Worm Breeder's Gazette 11(3): 37

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.

Identification of the Molecular Lesion of the smg-Suppressible lin-29 allele

Ann E. Rougvie and Victor Ambros

Figure 1

lin-29 is a C.  elegans heterochronic gene that activates a temporal 
developmental switch in the hypodermal cell lineage at the L4 molt.  
lin-29 loss-of-function mutations cause hypodermal 'seam' cells to 
fail to switch from larval to adult specific cell fates.  
Supernumerary molts occur as the seam cells continue to divide and 
synthesize larval cuticle.  We identified a ClaI RFLP in genomic lin-2(
n546) DNA using a partial lin-29 cDNA as probe.  Southern analysis 
demonstrated that this RFLP, detected as the fusion of two ClaI 
fragments, is due a point mutation or a deletion of less than 50 bp.  
Since the cDNA clone has been sequenced and since it hybridizes to 
both Cla fragments in wild type DNA, we were able to derive 
oligonucleotide primers that flanked the polymorphism for use in PCR 
experiments.  We then asymmetrically amplified (Gyllensten and Erlich, 
PNAS 85: 7652) this region from genomic N2 and n546 DNA by PCR and 
directly sequenced the resulting single stranded DNA.  The sequence of 
n546 and N2 was identical throughout the 385 bp amplified region 
except for a single T to C transition in the predicted ClaI 
restriction site (see below).  This mutation generates an opal stop 
codon in the lin-29 ORF.
The lin-29(n546) allele is especially interesting since it is 
suppressible by smg mutations (Hodgkin, et al., Genetics 123: 301).  
Mutations in the smg genes are informational suppressors that act on 
specific mutant alleles of a number of different genes.  Suppression 
of certain unc-54 mutations by smg alleles seems to occur by restoring 
unc-54 mRNA levels (Pulak and Anderson, WBG 11: 54).  Thus, one 
possibility is that the stop codon destabilizes the lin-29(n546) mRNA 
causing loss of function.  The smg mutations could then act to 
stabilize lin-29(n546)  mRNA, allowing a truncated, and possibly 
functional, protein to be translated.  Interestingly, the opal stop 
codon in lin-29(n546) is positioned just downstream of four Cys-His 
type zinc fingers.  Alternatively, increased stabilization of the mRNA 
could allow read through of the stop codon to occur.  We have yet, 
however, to detect an aberration in n546 lin-29 message levels by 
Northern analysis.  In one smg-suppressed n546 strain, mRNAs detected 
by the lin-29 cDNA were qualitatively similar to mRNA isolated from 
wild type or n546 animals at the L4 stage.  However, smg1; n546/mnDf87 
animals are not suppressed (Hodgkin, et al., Genetics 123: 301) 
suggesting there may be a sharp threshold for lin-29 activity, and 
careful quantitative Northerns may be necessary to discern 
functionally important difference in mRNA levels.
[See Figure 1]

Figure 1