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Comments on Tabuse Y et al. (1991) Worm Breeder's Gazette "Analysis of mutation sites in EMS-induced TPA-resistant mutants" (0)
Overview
Tabuse Y, Sano T, & Miwa J (1991). Analysis of mutation sites in EMS-induced TPA-resistant mutants. Worm Breeder's Gazette, 11(5), 40. Unpublished information; cite only with author permission.
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]
