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A Transmembrane Guanylyl Cyclase (DAF-11) and Hsp90 (DAF-21) Regulate a Common Set of Chemosensory Behaviors in Caenorhabditis elegans
Deborah A. Birnby1,2,a, Elizabeth Malone Link1,3,a, Jennifer J. Vowels2,a, Hong Tiana, Patrick L. Colacurcioa, and James H. Thomasaa Department of Genetics, University of Washington, Seattle, Washington 98195-7360
Corresponding author: James H. Thomas, Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195-7360., jht{at}genetics.washington.edu (E-mail)
Communicating editor: I. GREENWALD
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Caenorhabditis elegans daf-11 and daf-21 mutants share defects in specific chemosensory responses mediated by several classes of sensory neurons, indicating that these two genes have closely related functions in an assortment of chemosensory pathways. We report that daf-11 encodes one of a large family of C. elegans transmembrane guanylyl cyclases (TM-GCs). The cyclic GMP analogue 8-bromo-cGMP rescues a sensory defect in both daf-11 and daf-21 mutants, supporting a role for DAF-11 guanylyl cyclase activity in this process and further suggesting that daf-21 acts at a similar step. daf-11::gfp fusions are expressed in five identified pairs of chemosensory neurons in a pattern consistent with most daf-11 mutant phenotypes. We also show that daf-21 encodes the heat-shock protein 90 (Hsp90), a chaperone with numerous specific protein targets. We show that the viable chemosensory-deficient daf-21 mutation is an unusual allele resulting from a single amino acid substitution and that the daf-21 null phenotype is early larval lethality. These results demonstrate that cGMP is a prominent second messenger in C. elegans chemosensory transduction and suggest a previously unknown role for Hsp90 in regulating cGMP levels.
LIKE all free-living organisms, Caenorhabditis elegans responds to a variety of environmental stimuli. The presence of food affects locomotion, egg laying, and defecation (B. SAWIN, C. TRENT and H. R. HORVITZ, personal communication; 
In C. elegans, bilaterally symmetric pairs of ciliated sensory neurons in the head amphid sensilla mediate many chemosensory behaviors. For example, C. elegans is attracted to a variety of nonvolatile chemicals, including Cl-, cAMP, and biotin, which are sensed primarily by the ASE neurons (
The external environment also regulates formation of the C. elegans dauer larva, an alternative third-stage larva specialized for survival under harsh conditions (
Regulation of dauer formation is also mediated by particular classes of amphid sensory neurons, and the functions of these neurons have been determined by killing specified cells with a laser microbeam. When the ADF and ASI neurons are killed together, wild-type larvae form dauers constitutively (
The various shared properties of daf-11 and daf-21 mutants indicate that these genes have closely related functions in several types of sensory neurons. Here we present molecular analyses of these two genes. We show that DAF-11 is homologous to transmembrane guanylyl cyclases (TM-GCs), which catalyze the formation of cyclic GMP (cGMP) from GTP. cGMP is a widely used second messenger that regulates kinases, other nucleotide cyclases, cyclic nucleotide phosphodiesterases, and cGMP-gated ion channels (reviewed in
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culture, strains, and genetics:
C. elegans strain maintenance and genetic nomenclature were as described (
To map daf-21(p673) genetically, we crossed him-5 males to sqt-3 daf-21 unc-76/+ + + hermaphrodites, picked non-Sqt non-Unc hermaphrodite cross-progeny, and then picked Sqt non-Unc and Unc non-Sqt recombinants in the next generation. The identification of recombinants was not biased by the daf-21 genotype due to maternal rescue of the daf-21 phenotype. We isolated strains homozygous for each recombinant chromosome and then scored the Him and Daf-c phenotypes. Of 23 Sqt non-Uncs, 15 were Him non-Daf, 6 were non-Him non-Daf, and 2 were Daf non-Him. Of 39 Unc non-Sqts, 25 were Daf non-Him, 9 were Daf Him, and 5 were Him non-Daf. These results are summarized in Fig 5A, and they indicate that daf-21 is roughly two-thirds of the way between him-5 and unc-76.
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To test the nature of the daf-21(p673) mutation, we used deficiencies that delete the daf-21 gene. Wild-type males were crossed to heterozygous hermaphrodites carrying chromosome V deficiencies balanced by the nT1 n754 translocation. The dominant Unc phenotype of n754 was used to infer the genotype of the progeny. Non-Unc males (Df/+) were crossed to dpy-11 daf-21(p673) hermaphrodites and the progeny were raised and scored at 20° or 25°. Individuals were picked and daf-21(p673)/Df strains were identified based on the segregation of dead eggs. Dpy progeny were assumed to be daf-21(p673) homozygotes, since dpy-11 is linked to daf-21. We tested three deficiencies that delete the daf-21 region: nDf42, itDf2, and yDf8. All gave similar results.
The daf-21(nr2081) deletion was constructed in trans to the nT1 n754 balancer translocation for strain maintenance. Wild-type males were crossed to daf-21(nr2081)/+ heterozygotes and male progeny were crossed to nDf42/nT1 n754 hermaphrodites. Unc progeny were picked, allowed to have progeny, and screened by PCR for daf-21(nr2081). This procedure also served to outcross daf-21(nr2081) twice. We constructed daf-21(p673)/daf-21(nr2081) heterozygotes by crossing wild-type males to daf-21(nr2081)/nT1 n754 hermaphrodites and then crossing non-Unc male progeny to daf-21(p673) hermaphrodites. Non-dauer hermaphrodite progeny were picked individually and transferred to new plates each day for 3 days and were then used for PCR assays of nr2081. Three heterozygotes were obtained and in each case the progeny at 20° were similar. A total of 16% arrested as L1 or L2 larvae, 70% formed dauers, and 14% developed as non-dauers (n = 161).
To create worms with the genotype daf-21(p673)/daf-21(p673)/+, we used ctDp11, a free duplication containing part of chromosome V (including daf-21, her-1, and unc-42) and part of chromosome III (including dpy-17, ncl-1, and unc-36). We crossed dpy-17 ncl-1 unc-36; him-8; her-1 unc-24; ctDp11 males to dpy-17 ncl-1 unc-36; daf-21(p673) hermaphrodites. We picked wild-type progeny [dpy-17 ncl-1 unc-36him-8/+; her-1 unc-42 +/+ + daf-21(p673); ctDp11] and allowed them to self-fertilize. In the next generation, we picked non-Dpy non-Unc dauers (dpy-17 ncl-1 unc-36; daf-21; ctDp11) and then chose strains that segregated no males [him-8(+)]. To assay the Daf-c phenotype at 20°, we let parents carrying the duplication (p673/p673/+) lay eggs overnight and scored the progeny after 3 days. We found 7.2% of the progeny formed dauers (n = 568), in contrast to progeny of daf-21(p673)/+ mothers, which never form dauers.
PCR, DNA sequencing, and DNA oligonucleotides:
Sequencing was performed by the ABI dye terminator cycle sequencing method (Perkin-Elmer, Norwalk, CT) using either AmpliTaq DNA polymerase or AmpliTaq DNA polymerase FS. The PCR products were analyzed by the University of Washington Biochemistry and Pharmacology DNA Sequencing Facilities and by Axys Pharmaceuticals. PCR and sequencing primers were obtained from various sources, and sequences of all listed primers are available on request.
Molecular identification of daf-11:
daf-11(m597) was isolated by P. Albert and D. Riddle from a strain with active transposition of Tc1. Southern blots of genomic DNA from outcrossed m597 strains were probed with Tc1 DNA, and a 0.38-kb NdeI fragment was found to contain a 1.6-kb Tc1 insertion that cosegregated with m597 in recombinants. Genomic DNA from daf-11(m597) was digested with NdeI and used for nested inverse PCR with TC1 primers, and the products were ligated at low concentration to encourage intramolecular ligation. The PCR primers for the first round were oriented outward from the ends of Tc1 (OLG34 and OLG35). The primer for the second round (OLG23) is derived from the inverted repeat sequence at the ends of Tc1 and also oriented outward. The resulting PCR product was cloned into pBluescript II KS+ (Stratagene, La Jolla, CA) that had been digested with EcoRI and treated with Klenow to generate blunt ends to create plasmid pTJ277. We verified that pTJ277 contained DNA flanking the m597 Tc1 by using it as probe on Southern blots of genomic DNA from m597, from four phenotypic revertants of daf-11(m597), and from genetic recombinants that retained m597 but removed most of the rest of the Tc1-mutagenized chromosome V. In each case, polymorphisms were detected consistent with a Tc1 insertion only in the daf-11(m597) strains.
