The heterochronic genes
lin-4,
lin-14,
lin-28 and
lin-29 control the relative timing and sequence of many events during post-embryonic development, including the terminal differentiation of lateral hypodermal seam cells. In the course of performing a variety of genetic screens aimed at identifying additional members of the heterochronic gene pathway(1) we have isolated four alleles of
lin-42, a heterochronic gene for which a single allele has been reported(2).
lin-42(
n1089) mutants execute seam cell terminal differentiation precociously, during the L3-to-L4 molt. Epistasis analysis places
lin-42 downstream of
lin-4 and upstream of
lin-29, a position shared by
lin-14 and
lin-28. We set out to clone
lin-42 to better understand its role in the heterochronic gene pathway. Because
lin-42 resides in a gene-poor region of the left arm of LGII, we elected to use a transposon tagging approach to facilitate its cloning. We modified the gfp-based screen of Abrahante et al.(1) to allow use of transposons as a mutagen. We constructed the strain
lin-4;
mut-6; veIs13 [
col-19::gfp +
rol-6(
su1006)]. These animals do not express the
col-19::gfpfusion (expressed adult-specifically in wild type) due to the
lin-4 mutation that blocks the execution of the larval-to-adult switch in the hypodermis. We then screened for restoration of
col-19::gfp expression caused by Tc1-transposition events. At least two of more than 35 mutations isolated from this Tc1 screen are additional
lin-42 alleles. We detected a restriction fragment length polymorphism in DNA isolated from one of these,
ve27, using Tc1 sequences as probe. The polymorphic fragment was cloned and the DNA sequence flanking the Tc1 element was determined. Data base searches with this sequence showed identity to cosmid F47F6, a sequenced cosmid from LGII residing between RFLPs veP2 and nP48 in the position where we had previously mapped
lin-42. The Tc1 insertion site disrupts the fourth exon of a Genefinder predicted gene, F47F6.2. We have confirmed that this predicted gene corresponds to
lin-42. An 8.9 kb clone containing only this predicted open reading frame and approximately 3 kb 5'- and 1 kb 3'-flanking sequence rescues the
lin-42 mutant phenotype. In addition, we have sequenced several
lin-42 alleles and found lesions in Exons 2, 3, 4 and 5. We used RT-PCR to determine the structure of the
lin-42 mRNA, and found the Genefinder prediction to be accurate. Analysis of the predicted
lin-42 protein using BLAST revealed similarity to the PAS domain found in the family of proteins related to Drosophila Period (PER). The PER PAS domain has been shown to mediate protein-protein contact(3). LIN-42 shares highest identity with the PASB domain in the Per-like protein from our friend, the American cockroach Periplaneta americana. We are intrigued by the presence of the PAS domain in the heterochronic protein LIN-42 and in a variety proteins that time circadian rhythms including the PER family, the white collar proteins of bread mold(4), and the CLOCK protein of mice(5). This conservation of the PAS domain may reflect a molecular link between proteins that control timing in worms and other organisms. Expression studies are underway to test if the
lin-42 mRNA or protein products fluctuate during development. It is also possible that the sharing of this domain between the heterochronic and circadian rhythm classes of timing proteins simply reflects a conserved protein interaction domain. We note that PAS domains are also found other proteins, such as the aryl hydrocarbon receptor, where they also serve a role in protein-protein contact. Many of these proteins are transcription factors that include a basic helix-loop-helix domain, apparently lacking from PER and LIN-42. (1)Abrahante and Rougvie (1995). WBG 14, #1: 91; Abrahante, Miller and Rougvie, Manuscript in preparation. (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.