Harris BR et al. (1997) International C. elegans Meeting "Sequencing C. elegans YACs"

Woessner JP, Consortium TCeGS, Harris BR

[International C. elegans Meeting, 1997]

In order to obtain the maximum information in the shortest time, C. elegans sequencing was initiated within the gene-rich regions of the genome, which are estimated to contain over 80% of genes. Encompassing 60 MB, this region also had the advantage of being well represented in cosmid contigs. Sequencing of this 60 Mb region is now essentially complete. Of the remaining sequence approximately 20 Mb is only represented by YAC clones. Thus it was imperative that new sequencing strategies were developed so that YACs could be utilised as part of the initial sequencing substrate. During 1996, production scale methodologies were established in both labs to allow a shotgun sequencing approach to be efficiently applied to YAC clones. The major difficulties are handling the small amount of DNA that can be recovered from yeast hosts and elimination of yeast contamination. However, by double gel purification of YAC isolates, and the use of yeast 'window' strains where necessary, yeast sequence contamination can be reduced below 10%. pUC is better than M13 for providing adequate libraries from small amounts of DNA. (The Sanger Centre uses exclusively pUC, while the Genome Sequencing Center uses a mixture of pUC and M13). The pUC clones have the additional advantages of bridging inverted repeats (which M13 cannot handle) and of providing an extensive check on assembly from forward and reverse read pairs. Our strategy for sequencing the final 40 Mb will be to shotgun sequence YAC clones in conjunction with any underlying cosmid clones. This strategy is proceeding well and at the time of writing more than 40 YAC clones are being sequenced. We estimate that approximately 150 YACs will need to be shotgunned to complete the genomic sequence.

Carroll AS et al. (1996) East Coast Worm Meeting "Stabilization of the DNA-bound SKN-1 basic region by an adjacent a-helical domain"

Carroll AS, Wagner G, Ellenberger TE, Blackwell TK, Gunther CS, Gilbert DE, Liu XY, Cheung JW

[East Coast Worm Meeting, 1996]

During the earliest stages of embryogenesis in C. elegans, maternally-expressed SKN-1 protein directs cells to adopt fates that lead to formation of the pharynx, the gut, and part of the body-wall muscle. At the C-terminus of SKN-1 is a basic region (BR) like those of the basic-"leucine zipper" (bZIP) proteins. However, SKN-1 lacks a ZIP segment, which in bZIP proteins is essential for DNA binding because it mediates their dimerization, and may stabilize and orient the BR as an a-helix in the major groove. In contrast, SKN-1 binds to DNA as a monomer with nanomolar affinity, to a specific sequence consisting of a bZIP half-site adjacent to an AT-rich region. At the C-terminus of its 85 amino acid DNA-binding domain (the Skn domain) is its BR, and at the N-terminus is a sequence related to the flexible N-terminal "arm" (HD arm) with which homeodomain proteins bind to DNA in the minor groove. We have undertaken a series of mutagenesis, spectroscopic, and biochemical "footprinting" experiments to investigate how SKN-1 binds to DNA at high affinity as a monomer. Our findings indicate that the Skn domain inserts the BR into the major groove of the bZIP half-site, and places the HD arm in the minor groove of the AT-rich sequence. The intervening residues, which are distinct from homeodomains and ZIP segments, are both necessary and sufficient to promote sequence-specific DNA binding by the SKN-1 BR. When the Skn domain is not bound to DNA, the BR is unstructured, and the intervening residues are predominantly a-helical. However, surprisingly, this helical region is not rigidly ordered in structure, but exists in a partially-ordered state termed a "molten globule." Upon DNA binding, the BR forms an a-helix, the HD arm binds in the minor groove, and the intervening residues form a helical structure which is stably ordered, and which itself stabilizes the bound BR helix. Stabilization of the BR does not appear to involve intramolecular interactions with its side chains, but is impaired by disruption of residues in the helix immediately N-terminal to it. These findings demonstrate that the BR is not "trapped" in the major groove by side chain interactions, and indicate instead that its stability requires direct extension of a continuous a-helix from the adjacent helical structure. The SKN-1 DNA binding domain is thus unique in two respects: it represents the only known example of monomeric binding by a BR, and is the only such domain which is capable of sequence-specific binding but does not have at least some portion that is rigidly ordered when not bound to DNA. These results further imply that stability deriving from helix extension is likely an important factor in DNA binding by other BR-containing proteins.