Determination of the daf-11 cDNA sequence:
pTJ277 was used as a probe to isolate genomic and cDNA phage. Five phages with overlapping genomic inserts were isolated at a frequency of 1.8 x 10-4 from the Stratagene 
FIXII genomic library. Sequence analysis was performed on subclones from one of these phages to determine part of the daf-11 genomic sequence. This sequence was also compared to that generated by the C. ELEGANS SEQUENCING CONSORTIUM (1998), which sequenced the region containing daf-11 during the course of this work. daf-11 corresponds to the predicted gene B0240.3. From ~106 plaques from one cDNA library (A. FIRE, personal communication) and 3 x 105 plaques from another (
Determination of daf-21 cDNA sequence:
The 5.8-kb BamHI insert in pEM1 was used as a probe to isolate cDNA phage from a mixed-stage C. elegans cDNA library (Stratagene). Approximately 3% of plaques hybridized to this probe that contains C47E8.4 and daf-21/Hsp90. The inserts for 12 positive plaques were excised into pBluescript II using helper phage K07. On the basis of restriction digest with EcoRI, we found that 11 of the 12 were clearly related. We sequenced the longest, pEM29, with gene-specific primers designed on the basis of the Genefinder (P. GREEN, personal communication) prediction for the Hsp90 coding sequence. This analysis confirmed all of the intron-exon splicing predictions and revealed a 3' untranslated region of at least 117 bp. Our analysis of 12 publicly available 3' expressed sequence tags showed that the five most extensive sequences end at the same point as pEM29, suggesting this is the true 3' end of the mRNA. We did not find evidence of a poly(A) tail. The 5' end of pEM29 is exactly at the predicted ATG start codon. To determine the true 5' end of daf-21, we used the GIBCO-BRL 5' RACE system for RT-PCR (
Sequence of daf-11 and daf-21 mutations:
DNA was amplified by PCR from total genomic DNA (
Modification of Genefinder predictions of gcy genes:
We analyzed the Genefinder predictions for gcy-1 to gcy-18, all of the TM-GC genes fully sequenced at the time. The GCY-1 through GCY-18 proteins and a selected set of previously reported TM-GCs were subjected to an initial multiple alignment with CLUSTALW 1.4. We manually inspected the resulting alignments for regions in which specific Genefinder-predicted proteins appeared to have deletions of conserved sequence or insertions of unconserved sequence in a conserved region. Typically, these stood out dramatically in a multiple alignment, and all but one occurred precisely at Genefinder-predicted exon-intron boundaries. In these cases, we manually scanned genomic sequence for splice sites that would produce clearly improved alignments. In four cases, such an alternative splice was found. F23H12.6 (GCY-13) had an anomalous insertion of VSRHENP after predicted amino acid (aa) 614; use of a splice donor 21 nucleotides (nt) upstream (2215 nt from the predicted ATG, splice junction sequence AGT ^ GTGAGTC) precisely eliminated the anomaly. F23H12.6 (GCY-13) also had an anomalous deletion of 10 amino acids (following the F23H12.6 predicted sequence FFSDVVGFT); use of a splice donor 30 nt downstream (4495 nt from the predicted ATG, splice junction sequence CAG ^ GCGAGTT) added 10 amino acids (VLANKSTPLQ) that restored typical similarity to other TM-GCs. [This alteration is questionable because it requires the use of an unusual splice donor sequence. To test the validity of this change, we performed a BLASTP search of the nr GenBank data set with an 18-amino-acid sequence (VGFTVLANKSTPLQVVNL) centered on the inserted 10 amino acids. All 34 hits with P values <0.9 were TM-GCs.] ZK455.2 (GCY-9) had an anomalous insertion of VCKLRQKII after predicted aa941, which was precisely removed by using a splice donor 27 nt upstream (4742 nt from the predicted ATG, splice junction sequence CAG ^ GTTTGCC). B0024.6 (GCY-6) had an anomalous insertion of 18 amino acids (RKIF QKSTNISSSFHLFS) after predicted aa1161 that was precisely removed by creating a new 54-nt intron (starting 5798 nt from the predicted ATG, splice donor sequence AAC ^ GTAAAAT and splice acceptor sequence GTTTCAG ^ CTG). While this approach to amending Genefinder predictions is not rigorous and may have ignored some errors, we think it highly likely that the revised protein predictions more closely approximate the true structures. As a partial test of validity we performed TBLASTN searches (default parameters), using as the query the protein most closely related to the gene under analysis. This permits detection of conserved protein coding regions in a manner independent of Genefinder predictions. In each case, the TBLASTN match to the target gene verified our exon assignment.
The gcy names for TM-GCs correspond to cosmid names from the C. elegans Sequencing Consortium as follows (see also
8-Bromo-cGMP assays:
Plates (2 cm) were filled with 2 ml NGM agar (
In several 8-bromo-cGMP assays, many animals arrested as L1 or L2 larvae or formed larvae with only some characteristics of dauers. We interpret this to mean that 8-bromo-cGMP can affect non-dauer development as well as execution of the dauer developmental program. These animals generally constituted <25% of the total, and they were excluded from the data presented. The following were exceptions to this low frequency: daf-11(m87) 0.6 mM 8-bromo-cGMP (34% not counted); daf-11(sa195) 1.25 and 2.5 mM 8-bromo-cGMP (about 40% not counted); daf-21(p673) 1.25, 2.5, and 5 mM 8-bromo-cGMP (about 50% not counted); daf-4(e1364) 5 mM 8-bromo-cGMP (42% not counted); daf-8(e1393) 2.5 and 5 mM 8-bromo-cGMP (about 50% not counted); daf-2(e1370) 5 mM 8-bromo-cGMP (62% not counted); tax-4(ks11) 5 mM 8-bromo-cGMP (67% not counted); tax-4(p678) 5 mM 8-bromo-cGMP (43% not counted). Conclusions based on these data were essentially unchanged if the partial-dauer animals were counted as dauers.
Construction of daf-11::gfp fusions:
The 4.2 kb upstream of daf-11 plus the entire genomic coding region was cloned in two pieces from PCR products generated from the daf-11-containing cosmid W04E7. Taq polymerase was mixed 100:1 with Pfu polymerase to reduce the mutation rate (after
Construction of daf-21 subclones:
We constructed subclones of T10E3, a daf-21 rescuing cosmid, by digesting it with either BamHI, PstI, or StuI and shotgun cloning fragments into pBluescript SK+ (Stratagene). One fully rescuing subclone (pEM1) was recovered, which contained a 5.8-kb BamHI fragment. pEM1 was digested with XmaI and a 3.7-kb fragment (shown in Fig 5) extending from the insert XmaI site to the vector XmaI site was subcloned into pBluescript SK+ to create pEM12.
Construction and analysis of transgenic worms:
daf-11 transgenics were generated by injection (
For analysis of pheromone effects on GFP expression in daf-11 mutants, lin-15(n765); saEx238 or daf-11(sa195); saEx289 animals were grown on 50–80 µl of pheromone at 25° as described (
daf-21 transgenics were generated similarly except that the dominant rol-6(su1006dm) marker (pRF4, 200 ng/µl) was used as the marker. daf-21(p673) mutants have few progeny and injections in this background yielded few transformants, so we injected into the wild type with cosmids (5 ng/µl, individually or in pools) or plasmid subclones (15 or 20 ng/µl). We then crossed the heritable transgenic arrays into a daf-21(p673) background. We used a linked, visible marker to follow daf-21 in crosses because maternal rescue prohibited scoring the Daf-c phenotype in the progeny of daf-21/+ mothers. lon-3 daf-21/++ males were crossed to transgenic Rol hermaphrodites (saEx*), and Rol cross-progeny were allowed to self-fertilize. From plates that segregated Lons, we identified strains of the genotype lon-3 daf-21; saEx*. We found that the Lon phenotype is best scored in adults, when it largely suppresses the Rol phenotype. Therefore, in these strains there are Rol larvae, and most of the adults are Lon non-Rol. In all cases, some dauers segregated indicating that daf-21(p673) must be homozygous. To assay rescue, we determined the frequency of dauer formation in synchronous larval populations grown uncrowded at 25°. Strains with full rescue had only 10–30% dauers, strains with partial rescue had 40–50% dauers, and strains with no rescue had >80% dauers.
