 are nonsense mutations, encoding truncated proteins. sy161 is the only missense mutation, changing an R to a C residue. This arginine residue is conserved among all the homologe of the medium chain of clathrin adaptor complexes. We were unable to amplify by PCR any genomic DNA from the lethal allele sy216 ,suggesting that this allele is a deletion of the entire gene or more. These molecular data support our genetic conclusion that existing alleles severely reduce the function of unc-101 .
II. Mammalian AP47 rescues unc-101 mutant phenotypes. Since the amino acid sequence of unc-101 is 79% identical to that of the mammalian homolog AP47 ,it is conceivable that the function of the two homologs is conserved during evolution. To test this idea, we examined whether the mammalian homolog can rescue the phenotype of unc-101 animals. Because genomic clones are not available for mammalian AP47 ,we esamined a hybrid construct with unc-10 lgenomic DNA and a rat AP47 cDNA. As a control, we constructed an unc-101 hybrid construct which has the promoter region of the rescuing genomic plasmid pJL2 ,the 5' region of unc-101 coding region with two introns, unc-101 cDNA, and the 3' untranslated region. This construct rescues the Unc phenotype of unc-101 ( sy108 ).Then we constructed an AP47 hybrid identical to the unc-101 hybrid except that it has AP47 cDNA portion instead of unc-101 cDNA This AP47 hybrid also rescues the Unc phenotype. We then injected the AP47 hybrid into unc-101 ( sy108 ); let-23 ( sy1 )animals, and obtained a stable line of nUnc transgenic animals. Under Nomarski optics 10 transgenic animals had an average vulval differentiation of 63%, indicating that this construct does rescue the unc-101 suppression of let-23 ( sy1 )vulvaless phenotype. Thus, AP47 and unc-101 are not only very homologous in their sequences, but also functionally equivalent.