We have investigated the neurotransmitters used to control egg-laying in C. elegans. Previous studies suggested that 5-HT released by the HSN motor neurons stimulates egg laying, and that tricyclic antidepressants potentiate egg laying by blocking reuptake of 5-HT by the HSN neurons. We report studies of the wild type and a mutant that lacks detectable 5-HT that suggest 5-HT is not required for egglaying. Furthermore, we find that ACh is required for egg laying in response to 5-HT, suggesting that 5-HT is not sufficient to activate egg laying. The dominant egl-2(n693) mutation, which causes animals to lay eggs in response to tricyclics but not 5-HT, also conflicts with the model for egg laying. Experiments in which the HSN neurons or 5-HT are removed from egl-2 animals indicate that the action of tricyclics cannot be explained by a block of 5-HT reuptake. We find that D, family dopamine antagonists can also induce egg laying in egl-2(n693) mutants, and that dopamine inhibits egg laying in the wild type. These results suggest that dominant egl-2 mutations activate an inhibitory dopaminergic pathway that can be blocked by tricyclics and D, antagonists.We also find that these drugs stimulate egg laying in mutants lacking 5-HT or the HSN neurons, consistent with a target on the egg-laying muscles. In contrast to tricyclics, fluoxetine and other selective 5-HT reuptake inhibitors appear to be specific for 5-HT reuptake in C. e/egans egg laying.[Key words: C. elegans, dopamine, egg laying, fluoxetine, genetics, HSN neurons, imipramine, pharmacology, 5HT, tricyclic antidepressant] Genetic analysis can test the validity in viva of mechanisms of synaptic transmission and can identify genes that mediate synaptic transmission. Many of the neurotransmitters found in mammals are also found in the genetic model organism Caenorhab-

The glucocorticoid receptor (GR) activates transcription in certain glucocorticoid response element (GRE) contexts, and represses or displays no activity in others. We isolated point mutations in one GRE, plfG, at which GR activated transcription under conditions in which the wild-type element was inactive or conferred repression, implying that GREs may carry signals that are interpreted by bound receptors. Consistent with this notion, we identified a mutant rat GR, K461A, which activated transcription in all GRE contexts tested, implying that this residue is important in interpretation of GRE signals. In a yeast screen of 60,000 GR mutants for strong activation from plfG, all 13 mutants isolated contained substitutions at K461. This lysine residue is highly conserved in the zinc-binding region (ZBR) of the intracellular receptor (IR) superfamily; when it was mutated in MR and RARP, the resulting receptors similarly activated transcription at response elements that their wild-type counterparts repressed or were inactive. We suggest that IR response elements serve in part as signaling components, and that a critical lysine residue serves as an allosteric "lock" that restricts IRs to inactive or repressing configurations except in response element contexts that signal their conversion to transcriptional activators. Therefore, mutation of this residue produces altered receptors that activate in many or all response element contexts.[Key Words: Glucocorticoid receptor; transcriptional repression; DNA-binding domain; context-dependent activity; intracellular receptor] Received January 1, 1996; accepted in revised form March 27, 1996.Regulation of transcription typically commences with the specific binding of a regulatory protein to a response element close to the target promoter; the regulator also interacts with other regulatory factors, with the transcription machinery andlor chromatin components, therefore modulating the rate of transcription initiation (for review, see Sheldon and Reinberg 1995;Hori and Carey 1994; Tjian and Maniatis 1994). Numerous regulators have been shown to activate transcription in certain cell types and at certain promoters, and to repress in other settings (Meyer et al. 1980;Keleher et al. 1988;Pedersen et al. 1991). Therefore, it is apparent that the precise activity of a regulator can be determined largely by its context-cell type, promoter, response element, and extracellular signals. The mechanisms by which regulators sense and interpret their contexts and thereby display distinct activities in different settings remain to be determined.The glucocorticoid receptor (GR) is a member of the intracellular receptor (IR) superfamily, which encompasses a large group of transcriptional regulators (Tsai Present addresses: 2Department