Rescue of dauer formation and dauer recovery by saEx289:
Plates were prepared and assayed as for 8-bromo-cGMP assays (above), except that 80 µl of M9, pheromone solution, or a mixture (0, 10, or 20 units of pheromone) were added. Eggs were laid at room temperature for 3 hr and then plates were shifted to 25°. Dauers and L3 or older animals were counted and all L3 or older animals were removed once or twice per day starting at 48 hr. At 96 hr the number of dauers remaining on the plate was counted. The percentage of dauer formation was calculated as 100 x (no. of dauers)/(total no. of worms) at 48 hr, and the percentage of recovery was calculated as 100 x (total no. of recovered animals)/(total no. of recovered animals + dauers left on plate at 96 hr). Dauers that crawled up the side of the plate were not counted, as they did not have a chance to recover. Each data point was repeated on at least 2 days and on four to nine plates.
Chemotaxis assays:
Assays of chemotaxis to NaCl were performed as described (
Mosaic analysis:
Four gravid adults of genotype dpy-17(e164) ncl-1(e1865) unc-36(e251); daf-11(sa195); ctDp11 were placed on a plate at 25°. Three days later, non-Unc dauer mosaics were identified either visually or (in most cases) by flooding the plate with 1% SDS and picking animals that were alive and thrashed (1% SDS kills non-dauers and Unc dauers did not thrash). Mosaics were inspected by Nomarski microscopy, and cells were assayed for the presence or absence of the duplication by observing the Ncl (enlarged nucleolus) phenotype of several amphid cells. It was sometimes not possible to unambiguously identify all amphid neurons in each animal. In most cases, ASI, ADL, ASK, ASE, ASH, AWC, AUA, and ASJ neurons on each side were analyzed, and often several other cells were also analyzed.
Identification of the daf-21(nr2081) deletion mutation:
A library of mutagenized worms was screened by PCR with nested primers (
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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daf-11 encodes a transmembrane guanylyl cyclase:
We cloned daf-11 using m597, a transposon-tagged allele isolated from a strain with active Tc1 transposition (kindly provided by P. Albert and D. Riddle). Southern blot analysis revealed a Tc1 insertion present in strains containing m597 and absent in recombinants and revertants not containing m597 (data not shown). We cloned genomic DNA flanking this Tc1 insertion by inverse PCR and used the flanking DNA to isolate phages from cDNA and genomic DNA libraries. The two daf-11 cDNAs isolated were both incomplete, but together permitted determination of the last 3 kb of the poly(A)-terminated daf-11 mRNA. We completed sequence at the 5' end of the mRNA from RT-PCR products. We also determined genomic sequence for most of the gene from subclones of genomic phage. The Genome Sequence Consortium completed the sequence of this region during the course of our work. Comparison of the genomic sequence to the cDNA sequence indicated that the daf-11 mRNA contains 17 exons and spans almost 8 kb of genomic DNA (Fig 1A). The mRNA contains one long open reading frame predicted to encode a 1077-amino-acid protein that is a member of the TM-GC family (Fig 1B and Fig C).
Transmembrane guanylyl cyclases catalyze the production of cyclic GMP from GTP and have been identified in animals from C. elegans to humans (reviewed in
We used BLAST searches (
Seven other genes were identified with higher similarity to soluble guanylyl cyclases (not shown). Since over 95% of all C. elegans genes are included in this analysis (J. SULSTON and R. WATERSTON, personal communication), these observations suggest that all C. elegans guanylyl cyclases fall into one of these two previously defined families. Distinguishing between the TM and soluble GCs was straightforward based both on degree of identity in the cyclase domain and other structural features of each class of proteins.
Sequence changes in daf-11 mutants:
daf-11 alleles vary in their phenotypic effects (
Two other daf-11 alleles confer unusual phenotypes. The daf-11(sa203) mutant was strongly Daf-c at 25°, but also formed 88% dauers at 15° (N = 234). sa203 has a nonsense mutation at Q904, just at the end of the conserved residues of the cyclase domain (Fig 1B and Fig C). We speculate that the sa203 mRNA expresses a truncated protein product that interferes with the function of another protein. Alternatively, a second undetected mutation may be responsible for the unusual phenotype. The daf-11(m84) mutant is also unusual: it is very strongly Daf-c at both 15° and 25°, the dauers recover much more quickly and efficiently than do other daf-11 mutants, and m84 adults have only weak defects in response to the volatile attractant isoamyl alcohol (
cGMP signaling is perturbed in daf-11 and daf-21 mutants:
To test the biological relevance of the DAF-11 sequence homology to TM-GCs and to test directly for a role for cGMP in chemosensation, we assayed the effect on dauer formation of the membrane permeant cGMP analogue 8-bromo-cGMP (Fig 2A). If the daf-11 mutant phenotype is due to a loss of guanylyl cyclase activity, supplementing with 8-bromo-cGMP might suppress the Daf-c phenotype. When grown at 25° with plentiful food without 8-bromo-cGMP, over 95% of daf-11(sa195) and daf-11(m87) animals formed dauers. In contrast, when 5 mM 8-bromo-cGMP was added to the growth medium, <1% formed dauers. Intermediate concentrations of the drug caused intermediate suppression of the Daf-c phenotype. 8-Bromo-cGMP similarly suppressed pheromone-induced dauer formation in the wild type in a dose-dependent manner, directly implicating cGMP in normal dauer formation.
We hypothesized that 8-bromo-cGMP suppressed dauer formation daf-11 mutants by substituting for the cGMP normally synthesized by the DAF-11 protein. This hypothesis predicts that 8-bromo-cGMP would not suppress mutations in genes that act downstream of or in parallel to daf-11. As predicted, we found that 8-bromo-cGMP did not prevent constitutive dauer formation in mutants for daf-1, daf-4, daf-7, daf-8, daf-14, daf-2, or daf-28 (Fig 2B). On the basis of gene interactions, it is thought that all of these genes act downstream of or in parallel to daf-1 (
daf-11 expression in sensory neurons controlling dauer formation and dauer recovery:
To identify cells in which daf-11 is expressed, we constructed a fusion gene containing 4.2 kb upstream of daf-11 and the entire genomic coding region with GFP (
The GFP fusion protein was reproducibly expressed in the amphid neuron classes ASJ and ASI, both implicated in regulating dauer formation. Constitutive dauer formation in daf-11 mutants is dependent on the function of the ASJ neurons (
In contrast to the ASJ neurons, the ASI neurons function together with another neuron class, ADF, to repress dauer formation in the absence of dauer-inducing conditions (
Under appropriate environmental conditions, dauers recover and resume their normal life cycle (
The dauer-inducing pheromone does not affect daf-11::gfp:
Exposing animals to the dauer-inducing pheromone strongly reduces expression of daf-7::gfp fusions, suggesting that the effects of the pheromone on this TGF-ß pathway are mediated at the level of daf-7 expression (
daf-11 expression in cells regulating chemotaxis and 2-nonanone avoidance:
daf-11::gfp expression was also seen in AWC and ASK neurons (Fig 3E). The AWC class is required for chemotaxis toward the volatile attractants isoamyl alcohol and benzaldehyde. Since daf-11 mutants are defective in response to these attractants, expression in AWC neurons was predicted. There is evidence that the ASK neurons play a minor role in chemotaxis to lysine and possibly other nonvolatile attractants (
A shorter daf-11 fusion construct, with the same promoter region plus only the first 136 codons of daf-11 (up to the fifth exon) fused to gfp, was expressed consistently in the AWC and AWB neurons and was expressed occasionally in the ASI and ASK neurons (Fig 3E). (Another transgenic array containing the same construct had consistent expression in ASI neurons, but the strain stopped expressing detectable GFP before quantititative data were collected.) Reinspection of strains carrying the full-length daf-11 fusion also revealed occasional expression in AWB cells. Presumably the differences in expression among fusions result from internal daf-11 regulatory sequences, but we have not further investigated this. The AWB neurons are required for avoidance of the repulsive compound 2-nonanone (
In addition to their other phenotypes, daf-11 mutants are defective in chemotaxis toward nonvolatile chemicals, a process mediated primarily by the ASE neurons. However, daf-11::gfp expression in ASE cells was only rarely seen in larvae bearing the longer fusion and was never seen (0/21 cells) in adults grown at 20° (the temperature at which chemotaxis assays are performed). This lack of expression could be because the fusion lacks regulatory sequences or because the effect of the daf-11 mutation on chemotaxis is not mediated through ASE. We found that the longer fusion could partially rescue the daf-11 chemotaxis defect (Fig 3E), supporting the idea of an ASE-independent effect of daf-11 on chemotaxis. However, daf-11::gfp expression is weak and variable in some cells and it remains possible that a low level of daf-11 expression in ASE neurons was not identified by our reporter assay.
