Worm Breeder's Gazette 11(3): 24
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.
We previously summarized the genetic characterization of the function of let-60 in vulval induction (WBG 11(2): 105). Our data suggested that the level of let-60 activity controls the fates of the vulval precursor cells (VPCs) in response to the inductive signal from the gonadal anchor cell. Reduction of the activity of the gene causes a vulvaless (Vul) phenotype. We also argued that an increase of let- 60 activity (resulting from lin-34 mutations) causes a multivulva (Muv) phenotype. Moreover, let-60 acts after let-23 and lin-15, but before lin-1 and lin-12 in the genetic pathway of vulval cell type determination. We have carried out following experiments towards the goal of cloning let-60: (1). Determination of the left breakpoint of nDf27. let-60 maps between the left breakpoint of nDf27 and dpy-20 (see Figure 1). We located the breakpoint within the cosmids F58B3 and C50G2. (2). Chromosome walk by computer. We tried to find the two gaps between dpy-20 and the left breakpoint of nDf27 (Figure 1). Cosmid D1035 was the best candidate for the walk towards the left of dpy-20. We had a hard time isolating the intact cosmid from bacteria because of recombination. However, when we finally isolated the cosmid out of the original stock kindly provided by Alan Coulson and John Sulston, we found that the left half of the cosmid (three different fragments) hybridized with every colony of a genomic library. These results suggested that the left half of the cosmid is bacterial DNA. When Alan Coulson removed D1035 from the physical map, the computer rearranged the area and the gaps disappeared (compare the two figures). In addition, when we walked with another cosmid, ZK205, we were able to fish out new cosmids extending into the 'gaps' easily, suggesting that gaps in the area were unlikely. (3). RFLP mapping the let-60(lin-34) mutations. We have made genomic DNA from more than 20 EMS-generated mutants of let-60 or lin- 34 (some were provided by G. Beitel and S. Clark in R. Horvitz's laboratory). We detected no RFLPs by Southern analysis with more than 8 different restriction enzymes. (4). Transformation by microinjection. Mainly using the method reported by Mello et al. (WBG 11(1): 18-19), we have successfully suppressed the Let and Vul phenotypes by injecting cosmid DNAs into strains with dominant negative or putative null alleles of let-60. The results are shown in Figure 2. Strikingly, strains carrying the cosmids which complement the mutant phenotypes (about 20-50 copies per genome) often show a partial Muv phenotype. We interpret the Muv phenotype as resulting from additional copies of the let-60 gene. This result is thus consistent with our genetic argument that a high level of the let-60 activity specifies the vulval fates of VPCs. Further molecular analysis of the gene is in progress. [See Figure 1]