We have isolated two independent mutations in the DNA-binding domain of the rat glucocorticoid receptor, P493R and $459A, that implicate DNA binding in the control of attached transcriptional activation domains, either that of the receptor itself or of VP16. The mutants are capable of activating transcription normally, but unlike wild-type receptors, they interfere with particular transcriptional activators in yeast and mammalian cells, and inhibit growth when overexpressed in yeast. The mutant residues reside at positions within the three-dimensional structure of the receptor that could, in principle, transduce structural changes from the DNA-binding surface of the receptor to other functional domains. These findings, together with the salt dependence of specific and nonspecific DNA binding by these receptors, suggest that specific DNA acts as an aUosteric effector that directs the functional interaction of the receptor with targets of transcriptional activation and that the P493R and $459A mutants mimic the allosteric effect of specific DNA, allowing the receptor to interact with regulatory targets even in the absence of specific DNA binding. The interaction of DNA and site-specific DNA-binding proteins was classically considered as a "rigid body" association determined by the interaction of two preexisting and stable interfaces. However, it is now apparent that many DNA-protein interactions are accompanied by structural changes. DNA structure may be altered by interaction with proteins, and site-specific DNA-binding proteins may undergo conformational changes upon binding to DNA (Spolar and Record 1994). The dynamic nature of protein-DNA interactions raises the possibility that the final structure of the protein might depend upon the particular sequence to which it binds, that is, specific DNA sequences might act as allosteric effectors to determine the catalytic or regulatory activities of the protein. Restriction endonucleases, for example, are DNA-binding proteins whose catalytic functions are inactive on nonspecific DNA and active at specific sites. Allosteric effects of DNA sequence on endonuclease activity have been proposed for EcoRI (Heitman 1992) and demonstrated for NaeI, NarI, BspMI, HpaII, and SacII (Oller et al. 1991).Might the activities of transcriptional regulatory proteins similarly be governed or influenced by the DNA sequences to which they bind? The magnitude of activation or repression by a given factor commonly varies ~These authors contributed equally to this work.with "DNA context." In general, it has not been determined whether such context effects reflect the contributions of other transcriptional regulators bound nearby, or instead indicate a direct effect of DNA sequence on the disposition of a regulator. However, the correlation between site-specific conformations and site-specific activities of the transcription factors PRTF/MCM1 (Tan and Richmond 1990) and the p50 subunit of NF-KB (Fujita et al. 1992;Hay and Nicholson 1993) supports the idea that DNA-induced conformational chan...

Interstitial cells of Cajal (ICC) pace gastrointestinal phasic activity and transmit nerve activity. Gap junctions may couple these cells to smooth muscle, but no functional evidence exists. The objective of this study was to use uncouplers of gap junctions, 18 alpha-glycyrrhetenic acid and its water-soluble analogue carbenoxolone, to evaluate if gap junctions function in pacing and neurotransmission. After inhibition of nerve function with tetrodotoxin (TTX) and N(G)-nitro-L-arginine (L-NOARG), ionomycin- or carbachol-initiated regular phasic activities of circular muscle strips from canine colon and ileum. In some cases, the primary ICC network responsible for pacing was removed. The effects of inhibitors of gap junction conductance (10(-5)-10(-4) mol L(-1)) on frequencies and amplitudes of contraction were compared to appropriate time controls. Lower oesophageal sphincter (LOS) relaxations to nerve stimulation were studied before and after inhibition of gap junction functions. No major changes in LOS relaxations or frequencies of colonic or ileal contractions occurred, but amplitudes of contractions decreased from these agents. Similar results were obtained when the myenteric plexus-ICC network of ileum was removed. Regular phasic activity was not obtained after removal of the colon submuscular plexus ICC. These findings suggest that mechanisms other than gap junctions couple gut pacemaking activity and nerve transmission.