daf-21 encodes an Hsp90:
Genetic analysis has indicated that daf-21 acts at the same step as daf-11 in the dauer formation pathway (
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Hsp90 proteins consist of two highly conserved domains connected by a charged linker region (reviewed in
In vertebrates, there are two Hsp90 cytoplasmic isoforms, 
and ß. In addition there is a third cytosolic relative, Trap-1/Hsp75, that lacks the charged region ( and Hsp90ß, respectively, and is clearly most closely related to these proteins (Fig 6). There are no other predicted C. elegans genes with this degree of similarity to Hsp90, indicating that there is a single C. elegans Hsp90 ortholog. However, there are predicted C. elegans genes orthologous to Trap-1/Hsp75 (R151.7) and GRP94/GP96 (T05E11.3; Fig 6). Hsp90 is a highly abundant protein whose expression is increased further under conditions of stress. Consistent with abundant expression, we and others have identified many daf-21/Hsp90 cDNAs (
daf-21(p673) is not a loss-of-function mutation:
It is known from genetic studies in yeast and Drosophila that Hsp90 is required for viability (BORKOVITCH et al. 1989;
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To determine the null phenotype of daf-21, we obtained a deletion mutation kindly provided by Axys Pharmaceuticals, NemaPharm Group. The mutant was isolated according to the method of
Though gain-of-function mutations most often display some degree of dominance, daf-21(p673) is recessive in the sense that daf-21(p673)/+ heterozygotes never form dauers. We tested for weak dominance by using a duplication to create animals with three copies of the daf-21 locus (see MATERIALS AND METHODS). While daf-21(p673)/+ mothers made 0% dauer progeny at 25°, daf-21(p673)/daf-21(p673)/+ mothers grown in parallel made approximately 7% dauer progeny (Table 1). We conclude that two copies of the daf-21(p673) allele probably result in a stronger mutant phenotype, consistent with the model that daf-21(p673) is a weak gain-of-function mutation.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A variety of shared mutant phenotypes suggest that the C. elegans genes daf-11 and daf-21 act at the same step to regulate chemosensory transduction in several of the exposed, ciliated amphid neurons. First, mutations in both genes cause defects in chemotaxis to the nonvolatile attractants Cl-, cAMP, and biotin (
C. elegans dauer formation provides a genetic model for TM-GC function in sensory transduction:
In addition to daf-11, several other genes are candidates for functioning in sensory transduction events controlling dauer formation. A model for this transduction process is provided by mammalian visual transduction, in which a TM-GC (RetGC) is known to function (reviewed in KOUTALOS and YAO 1993; subunits, gpa-2 and gpa-3, are implicated in dauer formation. Loss-of-function mutations in these genes cause a Daf-d (dauer formation defective) phenotype, and animals carrying activated gpa-2 or gpa-3 transgenes have a Daf-c phenotype (
In visual transduction, cGMP acts directly on an excitatory cyclic nucleotide gated ion channel ( and ß subunits of a cyclic nucleotide gated ion channel (
This genetic model system can be used to determine in vivo the functions of the three domains of the TM-GC molecule as well as the relationships among the components of a cGMP transduction pathway. Mutations already exist in many of these genes and gene disruptions could be generated in the phosphodiesterase and in other potential components discovered by sequence analysis (
The KHD of the transmembrane-guanylyl cyclase gene family:
We analyzed the sequence of 14 of the C. elegans TM-GC genes in more detail (all the genes with complete sequence at the time of the analysis). With the exception of DAF-11, all of our protein sequence analysis was done with gene products predicted by the Genefinder program (P. GREEN, personal communication), with minor amendments described in MATERIALS AND METHODS. Thirteen of the 14 analyzed TM-GCs have the standard TM-GC structure, a transmembrane domain separating a large extracellular domain from the intracellular kinase homology and cyclase domains. The one exception, GCY-11, is quite similar to other TM-GCs except that it appears to lack a predicted transmembrane domain. Since this difference might be due to a Genefinder error or gcy-11 might be a pseudogene, it was not further analyzed.
Though all previously reported TM-GCs have large extracellular domains, these domains are highly divergent from one another and in most cases a function has not been identified. Two features of the C. elegans genes nevertheless suggest that these extracellular domains consistently have important functions. First, 27 of the 28 C. elegans genes in the completed genome sequence are predicted to have a large extracellular domain of similar size. Second, a limited amount of homology is apparent in this domain between nearly all of the C. elegans TM-GCs and those of other organisms. This ranges as high as 28% identity between GCY-12 and the Drosophila DrGC-1 (
The sequencing of a large set of divergent TM-GCs affords important new insight into the conserved features of the kinase homology domain. An alignment of the KHD of the 13 analyzed C. elegans proteins and 5 from other organisms is shown in Fig 7, with annotations showing the blocks of conservation observed in bona fide protein kinases (
|
Some conserved regions of the guanylyl cyclase KHD are not particularly conserved in true protein kinases, especially C-terminal to block III and N-terminal to block I (Fig 7). These facts suggest that the KHD diverged from true protein kinases before the divergence of known TM-GCs from one another. Despite the differences between protein kinases and the KHD of TM-GCs, there is a clear general parallel between their most conserved stretches (Fig 7). These parallels in sequence conservation indicate that the TM-GC and protein kinase groups have been subject to related selective pressure. We speculate that this conservation reflects selection for a common protein binding domain rather than enzymatic kinase activity. One possible binding partner for this domain is DAF-21/Hsp-90.
DAF-21 is Hsp90:
The previously described phenotypic similarities between daf-11 mutants and the daf-21(p673) mutant suggested that the two gene products have closely related biochemical functions. Furthermore, our new finding that a cGMP analogue rescues both daf-11 and daf-21 mutants strengthens this hypothesis. On the basis of the fact that daf-11 encodes a TM-GC, we speculated that the DAF-21 protein might also regulate cGMP levels. Detailed knowledge of the role of cGMP in other transduction pathways (see above) suggested such candidates as a phosphodiesterase, a guanylyl cyclase activating protein (GCAP), a G protein, another GC, or a cyclic nucleotide gated channel. Instead, our molecular analysis indicates that daf-21 encodes the C. elegans ortholog of Hsp90, a chaperone protein. This very unexpected result suggests a previously undescribed role for Hsp90 in regulating one or more proteins involved in cGMP signaling.
Three lines of evidence support our conclusion that daf-21 encodes Hsp90. Initially transformation rescue with small genomic subclones suggested the Hsp90-encoding gene is daf-21. However, as shown in Fig 5, there is only a 612-bp segment separating the Hsp90 coding region and C47E8.4, a divergently transcribed gene that is homologous to human XAP-5 (
In other organisms, Hsp90 is reported to be one of the most highly expressed proteins (1–2% of total). In accord with this, we found that ~3% of C. elegans cDNA clones hybridized to an Hsp90 probe. Surprisingly, reduction of Hsp90 expression to 5% of normal was shown to have little effect in Saccharomyces cerevisiae (
A possible role of Hsp90 in chemosensory transduction:
Although Hsp90 function is not entirely understood, two related functions have been ascribed to this chaperone protein (reviewed in
The rules governing the specificity of Hsp90 interaction with target proteins seem elusive. Hsp90 is promiscuous in that it interacts with targets as varied as nuclear hormone receptors, kinases, tubulin, nitric oxide synthase, and telomerase (see
On the basis of the known functions of Hsp90, we hypothesize that DAF-21/Hsp90 associates with the DAF-11 TM-GC, perhaps to stabilize an inactive form of the cyclase. This model of physical association can be tested, for example, by coimmunoprecipitation of Hsp90 and DAF-11. An alternative explanation for the shared phenotypes of daf-11 and daf-21 mutants is that Hsp90 stabilizes an unidentified protein required for DAF-11-dependent chemosensory transduction. The daf-21(p673) E292K mutation causes relatively limited phenotypes (specific sensory defects and reduced fertility) and is not a null mutation. Therefore, the daf-21(p673) mutation may interfere relatively specifically with stabilization of a chemosensory transduction component (such as DAF-11) without seriously impairing other Hsp90 functions. However, a more detailed knowledge of how Hsp90 binds to its targets and cochaperones is needed to understand the consequences of this change.
