Worm Breeder's Gazette 11(3): 34
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.
As is well known,C. elegans must choose between two alternative L3 larval fates at the L2 to L3 molt. If conditions are favorable (ie. food and space are abundant) the animal molts into an L3 committed to becoming an adult within a day. However, unfavorable environmental conditions induce the developmentally arrested dauer larva . Dauers can survive for more than a month as they search for greener pastures in which to resume development toward adulthood. Mutations defining the signal transduction pathway leading to dauer, or non-dauer development have been identified, and fall into two categories. Constitutive mutants form dauers at a restrictive temperature regardless of the presence of food. Dauer-defectives, on the other hand, will not form dauers even when starved. These isolated mutants and their interactions have been extensively characterized (Nature vol. 290, p. 688). At this time, analysis of dauer induction at the molecular level is proceeding at breakneck speed. daf-4, a late-acting dauer-constitutive gene in the genetic pathway, confers several curious phenotypes. Like other dauer-constitutive mutants, daf-4 forms 100% dauers at 25 C. In addition, daf-4 exhibits a small phenotype, and has been reported by Lewis Jacobson's lab to be defective in intestinal endocytosis. It is also interesting to note that daf-12, a dauer-defective with a long phenotype, is epistatic to daf-4's constitutive phenotype, but not its small phenotype. This might imply a role for the daf-4 gene product in pathways other than dauer induction. Although genetic studies have provided insight into daf-4's relative place in dauer induction, molecular analyses of the gene will be required in order to further elucidate its function. Toward this end, we have isolated two Tc1 induced daf-4 mutant alleles, and a revertant of each. Genomic Southerns of DNA from the two mutants and their revertants were probed with Tc1 in the hope of finding a Tc1 band in the mutant alleles that was absent in the revertants. An XbaIS) digest revealed an extra Tc1-containing band approximately 2.4 kb in size, and an EcoRIB) digest revealed an extra band approximately 1.9 kb in size. Both fragments were cloned. Using sequences flanking Tc1 from either fragment to probe Southern blots of mutant and revertant DNA's revealed a 1.6 kb upshift in the detected mutant bands relative to the revertant bands (this is consistent with the insertion of one Tc1). The flanking sequences from both fragments detect the same bands on these Southern blots. Sequencing of the XB fragments is now underway in order to identify the sites of Tc1 insertion in the two mutant strains. Open reading frames have been found, but initial database searches have not revealed obvious homologies to known proteins. Genomic and cDNA library screens have yielded 4 genomic clones, and 5 cDNA clones. Sequencing of these clones will begin shortly.