Toshihiro Sassa et al. (2006) East Asia C. elegans Meeting "The F09G2.9 gene encoding a novel transcription factor positively regulates neuron-specific gene expression"

Shin-ichi Harada, Toshihiro Sassa, Ryuji Hosono, Takashi Kano

[East Asia C. elegans Meeting, 2006]

cis-acting element in promoter and its binding factors play central role in gene expression. We characterized the promoter region of the unc-18 gene, one of neuronal genes, by deletion analyses and identified the minimal regulatory 19-base element between -341 to -359. By screening the expression library, we identified factors binding to the 19-base element. One of these, the F09G2.9 gene product, is a member of the high mobility group(HMG) family of proteins.<Br> F09G2.9(RNAi) treatment caused reduction of the unc-18 gene expression, suggesting that F09G2.9 positively regulates the unc-18 gene expression. C. elegans shows chemotaxis to NaCl but avoids NaCl when they are kept under the starved condition with NaCl. However, F09G2.9(RNAi) animals showed chemotaxis toward NaCl irrespective of the conditioning. A F09G2.9::GFP promoter fusion with 0.7 kb of upstream sequence was expressed in head neurons, ventral nerve cord and tail neurons, whose expression pattern was similar to that of UNC-18::GFP. A recombinant C-terminal fragment in the F09G2.9 product containing HMG domain could bind to the 19-base element in unc-18 promoter. Its in vivo binding to the 19-base element was also confirmed by the chromatin immunoprecipitation(ChIP) assay.<Br> To identify genes other than unc-18 regulated by F09G2.9, we searched with the ChIP method. We cloned 14 genes containing binding site within 2 kb upstream from the ATG start codon. The F09G2.9 gene had the binding site in its promoter. Of genes detected, we especially noticed Y53C10A.5 gene encoding ubiquitin-conjugating enzyme and kallmann syndrome related gene, K03D10.1. Because both genes are implicated in neural functions. We are characterizing expression patterns of these genes by constructing GFP fusion proteins and observing phenotypes of these gene mutants. <Br> To further explore the role of the F09G2.9 gene in neural function, we are trying to isolate the deletion mutant with a PCR-based screening approach.

M Tijsterman et al. (1999) International C. elegans Meeting "A genetic approach to identify the molecular target of SB-204269 in Caenorhabditis elegans"

M Tijsterman, GP Livi, J Ahringer, RH Plasterk, S Rastan, H Herdon

[International C. elegans Meeting, 1999]

The compound SB-204269 shows potent anticonvulsant activity in a range of animal seizure models without any neurological or cardiovasular side-effects 1 . A stereospecific binding site has been identified in the rat brain 2 but the molecular nature of this unique binding site remains to be elucidated. We have started to use the strength of C. elegans genetics in an attempt to identify the molecular target of SB-204269. Exposure of mothers to SB-204269 (micromolar range) results in embryonic lethality in this organism whereas its enantiomer SB-204268 does not. A C. elegans strain with a general increased sensitivity to drugs ( pgp-1; pgp-3; mrp-1 ) are also more sensitive to SB-204269 than Bristol N2 and the obtained level of killing allows a large-scale selection for mutants that have gained resistance specifically to the compound. Besides the use of EMS as a source to introduce mutations into the C. elegans genome we crossed a mutator allele ( mut-7 ) into this genetic background to allow a more easy detection of the causal mutation via a transposon display experiment. Both EMS- and "mutator"-screens are currently ongoing. 1 Upton et al. (1997) Br. J. Pharmacol., 121, 1679-1686 2 Herdon et al. (1997) Br. J. Pharmacol. 121, 1687-1691

Christopher Ortiz et al. (2007) International Worm Meeting "Laterality in the gustatory system of C. elegans is mediated by specific guanylyl cyclases."