| FOOTNOTES |
|---|
1 These authors contributed equally to this work. 
2 Present address: QIAGEN Inc., 28159 Avenue Stanford, Valencia, CA 91355.
3 Present address: Cambria Biosciences, LLC, 2 Preston Ct., Medford, MA 01730.
| ACKNOWLEDGMENTS |
|---|
We thank D. Riddle and P. Albert for the daf-11(m597) allele; S. Xu, J. Ahnn, G. Seydoux, and Andy Fire for GFP vectors; Jing Chen and Jill Spoerke (Axys Pharmaceuticals, NemaPharm Group) for generation of the daf-21(nr2081) deletion mutation; the Caenorhabditis Genetics Center for strains; the C. elegans Genome Sequencing Consortium for sequence data and for cosmids; R. BARSTEAD and A. FIRE for cDNA libraries; Axys Pharmaceuticals, NemaPharm Group and Cambria Biosciences LLC for resources and equipment; K. Iwasaki for RNA; J. Beavo for advice about 8-bromo-cGMP; S. Yu and D. Garbers for sharing data prior to publication; B. Sawin, C. Trent, H. R. Horvitz, C. Bargmann, M. Ailion, D. Weinshenker, P. Green, D. Pilgrim, K. Kemphues, M. Hengartner, and M. Gilbert for sharing unpublished information. We also thank M. Ailion for helpful comments on the manuscript and C. Bargmann and all members of our laboratory for fruitful conversation. D.A.B. and J.J.V. were supported by National Institutes of Health (NIH) Training Grant T32 GM-07735. E.M.L. was supported by a grant from the American Cancer Society. This work was also supported by NIH grant GM-48700 to J.H.T.
Manuscript received November 4, 1999; Accepted for publication January 10, 2000.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ALTSCHUL, S. F., W. GISH, W. MILLER, E. W. MYERS, and D. J. LIPMAN, 1990 Basic local alignment search tool. J. Mol. Biol. 215:403-410[Medline].
BAEHR, W., E. A. MORITA, R. J. SWANSON, and M. L. APPLEBURY, 1982 Characterization of bovine rod outer segment G-protein. J. Biol. Chem. 257:6452-6460
BARGMANN, C. I. and H. R. HORVITZ, 1991a Control of larval development by chemosensory neurons in Caenorhabditis elegans.. Science 251:1243-1246
BARGMANN, C. I. and H. R. HORVITZ, 1991b Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.. Neuron 7:729-742[Medline].
BARGMANN, C. I., E. HARTWIEG, and H. R. HORVITZ, 1993 Odorant-selective genes and neurons mediate olfaction in C. elegans.. Cell 74:515-527[Medline].
BARNES, W. M., 1994 PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91:2216-2220
BARSTEAD, R. J. and R. H. WATERSTON, 1989 The basal component of the nematode dense-body is vinculin. J. Biol. Chem. 264:10177-10185
BLOOM, L. and H. R. HORVITZ, 1997 The Caenorhabditis elegans gene unc-76 and its human homologs define a new gene family involved in axonal outgrowth and fasciculation. Proc. Natl. Acad. Sci. USA 94:3414-3419
BORKOVICH, K. A., F. W. FARRELLY, D. B. FINKELSTEIN, J. TAULIEN, and S. LINDQUIST, 1989 hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9:3919-3930
BRENNER, S., 1974 The genetics of Caenorhabditis elegans.. Genetics 77:71-94
BUCHNER, J., 1999 Hsp90 & Co.—a holding for folding. Trends Biochem. Sci. 24:136-141[Medline].
Genome sequence of the nematode C. elegans: a platform for investigating biology. (1998) Science 282:2012-2018
CAPLAN, A. J., 1999 Hsp90's secrets unfold: new insights from structural and functional studies. Trends Cell Biol. 9:262-268[Medline].
CASSADA, R. and R. L. RUSSELL, 1975 The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.. Dev. Biol. 46:326-342[Medline].
CHALFIE, M., Y. TU, G. EUSKIRCHEN, W. W. WARD, and D. C. PRASHER, 1994 Green fluorescent protein as a marker for gene expression. Science 263:802-805
CHEN, C. F., Y. CHEN, K. DAI, P. L. CHEN, and D. J. RILEY et al., 1996 A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock. Mol. Cell. Biol. 16:4691-4699[Abstract].
CHINKERS, M., 1994 Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor. Proc. Natl. Acad. Sci. USA 91:11075-11079
CHINKERS, M. and D. L. GARBERS, 1989 The protein kinase domain of the ANP receptor is required for signaling. Science 245:1392-1394
CHINKERS, M., D. L. GARBERS, M. S. CHANG, D. G. LOWE, and H. M. CHIN et al., 1989 A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338:78-83[Medline].
CHINKERS, M., S. SINGH, and D. L. GARBERS, 1991 Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system. J. Biol. Chem. 266:4088-4093
COBURN, C. M. and C. I. BARGMANN, 1996 A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans.. Neuron 17:695-706[Medline].
COBURN, C. M., I. MORI, Y. OHSHIMA, and C. I. BARGMANN, 1998 A cyclic nucleotide-gated channel inhibits sensory axon outgrowth in larval and adult Caenorhabditis elegans: a distinct pathway for maintenance of sensory axon structure. Development 125:249-258[Abstract].
CUTFORTH, T. and G. M. RUBIN, 1994 Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila. Cell 77:1027-1036[Medline].
DALLEY, B. K. and M. GOLOMB, 1992 Gene expression in the Caenorhabditis elegans dauer larva: developmental regulation of Hsp90 and other genes. Dev. Biol. 151:80-90[Medline].
DREWETT, J. G. and D. L. GARBERS, 1994 The family of guanylyl cyclase receptors and their ligands. Endocr. Rev. 15:135-162
DUSENBERY, D. B., 1974 Analysis of chemotaxis in the nematode Caenorhabditis elegans by countercurrent separation. J. Exp. Zool. 188:41-48[Medline].
ESTEVEZ, M., L. ATTISANO, J. L. WRANA, P. S. ALBERT, and J. MASSAGUE et al., 1993 The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development. Nature 365:644-649[Medline].
FESENKO, E. E., S. S. KOLESNIKOV, and A. L. LYUBARSKY, 1985 Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313:310-313[Medline].
FREEMAN, B. C. and R. I. MORIMOTO, 1996 The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J. 15:2969-2979[Medline].
FROHMAN, M. A., M. K. DUSH, and G. R. MARTIN, 1988 Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998-9002
GEORGI, L. L., P. S. ALBERT, and D. L. RIDDLE, 1990 daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase. Cell 61:635-645[Medline].
GOLDEN, J. W. and D. L. RIDDLE, 1982 A pheromone influences larval development in the nematode Caenorhabditis elegans.. Science 218:578-580
GOLDEN, J. W. and D. L. RIDDLE, 1984a The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature. Dev. Biol. 102:368-378[Medline].
GOLDEN, J. W. and D. L. RIDDLE, 1984b A Caenorhabditis elegans dauer-inducing pheromone and an antagonistic component of the food supply. J. Chem. Ecol. 10:1265-1280.
GOTTLIEB, S. and G. RUVKUN, 1994 daf-2, daf-16 and daf-23: genetically interacting genes controlling dauer formation in Caenorhabditis elegans.. Genetics 137:107-120[Abstract].
GOY, M. F., 1991 cGMP: the wayward child of the cyclic nucleotide family. Trends Neurosci. 14:293-299[Medline].
GRENERT, J. P., B. D. JOHNSON, and D. O. TOFT, 1999 The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J. Biol. Chem. 274:17525-17533
HANKS, S. K., A. M. QUINN, and T. HUNTER, 1988 The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42-52
HORVITZ, H. R., S. BRENNER, J. HODGKIN, and R. K. HERMAN, 1979 A uniform genetic nomenclature for the nematode Caenorhabditis elegans.. Mol. Gen. Genet. 175:129-133[Medline].
