The
daf-21!gene is of interest to us due to its role in chemosensory signal transduction.
daf-21 is important in dauer formation, a developmental decision regulated by the environment and controlled by both dauer-activating and dauer-repressing sensory neurons.1,2
daf-21(
p673), the only mutant allele, confers a dauer-constitutive (Daf-c) phenotype that is suppressed by mutations that disrupt the sensory cilia. This suggests that DAF-21 acts in the sensory endings.3
daf-21 mutants are also defective in thermotaxis and response to non-volatile and volatile attractants, all behaviors mediated by separate sensory neurons.4 Thus
daf-21 functions in several sensory transduction pathways. Most of these characteristics are shared by
daf-11 mutants,4,5 suggesting that
daf-21 and
daf-11 function together.
daf-11 encodes a trans-membrane guanylyl cyclase (GC),6 which produces cGMP from GTP. A poorly hydrolyzed cGMP analog (8-bromo-cGMP) rescues the Daf-c phenotype of
daf-11 and
daf-21, but not other Daf-c mutants.6 This indicates that cGMP is an important second messenger in this transduction system and suggests that DAF-21 is involved in the synthesis of cGMP by DAF-11. Previous analysis of
daf-21(
p673)/Df suggested that
p673 is a gain-of-function mutation.7 Initial attempts to screen for additional
daf-21 alleles proved to be laborious and unfruitful. In order to understand the function of
daf-21 and to clarify its relationship to
daf-11, we have cloned the
daf-21 gene. Based on its relative position between
him-5 and
unc-76, we chose several cosmids to inject. Two rescue the Daf-c phenotype (M05D1 and T10E3) and a third rescues partially (R08A2). By subcloning we identified a 5.8 kb fragment that rescues completely. The sequence of this fragment (determined by the genome project) is predicted to include two divergently transcribed genes. One (C47E8.4) is similar to XAP-5, a human gene of unknown function. The second (C47E8.5) encodes heat shock protein 90 (HSP-90). Based on three lines of evidence, we conclude that
daf-21!encodes HSP-90. First. a 3.7 kb subclone containing HSP-90 and only the N-terminal third of XAP-5 rescues
daf-21 partially. Second, a frameshift mutation engineered in the HSP-90 coding sequence partially impairs rescue by the 5.8 kb fragment. Finally, an HSP-90 mutation exists in the
daf-21(
p673) mutant (E292 > K). Characterization the
daf-21 (HSP-90) RNA indicates that the 2.5 kb RNA is trans-spliced to SL1 and follows the splicing pattern predicted by the genome project. Interestingly, it was previously shown that HSP-90 transcripts accumulate in dauers and are depleted as dauers recover.8 HSP-90 binds to and influences the activity of several classes of proteins including specific steroid hormone receptors and certain kinases such as
pp60v-src, Raf, and casein kinase II.9 All trans-membrane GC's, including DAF-11, contain an intracellular domain of unknown function with sequence similarity to protein kinases.10 We are planning two-hybrid studies to address the possibility that DAF-21 (HSP-90) interacts with and regulates the DAF-11 GC. Interactions between proteins of these two types have not been described. If we demonstrate a physical association, it will be interesting to determine the affect of the
p673 mutation and to identify the parts of the proteins required for the interaction. 1 Golden & Riddle, 1982, Science 218: 578; Golden & Riddle, 1984, Dev. Biol. 102: 368; Golden & Riddle, 1984, J. Chem. Ecol. 10:1265 2 W. Schackwitz & J. Thomas, Neuron, in press Bargmann & Horvitz, 1991, Science 251: 1243 3 Thomas et al., 1993, Genetics 134: 1105 4 Vowels & Thomas, 1994, Genetics 138: 303 I. Mori, pers. comm. 5 Vowels & Thomas, 1992, Genetics 130: 105 6 Birnby et al., 1995 ICEM abstract 7 Malone et al., 1993 ICEM abstract 8 Daley & Golomb, 1992, Dev. Biol. 151:80 9 Pratt, 1992, Bioessays 14: 841; Brugge, 1986, Curr. Top. Microbiol. Immunol. 123: 1; Stancato et al., 1993, JBC 268: 21711; Miyata & Yahara, 1992, JBC267: 7042; 10 Garbers & Lowe, 1994, JBC 269: 30741