Oliver Hobert, Heidi Smith, Christopher Ortiz

[International Worm Meeting, 2007]

Previous work by our group has identified a regulatory network of transcription factors and miRNAs that regulates asymmetric gene expression in the morphologically symmetric gustatory neuron pair ASEL/R. We recently undertook an analysis of the receptor-type guanylyl cyclase (gcy) gene family and found that 12/27 are expressed in ASE, 9 of which display lateralized expression. The preponderance of gcy gene expression in ASE suggests that these proteins play a role in the chemosensory function of these neurons, and that their asymmetric expression is required to both increase the chemosensory repertoire and the ability of these neurons to discriminate between multiple gustatory cues. In order to identify more ASE-sensed gustatory cues that could be potential ligands for GCY receptor proteins, we tested the chemotaxis response of wild-type and che-1(ot66) worms to a panel of inorganic salts. Several ions were found to elicit attraction in wild-type worms but not in che-1, indicating that ASEL/R are required for the detection of a wider panel of taste cues than had been previously known. To determine if any of these salts are asymmetrically detected, we conducted chemotaxis assays with lsy-6(ot71), a 2-ASER mutant, and lsy-18(ot192), a 2-ASEL mutant. To date we have identified several ions that are asymmetrically detected, including Li+ (ASEL), I- (ASER), and Br- (ASER). We plan to conduct laser ablation experiments to further confirm these results. The chemotaxis analysis of gcy mutants suggests that several of the proteins encoded by this family play a role in salt detection. gcy-22(tm2364) shows broad chemotaxis defects to several salts, including Cl-, I-, Br-, K+, and Mg++. Additionally, gcy-6(tm1449) and gcy-4(tm1653) animals are defective for Mg++ and I- sensation, respectively. Rescue experiments for these mutant phenotypes are currently underway. We are also studying the extent to which the worm gustatory system is able to discriminate between different taste cues. To address this, we challenged wild-type animals to locate the peak of a gradient of several different salts, presented in a saturating background concentration of other salts. Results of these assays will be presented. Overall this work indicates that functional lateralization in the nematode gustatory system is an adaptation that has allowed the worm to make the most use of its limited number of sensory neurons to mediate the critical sensory modality of chemosensation. Furthermore, it provides strong evidence that receptor-type GCYs play an important role in the detection of salt taste cues.

James B Endres et al. (2002) West Coast Worm Meeting "A Wnt gradient may guide HSN migration."

Nancy Hawkins, Gian Garriga, Shaun Cordes, James B Endres

[West Coast Worm Meeting, 2002]

The migration of neuronal cell bodies is a critical feature of neural development in all animal phyla. To investigate the molecular mechanisms of this process, we study the migrations of the hermaphrodite specific neurons (HSNs) of Caenorhabditis elegans. The HSNs are born in the tail of the comma-stage embryo and migrate anteriorly to the midbody. Later, during postembryonic development, the HSNs extend axons and synapse onto vulval muscles to control egg laying. Migrations of both the HSNs (Desai et al. 1988) and the Q neuroblasts (Harris et al. 1996) require the function of the egl-20 gene, which encodes a Wnt homolog. Wnts are secreted glycoproteins with diverse functions in many developmental processes. Desai et al. (1988) demonstrated that loss of egl-20 leads to posterior displacement (undermigration) of the HSNs, and we have observed that EGL-20 overexpression leads to anterior displacement (overmigration). Endogenous EGL-20 is expressed in the embryonic tail during the stage when the HSNs are migrating, raising the possibility that an EGL-20 gradient guides HSN migration. In contrast, EGL-20 plays a permissive role in Q cell migration by controlling mab-5 expression (Maloof et al. 1999). Supporting the possibility that an EGL-20 gradient is essential for HSN migration, global overexpression of EGL-20 from a heat-shock promoter leads to both overmigration and undermigration of the HSNs.

Hapiak, Vera et al. (2009) International Worm Meeting "Multiple monoamine receptors modulate nose touch in C. elegans."