HUANG, L. S., P. TZOU, and P. W. STERNBERG, 1994 The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development. Mol. Biol. Cell 5:395-412[Abstract].
HURLEY, J. B., 1987 Molecular properties of the cGMP cascade of vertebrate photoreceptors. Annu. Rev. Physiol. 49:793-812[Medline].
INOUE, T. and J. H. THOMAS, 2000 Targets of TGF-ß signaling in C. elegans dauer formation. Dev. Biol. 217:192-204[Medline].
JAKOB, U., H. LILIE, I. MEYER, and J. BUCHNER, 1995 Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J. Biol. Chem. 270:7288-7294
KIMURA, Y., S. MATSUMOTO, and I. YAHARA, 1994 Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae.. Mol. Gen. Genet. 242:517-527[Medline].
KOMATSU, H., I. MORI, J. S. RHEE, N. AKAIKE, and Y. OHSHIMA, 1996 Mutations in a cyclic nucleotide-gated channel lead to abnormal thermosensation and chemosensation in C. elegans.. Neuron 17:707-718[Medline].
KOUTALOS, Y. and K. W. YAU, 1993 A rich complexity emerges in phototransduction. Curr. Opin. Neurobiol. 3:513-519[Medline].
LIU, D. W. C. and J. H. THOMAS, 1994 Regulation of a periodic motor program in C. elegans.. J. Neurosci. 14:1953-1962[Abstract].
LIU, L. X., J. M. SPOERKEM, E. L. MULLIGAN, J. CHEN, and B. REARDON et al., 1999 High-throughput isolation of Caenorhabditis elegans deletion mutants. Genome Res. 9:859-867
MALONE, E. A., T. INOUE, and J. H. THOMAS, 1996 Genetic analysis of the roles of daf-28 and age-1 in regulating Caenorhabditis elegans dauer formation. Genetics 143:1193-1205[Abstract].
MAYER, M. P. and B. BUKAU, 1999 Molecular chaperones: the busy life of Hsp90. Curr. Biol. 9:R322-R325[Medline].
MAZZARELLA, R., G. PENGUE, J. YOON, J. JONES, and D. SCHLESSINGER, 1997 Differential expression of XAP5, a candidate disease gene. Genomics 45:216-219[Medline].
MCNEIL, L., M. CHINKERS, and M. FORTE, 1995 Identification, characterization, and developmental regulation of a receptor guanylyl cyclase expressed during early stages of Drosophila development. J. Biol. Chem. 270:7189-7196
MELLO, C. C., J. M. KRAMER, D. STINCHCOMB, and V. AMBROS, 1991 Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10:3959-3970[Medline].
MENG, X., J. DEVIN, W. P. SULLIVAN, D. TOFT, and E. E. BAULIEU et al., 1996 Mutational analysis of Hsp90 alpha dimerization and subcellular localization: dimer disruption does not impede "in vivo" interaction with estrogen receptor. J. Cell Sci. 109:1677-1687[Abstract].
MIKI, N., J. M. BARABAN, J. J. KEIRNS, J. J. BOYCE, and M. W. BITENSKY, 1975 Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. J. Biol. Chem. 250:6320-6327
MILLER, L. M., J. D. PLENEFISCH, L. P. CASSON, and B. J. MEYER, 1988 xol-1: a gene that controls the male modes of sex determination and X chromosome dosage compensation in C. elegans.. Cell 55:167-183[Medline].
MORRIS, J. Z., H. A. TISSENBAUM, and G. RUVKUN, 1996 A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382:536-539[Medline].
NATHAN, D. F., M. H. VOS, and S. LINDQUIST, 1997 In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc. Natl. Acad. Sci. USA 94:12949-12956
NEMOTO, T. and N. SATO, 1998 Oligomeric forms of the 90-kDa heat shock protein. Biochem. J. 330:989-995.
NEMOTO, T., Y. OHARA-NEMOTO, M. OTA, T. TAKAGI, and K. YOKOYAMA, 1995 Mechanism of dimer formation of the 90-kDa heat-shock protein. Eur. J. Biochem. 233:1-8[Medline].
OBERMANN, W. M., H. SONDERMANN, A. A. RUSSO, N. P. PAVLETICH, and F. U. HARTL, 1998 In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J. Cell Biol. 143:901-910
PARKINSON, S. J., S. L. CARRITHERS, and S. A. WALDMAN, 1994 Opposing adenine nucleotide-dependent pathways regulate guanylyl cyclase C in rat intestine. J. Biol. Chem. 269:22683-22690
PICARD, D., B. KHURSHEED, M. J. GARABEDIAN, M. G. FORTIN, and S. LINDQUIST et al., 1990 Reduced levels of hsp90 compromise steroid receptor action in vivo.. Nature 348:166-168[Medline].
PRODROMOU, C., S. M. ROEM, P. W. PIPER, and L. H. PEARL, 1997a A molecular clamp in the crystal structure of the N-terminal domain of the yeast Hsp90 chaperone. Nat. Struct. Biol. 4:477-482[Medline].
PRODROMOU, C., S. M. ROE, R. O'BRIEN, J. E. LADBURY, and P. W. PIPER et al., 1997b Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90:65-75[Medline].
REN, P., C. S. LIM, R. JOHNSEN, P. S. ALBERT, and D. PILGRIM et al., 1996 Control of C. elegans larval development by neuronal expression of a TGF-ß homolog. Science 274:1389-1391
RIDDLE, D. L., and P. S. ALBERT, 1997 Genetic and environmental regulation of dauer larva development, pp. 739–768 in C. elegans II, edited by D. L. RIDDLE, T. BLUMENTHAL, J. MEYER and J. R. PRIESS. Cold Spring Harbor Laboratory Press, Plainview, NY.
RIDDLE, D. L., M. M. SWANSON, and P. S. ALBERT, 1981 Interacting genes in nematode dauer formation. Nature 270:668-671.
SCHACKWITZ, W. S., T. INOUE, and J. H. THOMAS, 1996 Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans.. Neuron 17:719-728[Medline].
SCHEIBEL, T. and J. BUCHNER, 1998 The Hsp90 complex—a super-chaperone machine as a novel drug target. Biochem. Pharmacol. 56:675-682[Medline].
SCHEIBEL, T., T. WEIKL, and J. BUCHNER, 1998 Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence. Proc. Natl. Acad. Sci. USA 95:1495-1499
SCHEIBEL, T., H. I. SIEGMUND, R. JAENICKE, P. GANZ, and H. LILIE et al., 1999 The charged region of Hsp90 modulates the function of the N-terminal domain. Proc. Natl. Acad. Sci. USA 96:1297-1302
SCHUMACHER, R. J., W. J. HANSEN, B. C. FREEMAN, E. ALNEMRI, and G. LITWACK et al., 1996 Cooperative action of Hsp70, Hsp90, and DnaJ proteins in protein renaturation. Biochemistry 35:14889-14898[Medline].
SINGH, S., D. G. LOWE, D. S. THORPE, H. RODRIGUEZ, and W. J. KUANG et al., 1988 Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases. Nature 334:708-712[Medline].
SONG, H. Y., J. D. DUNBAR, Y. X. ZHANG, D. GUO, and D. B. DONNER, 1996 Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor. J. Biol. Chem. 24:3574-3581.
STEBBINS, C. E., A. A. RUSSO, C. SCHNEIDER, N. ROSEN, and F. U. HARTL et al., 1997 Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89:239-250[Medline].
THOMAS, J. H., D. A. BIRNBY, and J. J. VOWELS, 1993 Evidence for parallel processing of sensory information controlling dauer formation in Caenorhabditis elegans.. Genetics 134:1105-1117[Abstract].
TROEMEL, E. R., J. H. CHOU, N. D. DWYER, H. A. COLBERT, and C. I. BARGMANN, 1995 Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans.. Cell 83:207-218[Medline].
TROEMEL, E. R., B. E. KIMMEL, and C. I. BARGMANN, 1997 Preprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.. Cell 91:161-169[Medline].
