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]