Worm Breeder's Gazette 9(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.
A phorbol ester tumor promoter, TPA, causes severe perturbations in the development and behavior of C.elegans. From the genetic analysis of TPA resistant mutants, the gene tpa-1 was shown to be involved in the TPA action. In order to know the structure and function of the tpa- 1 product, we are attempting to clone the tpa-1 gene by the transposon tagging method. The mutator strain NJ82, which is a gift from Dr.E. Hedgecock and shows a high incidence of Tc1 induced mutations, was incubated in the presence of 1 ug/ml TPA, and many spontaneous TPA resistant mutants which could grow there were isolated. Among five mutants so far tested, one mutant (MJ562) showed resistance as strong as the EMS induced tpa-1 reference mutant MJ500 and the mutation defined by this mutant was assigned to the tpa-1 gene by complementation analysis. The other four were weakly resistant mutants and fell into other linkage groups. MJ562 was back-crossed 10 times using N2, dpy-9 IV and unc-17 IV, and DNA was isolated, digested with restriction enzymes, gel-electrophoresed, and transferred to nylon membranes. The membranes were hybridized with biotinlabelled Tc1 and then incubated with avidin-phosphatase complex. In the HindIII restriction pattern, we could find two extra bands, 3.2 kb and 3.4 kb, unique to MJ562. From three-factor cross, MJ562 x dpy-9 egregating only Dpy progeny and F2 Unc nonDpy segregating only Unc progeny were cloned and analyzed by Southern hybridization. The TPA resistant mutation co-segregated with unc-17(15/15) but not with dpY-9(0/10). And the 3.2 kb fragment was detected in all of the Unc clones and none of the Dpy clones suggesting that the Tc1 insertion occurred in or near (<0.9% distance) tpa-1. Reversion analysis is in progress to determine the relationship between the 3.2 kb fragment and the TPA resistant phenotype. Also, we are searching the genomic library for a clone corresponding to the flanking region of the Tc1 sequence in the 3.2 kb fragment. We want to thank Dr.T.Otsuka for giving us valuable advice during his visit to our laboratory.