The
daf-1 and
daf-4 genes encode transmembrane receptor serine/threonine kinases (S/TK) that are members of the Transforming Growth Factor B (TGF-B) receptor superfamily. In C. elegans, these receptors link signals transduced from chemosensory amphid neurons, which detect a dauer-inducing pheromone, to cells that employ alternative patterns of gene expression during dauer or non-dauer development. The phenotype of
daf-1 and
daf-4 mutants is temperature-sensitive dauer-constitutive, indicating that the normal function of these receptors is to transduce growth-promoting signals. Our goal is to identify proteins that interact with DAF-1 and DAF-4 and to determine the molecular mechanism of signal transduction that regulates the dauer developmental switch. A large number of
daf-1 and
daf-4 mutant alleles have been isolated in genetic screens for dauer-constitutive mutants. To determine what types of defects in DAF-1 and DAF-4 confer a dauer-constitutive phenotype, we have sequenced mutations in the coding regions of
daf-1 and
daf-4 alleles. Heteroduplex DNA molecules formed between PCR-generated fragments of coding sequence from wild-type (N2) and
daf-1 or
daf-4 mutants were analyzed for the presence and location of single base-pair mismatches or small deletions using the cytidine-modifying agent hydroxylamine. Sequence analysis at these sites of mismatch revealed base-pair changes that encode amino acid substitutions or premature termination codons in the receptor proteins. Among twelve alleles of
daf-1, six encode single base transitions that result in translation-terminating codons; the remaining six alleles are missense mutants. In the case of
daf-4, seven dauer constitutive alleles are missense mutants and the remaining two are nonsense mutants. The location of a mutation within DAF-1 or DAF-4 provides information about the probable mechanistic defect in that mutant receptor. Among six DAF-1 missense mutants, two lesions were present in the extracellular ligand-binding domain of the receptor and four others were located within the S/TK catalytic domain. All dauer constitutive
daf-4 mutations analyzed localized to sequences encoding the S/TK catalytic domain. For mutations in the kinase domain, further insights into mutant protein defects were provided by aligning the
daf-1 and
daf-4 amino acid sequences with other S/T kinases, such as cAMP-dependent protein kinase, and additional receptor S/TKs in the TGF-B receptor superfamily. Based on these analyses, mutations in
daf-1 and
daf-4 kinases belong to one of three classes: mutations in amino acids that are conserved in all S/TKs (Class I), mutations in amino acids that are conserved only among TGF-B receptor S/TKs (Class II), mutations in amino acids that are unique to the
daf-1 or
daf-4 receptors (Class III). This study provides a molecular basis for understanding the functional defects in genetically-derived signal transduction mutants. These findings will be useful in the analysis of interaction targets identified in genetic and biochemical screens for
daf-1- and
daf-4-associated signalling components that are presently underway in this laboratory.