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.

Molecular Analysis of the tpa-1 Locus

Y. Tabuse, K. Nishiwaki and J. Miwa

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.