VAANDRAGER, A. B., E. VAN DER WIEL, and H. R. DE JONGE, 1993a Heat-stable enterotoxin activation of immunopurified guanylyl cyclase C: modulation by adenine nucleotides. J. Biol. Chem. 268:19598-19603
VAANDRAGER, A. B., S. SCHULZ, H. R. DE JONGE, and D. L. GARBERS, 1993b Guanylyl cyclase C is an N-linked glycoprotein receptor that accounts for multiple heat-stable enterotoxin-binding proteins in the intestine. J. Biol. Chem. 268:2174-2179
VAN DER KEYL, H., H. KIM, R. ESPEY, C. V. OKE, and M. K. EDWARDS, 1994 Caenorhabditis elegans sqt-3 mutants have mutations in the col-1 collagen gene. Dev. Dyn. 201:86-94[Medline].
VAN DER STRATEN, A., C. ROMMEL, B. DICKSON, and E. HAFEN, 1997 The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J. 16:1961-1969[Medline].
VOWELS, J. J., 1994 Genes mediating chemosensory responses in Caenorhabditis elegans. Ph.D. Thesis, University of Washington, Seattle.
VOWELS, J. J. and J. H. THOMAS, 1992 Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans.. Genetics 130:105-123[Abstract].
VOWELS, J. J. and J. H. THOMAS, 1994 Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans.. Genetics 138:303-316[Abstract].
WARD, S., 1973 Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants. Proc. Natl. Acad. Sci. USA 70:817-821
WATERSTON, R., C. MARTIN, M. CRAXTON, C. HUYNH, and A. COULSON et al., 1992 A survey of expressed genes in C. elegans.. Nat. Genet. 1:114-123[Medline].
WATERSTON, R. H., J. E. SULSTON and A. R. COULSON, 1997 The genome, pp. 23–45 in C. elegans II, edited by D. L. RIDDLE, T. BLUMENTHAL, B. J. MEYER and J. R. PRIESS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
WEICH, H., J. BUCHNER, R. ZIMMERMAN, and U. JAKOB, 1992 Hsp90 chaperones protein folding in vitro.. Nature 358:169-170[Medline].
WELCH, W. J. and J. R. FERAMISCO, 1982 Purification of the major mammalian heat shock proteins. J. Biol. Chem. 257:14949-14959
WHITE, J. G., E. SOUTHGATE, J. N. THOMSON, and S. BRENNER, 1986 The structure of the nervous system of the nematode Caenorhabditis elegans.. Philos. Trans. R. Soc. Lond. B 314:1-340.
WOOD, W. B. (Editor), 1988 The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
XU, Y. and S. LINDQUIST, 1993 Heat-shock protein hsp90 governs the activity of pp60v-src kinase. Proc. Natl. Acad. Sci. USA 90:7074-7078
XU, Y., M. A. SINGER, and S. LINDQUIST, 1999 Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90. Proc. Natl. Acad. Sci. USA 96:109-114
YOUNG, J. C., C. SCHNEIDER, and F. U. HARTL, 1997 In vitro evidence that hsp90 contains two independent chaperone sites. FEBS Lett. 418:139-143[Medline].
YU, S., L. AVERY, E. BAUDE, and D. L. GARBERS, 1997 Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci. USA 94:3384-3387
YUEN, P. S. T. and D. L. GARBERS, 1992 Guanylyl cyclase-linked receptors. Annu. Rev. Neurosci. 15:193-225[Medline].
ZWAAL, R. R., A. BROEKS, J. VAN MEURS, J. T. GROENEN, and R. H. PLASTERK, 1993 Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc. Natl. Acad. Sci. USA 90:7431-7435
ZWAAL, R. R., J. E. MENDEL, P. W. STERNBERG, and R. H. A. PLASTERK, 1997 Two neuronal G proteins are involved in chemosensation of the C. elegans dauer inducing pheromone. Genetics 145:715-727[Abstract].
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 A. M. van der Linden, S. Wiener, Y.-j. You, K. Kim, L. Avery, and P. Sengupta The EGL-4 PKG Acts With KIN-29 Salt-Inducible Kinase and Protein Kinase A to Regulate Chemoreceptor Gene Expression and Sensory Behaviors in Caenorhabditis elegans Genetics, November 1, 2008; 180(3): 1475 - 1491. [Abstract] [Full Text] [PDF]
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|
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 R. A. Butcher, J. R. Ragains, E. Kim, and J. Clardy A potent dauer pheromone component in Caenorhabditis elegans that acts synergistically with other components PNAS, September 23, 2008; 105(38): 14288 - 14292. [Abstract] [Full Text] [PDF]
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N. Fielenbach and A. Antebi C. elegans dauer formation and the molecular basis of plasticity Genes & Dev., August 15, 2008; 22(16): 2149 - 2165. [Abstract] [Full Text] [PDF]
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 |
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 K. L. Banfield, T. A. Gomez, W. Lee, S. Clarke, and P. L. Larsen Protein-Repair and Hormone-Signaling Pathways Specify Dauer and Adult Longevity and Dauer Development in Caenorhabditis elegans J. Gerontol. A Biol. Sci. Med. Sci., August 1, 2008; 63(8): 798 - 808. [Abstract] [Full Text] [PDF]
|
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 |
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 J. Ouellet, S. Li, and R. Roy Notch signalling is required for both dauer maintenance and recovery in C. elegans Development, August 1, 2008; 135(15): 2583 - 2592. [Abstract] [Full Text] [PDF]
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E. A. Hallem and P. W. Sternberg Acute carbon dioxide avoidance in Caenorhabditis elegans PNAS, June 10, 2008; 105(23): 8038 - 8043. [Abstract] [Full Text] [PDF]
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 A. Leskovar, H. Wegele, N. D. Werbeck, J. Buchner, and J. Reinstein The ATPase Cycle of the Mitochondrial Hsp90 Analog Trap1 J. Biol. Chem., April 25, 2008; 283(17): 11677 - 11688. [Abstract] [Full Text] [PDF]
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M. A. Alaamery and C. S. Hoffman Schizosaccharomyces pombe Hsp90/Git10 Is Required for Glucose/cAMP Signaling Genetics, April 1, 2008; 178(4): 1927 - 1936. [Abstract] [Full Text] [PDF]
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N. Wayne and D. N. Bolon Dimerization of Hsp90 Is Required for in Vivo Function: DESIGN AND ANALYSIS OF MONOMERS AND DIMERS J. Biol. Chem., November 30, 2007; 282(48): 35386 - 35395. [Abstract] [Full Text] [PDF]
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T. Inoue, M. Ailion, S. Poon, H. K. Kim, J. H. Thomas, and P. W. Sternberg Genetic Analysis of Dauer Formation in Caenorhabditis briggsae Genetics, October 1, 2007; 177(2): 809 - 818. [Abstract] [Full Text] [PDF]
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V. L. Jensen, P. S. Albert, and D. L. Riddle Caenorhabditis elegans SDF-9 Enhances Insulin/Insulin-Like Signaling Through Interaction With DAF-2 Genetics, September 1, 2007; 177(1): 661 - 666. [Abstract] [Full Text] [PDF]
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K. Yook and J. Hodgkin Mos1 Mutagenesis Reveals a Diversity of Mechanisms Affecting Response of Caenorhabditis elegans to the Bacterial Pathogen Microbacterium nematophilum Genetics, February 1, 2007; 175(2): 681 - 697. [Abstract] [Full Text] [PDF]
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 M. E. Baker Evolution of metamorphosis: role of environment on expression of mutant nuclear receptors and other signal-transduction proteins Integr. Comp. Biol., December 1, 2006; 46(6): 808 - 814. [Abstract] [Full Text] [PDF]
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H. Lans and G. Jansen Noncell- and Cell-Autonomous G-Protein-Signaling Converges With Ca2+/Mitogen-Activated Protein Kinase Signaling to Regulate str-2 Receptor Gene Expression in Caenorhabditis elegans Genetics, July 1, 2006; 173(3): 1287 - 1299. [Abstract] [Full Text] [PDF]
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F. Shinozaki, M. Minami, T. Chiba, M. Suzuki, K. Yoshimatsu, Y. Ichikawa, K. Terasawa, Y. Emori, K. Matsumoto, T. Kurosaki, et al. Depletion of Hsp90beta Induces Multiple Defects in B Cell Receptor Signaling J. Biol. Chem., June 16, 2006; 281(24): 16361 - 16369. [Abstract] [Full Text] [PDF]
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C. O. Ortiz, J. F. Etchberger, S. L. Posy, C. Frokjaer-Jensen, S. Lockery, B. Honig, and O. Hobert Searching for Neuronal Left/Right Asymmetry: Genomewide Analysis of Nematode Receptor-Type Guanylyl Cyclases Genetics, May 1, 2006; 173(1): 131 - 149. [Abstract] [Full Text] [PDF]
