The heterochronic genes control the relative timing of several post-embryonic developmental events including the terminal differentiation of lateral hypodermal seam cells during the L4-to-adult molt. Mutation of the heterochronic gene
lin-42 results in the execution of the seam cell terminal differentiation one stage earlier than wild type, during the L3-to-L4 molt. We wish to determine the role of
lin-42 relative to other known heterochronic genes,
lin-4,
lin-14,
lin-28, and
lin-29 in timing seam cell terminal differentiation. We have initiated the genetic and molecular analysis of
lin-42 to better understand its function in the heterochronic gene pathway. We have isolated four new alleles of
lin-421, adding to the single allele previously reported2. Epistasis analysis places
lin-42 downstream of
lin-4 and upstream of
lin-29. We have cloned the locus using a transposon tagging approach. We constructed the strain
lin-4;
mut-6; veIs13 [
col-19::gfp+
rol-6(
su1006)], a strain where
col-19::gfp expression is suppressed by the
lin-4 mutation. We screened the progeny of this strain for mutations that restored GFP expression. Two of 35 mutations isolated from this screen were Tc1-tagged
lin-42 alleles. We detected a RFLP in one of these alleles,
ve27. This polymorphic fragment was cloned, and the sequence flanking the Tc1 element showed identity to a predicted gene in cosmid F47F6, from LGII. An 8.9 kb clone consisting of only this predicted open reading frame and approximately 3 kb of 5'- and 1 kb of 3'-flanking sequences rescued the
lin-42 mutant phenotype. We sequenced several
lin-42 alleles and found lesions in four of its five exons, confirming that the predicted gene indeed corresponds to
lin-42. The mRNA structure was determined by RT-PCR, and was found to correspond to Genefinder's prediction. BLAST analysis of the
lin-42 protein sequence identified similarity to the PAS domain found in the family of proteins related to Drosophila Period (PER). The PAS domain has been shown to mediate protein-protein interaction3. It is intriguing that the PAS domain in the developmental timing protein LIN-42 is shared by a growing family of proteins involved in the regulation of another timing mechanism, namely that which controls circadian rhythms. These proteins include members of the PER family, the white collar proteins of Neurospora, and the CLOCK protein of mice5. To test if this conservation of the PAS domain in LIN-42 reflects a functional conservation of PER's characteristic oscillating expression patterns, studies are underway to analyze a variety of
lin-42::gfp fusions, as well as
lin-42 message levels throughout development6. We plan to test if
lin-42 expression patterns are affected by light-dark regimens. 1 Abrahante, Miller and Rougvie, Genetics, in press. 2 Liu, Z. (1990). Ph.D. Thesis, Harvard University, Cambridge, MA. 3 Nambu et al. (1991). Cell 67:1157; Huang et al. (1993). Nature 364:259. 4 Crosthwaite et al. (1997). Science 276:763. 5 King et al. (1997). Cell 89:641. 6 Gardner et al. (1998). Midwest C. elegans Meeting Abstract.