Worm Breeder's Gazette 14(4): 52 (October 1, 1996)

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.

daf-21, A Gene Required in Several Chemosensory Transduction Pathways, Encodes HSP-90

Elizabeth A. Malone, James H. Thomas

Department of Genetics University of Washington Seattle, WA 98195

        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
        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