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H. Inada, H. Ito, J. Satterlee, P. Sengupta, K. Matsumoto, and I. Mori Identification of Guanylyl Cyclases That Function in Thermosensory Neurons of Caenorhabditis elegans Genetics, April 1, 2006; 172(4): 2239 - 2252. [Abstract] [Full Text] [PDF]
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J. Li, G. Brown, M. Ailion, S. Lee, and J. H. Thomas NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways Development, November 15, 2004; 131(22): 5741 - 5752. [Abstract] [Full Text] [PDF]
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 P. Syntichaki and N. Tavernarakis Genetic Models of Mechanotransduction: The Nematode Caenorhabditis elegans Physiol Rev, October 1, 2004; 84(4): 1097 - 1153. [Abstract] [Full Text] [PDF]
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C. V. Gunther and D. L. Riddle Alternative Polyadenylation Results in a Truncated daf-4 BMP Receptor That Antagonizes DAF-7-mediated Development in Caenorhabditis elegans J. Biol. Chem., September 17, 2004; 279(38): 39555 - 39564. [Abstract] [Full Text] [PDF]
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 Y. Nakano, Y. Nagamatsu, and Y. Ohshima cGMP and a germ-line signal control body size in C. elegans through cGMP-dependent protein kinase EGL-4 Genes Cells, September 1, 2004; 9(9): 773 - 779. [Abstract] [Full Text] [PDF]
|
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H. Lans, S. Rademakers, and G. Jansen A Network of Stimulatory and Inhibitory G{alpha}-Subunits Regulates Olfaction in Caenorhabditis elegans Genetics, August 1, 2004; 167(4): 1677 - 1687. [Abstract] [Full Text] [PDF]
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 K. K. Langlais, J. A. Stewart, and D. B. Morton Preliminary characterization of two atypical soluble guanylyl cyclases in the central and peripheral nervous system of Drosophila melanogaster J. Exp. Biol., June 1, 2004; 207(13): 2323 - 2338. [Abstract] [Full Text] [PDF]
|
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O. Hainzl, H. Wegele, K. Richter, and J. Buchner Cns1 Is an Activator of the Ssa1 ATPase Activity J. Biol. Chem., May 28, 2004; 279(22): 23267 - 23273. [Abstract] [Full Text] [PDF]
|
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B. Gerisch and A. Antebi Hormonal signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer diapause in response to environmental cues Development, April 15, 2004; 131(8): 1765 - 1776. [Abstract] [Full Text] [PDF]
|
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H. Y. Mak and G. Ruvkun Intercellular signaling of reproductive development by the C. elegans DAF-9 cytochrome P450 Development, April 15, 2004; 131(8): 1777 - 1786. [Abstract] [Full Text] [PDF]
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T. Cai, T. Fukushige, A. L. Notkins, and M. Krause Insulinoma-Associated Protein IA-2, a Vesicle Transmembrane Protein, Genetically Interacts with UNC-31/CAPS and Affects Neurosecretion in Caenorhabditis elegans J. Neurosci., March 24, 2004; 24(12): 3115 - 3124. [Abstract] [Full Text] [PDF]
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X. Huang, J. A. Powell-Coffman, and Y. Jin The AHR-1 aryl hydrocarbon receptor and its co-factor the AHA-1 aryl hydrocarbon receptor nuclear translocator specify GABAergic neuron cell fate in C. elegans Development, February 15, 2004; 131(4): 819 - 828. [Abstract] [Full Text] [PDF]
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 J. F. Morley and R. I. Morimoto Regulation of Longevity in Caenorhabditis elegans by Heat Shock Factor and Molecular Chaperones Mol. Biol. Cell, February 1, 2004; 15(2): 657 - 664. [Abstract] [Full Text] [PDF]
|
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M. Ailion and J. H. Thomas Isolation and Characterization of High-Temperature-Induced Dauer Formation Mutants in Caenorhabditis elegans Genetics, September 1, 2003; 165(1): 127 - 144. [Abstract] [Full Text] [PDF]
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K. Ohkura, N. Suzuki, T. Ishihara, and I. Katsura SDF-9, a protein tyrosine phosphatase-like molecule, regulates the L3/dauer developmental decision through hormonal signaling in C. elegans Development, July 15, 2003; 130(14): 3237 - 3248. [Abstract] [Full Text] [PDF]
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J. Wang and S. K. Kim Global analysis of dauer gene expression in Caenorhabditis elegans Development, April 15, 2003; 130(8): 1621 - 1634. [Abstract] [Full Text] [PDF]
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O. Uchida, H. Nakano, M. Koga, and Y. Ohshima The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons Development, April 1, 2003; 130(7): 1215 - 1224. [Abstract] [Full Text] [PDF]
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W. Li, S. G. Kennedy, and G. Ruvkun daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 signaling pathway Genes & Dev., April 1, 2003; 17(7): 844 - 858. [Abstract] [Full Text] [PDF]
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B. van Swinderen, L. B. Metz, L. D. Shebester, and C. M. Crowder A Caenorhabditis elegans Pheromone Antagonizes Volatile Anesthetic Action Through a Go-Coupled Pathway Genetics, May 1, 2002; 161(1): 109 - 119. [Abstract] [Full Text] [PDF]
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 A. H. Hutagalung, M. L. Landsverk, M. G. Price, and H. F. Epstein The UCS family of myosin chaperones J. Cell Sci., January 11, 2002; 115(21): 3983 - 3990. [Abstract] [Full Text] [PDF]
|
|
|
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K. Jia, P. S. Albert, and D. L. Riddle DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity Development, January 1, 2002; 129(1): 221 - 231. [Abstract] [Full Text] [PDF]
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J.-C. Labbé, J. Burgess, L. A. Rokeach, and S. Hekimi ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation PNAS, November 2, 2000; (2000) 230284297. [Abstract] [Full Text]
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T. Inoue and J. H. Thomas Suppressors of Transforming Growth Factor-{beta} Pathway Mutants in the Caenorhabditis elegans Dauer Formation Pathway Genetics, November 1, 2000; 156(3): 1035 - 1046. [Abstract] [Full Text]
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M. Ailion and J. H. Thomas Dauer Formation Induced by High Temperatures in Caenorhabditis elegans Genetics, November 1, 2000; 156(3): 1047 - 1067. [Abstract] [Full Text]
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S. A. Daniels, M. Ailion, J. H. Thomas, and P. Sengupta egl-4 Acts Through a Transforming Growth Factor-{beta}/SMAD Pathway in Caenorhabditis elegans to Regulate Multiple Neuronal Circuits in Response to Sensory Cues Genetics, September 1, 2000; 156(1): 123 - 141. [Abstract] [Full Text]
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 T. M. Rogalski, G. P. Mullen, M. M. Gilbert, B. D. Williams, and D. G. Moerman The UNC-112 Gene in Caenorhabditis elegans Encodes a Novel Component of Cell-Matrix Adhesion Structures Required for Integrin Localization in the Muscle Cell Membrane J. Cell Biol., July 11, 2000; 150(1): 253 - 264. [Abstract] [Full Text] [PDF]
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R. Kumar, N. Grammatikakis, and M. Chinkers Regulation of the Atrial Natriuretic Peptide Receptor by Heat Shock Protein 90 Complexes J. Biol. Chem., March 30, 2001; 276(14): 11371 - 11375. [Abstract] [Full Text] [PDF]
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J.-C. Labbe, J. Burgess, L. A. Rokeach, and S. Hekimi ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation PNAS, November 21, 2000; 97(24): 13233 - 13238. [Abstract] [Full Text] [PDF]
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 S. J.M. Jones, D. L. Riddle, A. T. Pouzyrev, V. E. Velculescu, L. Hillier, S. R. Eddy, S. L. Stricklin, D. L. Baillie, R. Waterston, and M. A. Marra Changes in Gene Expression Associated with Developmental Arrest and Longevity in Caenorhabditis elegans Genome Res., August 1, 2001; 11(8): 1346 - 1352. [Abstract] [Full Text] [PDF]
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