Worm Breeder's Gazette 11(5): 40

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Analysis of Mutation Sites in EMS-Induced TPA-Resistant Mutants

Y. Tabuse, T. Sano and J. Miwa

Figure 1

The tumor promoting phorbol ester TPA causes severe disturbance in 
movement and growth of C.  elegans.  The gene tpa-1, which is involved 
in TPA-induced effects, encodes a protein highly homologous to protein 
kinase C.
In order to know how mutations in the tpa-1 gene confer TPA 
resistance on C.  elegans, we have been analyzing mutation sites in 
TPA resistant mutants.  As for Tc1-induced TPA-resistant mutants, we 
reported on Tc1 insertion sites in three independent mutants in the 
last C.  elegans meeting.  All of the three mutants have their Tc1 
inserted in almost the same position in the exon coding the possible 
kinase domain.
We are now trying to identify the mutation sites in EMS-induced TPA-
resistant mutants.  First, we searched for RFLP in Southern 
hybridization analysis of genomic DNA using cDNA#1 as a probe.  We 
screened more than 20 strains and found loss of the EcoRV site in 
MJ514 and the PstI site in MJ534.  From the sequence of cDNA#1 and 
further Southern analyses in which cDNA#1 divided into three parts was 
used as probes, both restriction sites were supposed to reside on 
cDNA#1.  Small regions containing each site were amplified by PCR and 
sequenced directly.  In MJ514, the mutation causes amino acid change (
G46->E) in a cysteine-rich sequence, which has a zinc-finger like 
motif, in the amino-terminal regulatory domain.  The mutation of MJ534 
generates a stop codon at Q340 in the kinase domain.
Although it is difficult to describe the mechanism of TPA-resistance 
from the results obtained so far, the cys-rich sequence in the 
regulatory domain might have a role in interaction with TPA.  The cys-
rich sequence is thought to be important for binding phorbol esters in 
mammalian systems.  The results with MJ534 and Tc1-induced mutants 
suggest that structural change induced by immature termination in the 
kinase domain also results in TPA-resistance.  We are trying to 
identify and examine the tpa-1 product in both the wild type and these 
mutant strains to understand the relationship between mutation sites 
and the activity of mutant products.
[See Figure 1]

Figure 1