Schafer, William, Chatzigeorgiou, Marios, Komuniecki, Richard, Wragg, Rachel, Hapiak, Vera, Harris, Gareth

[International Worm Meeting, 2009]

The ASH sensory neuron in C. elegans is polymodal, responding to both noxious (high osmolarity/volatile repellents) and mechanical (nose touch) stimuli to initiate backward locomotion. Examination of one ASH-mediated locomotory behavior, avoidance to octanol, has revealed that modulation of the ASH neural circuit is complex and involves multiple monoamines and distinct amine receptors (Wragg et al., 2007; Harris et al., 2009). In the present study, we have identified the monoamine receptors involved in another 5-HT stimulated ASH-mediated aversive response, nose touch. Like responses to octanol, multiple 5-HT receptors also have been identified in the food/5-HT sensitization of nose touch. For example, the expression of ser-5 in the ASHs appears to be essential for 5-HT dependent increases in aversive responses to nose touch. In contrast, both TA and OA inhibit nose touch and two monoamine receptors (F14D12.6 and SER-3) appear to be involved in octopaminergic inhibition. These receptors are currently being localized in the ASH locomotory circuit. Using the calcium indicator cameleon, we analyzed ASH Ca2+ responses to nose touch in ser-5 null animals. ASH Ca2+ transients were independent of exogenous 5-HT and more robust in ser-5 null animals than in wild-type or ser-4; mod-1; ser-7 ser-1 quadruple null animals that presumably only express ser-5. Since we predict that SER-5 stimulates neurotransmitter release at the ASH synapse, the increased Ca2+ signaling in the ASH soma was surprising. We are currently exploring whether SER-5 signaling has other effects on the ASH or whether SER-5 also modulates the release of other ligands, perhaps peptides, that inhibit ASH Ca2+ dynamics.

Gallotta I et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

D'Orta M, Vitale M, Castro S, Esposito G, Hilliard M A, Di Schiavi E, Gallotta I

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

An important aspect in the study of gene function is the possibility to dissect the role exerted by a gene of interest selectively in specific cells or groups of cells. In C. elegans the available approaches are time consuming and largely restricted to non-essential genes for which mutants are available. We have developed a simple reverse genetics approach to reduce the function of specific genes in chosen C. elegans neurons (Esposito et al., 2007). By expressing sense and antisense RNA corresponding to a gene of interest under cell-specific promoters, we obtain an efficient, heritable and cell autonomous knock-down of the targeted gene function in the specific neurons. This strategy has now been used by several other labs, with a variety of genes and on different neurons (Harris et al. 2009; Yamada et al. 2009; Jose et al. 2009; Ezak et al. 2010). To optimize the efficiency of this approach we have tested the influence of genetic backgrounds that affect RNA interference. We show that in a rrf-3(pk1426) background silencing is more efficient. Although this result indicates the involvement of RNAi pathways in the silencing mechanism we have shown that there is no spreading of the silencing effect at least among amphidial chemosensory neurons and within the motorneurons of the ventral cord. We are now targeting chemosensory and mechanosensory neurons and two classes of motorneurons (GABAergic and HSN) using a variety of different promoters. We are testing the effect of silencing several essential genes on the development, morphology and survival of these neurons as well as on the behavior that they control.

Chung, Kevin et al. (2015) International Worm Meeting "The neuronal basis of bacterial food odor preference."

Kan, Emily, Glater, Elizabeth, Haynes, Lillian, Ota, Ryan, Chambers, Melissa, Chung, Kevin

[International Worm Meeting, 2015]

Caenorhabditis elegans uses chemosensation to distinguish among various species of bacteria, their major food source 1-3. Although the neurons required for the detection of specific food-odors have been well-defined, less is known about the sensory circuits underlying the discrimination among the mixtures of odors released by different kinds of bacteria. We are examining the neural machinery underlying bacterial preference among a diverse set of bacterial species. Specifically, we are testing the food preferences of C. elegans for bacteria found in their natural habitats (kindly provided by Marie-Anne Felix, Institute of Biology of Ecole Normale Superieure, Paris, France). We have found that C. elegans prefers the odors of most species tested over E. coli. We have identified that the olfactory neuron AWC is involved in many preferences. We also find that some bacterial choices involve multiple sensory neurons with opposing roles. For example, in one choice in which wild-type animals prefer Providencia sp. (JUb39) over E. coli, the AWC neuron appears to be involved in increased preference for Providencia and the AWA neuron in increased preference for E. coli. In the future we will extend our analysis to additional bacterial species to determine the diversity of the underlying neuronal mechanisms.1. Harris, G. et al. Dissecting the Signaling Mechanisms Underlying Recognition and Preference of Food Odors. J. Neurosci. 34, 9389-9403 (2014).2. Ha, H. et al. Functional Organization of a Neural Network for Aversive Olfactory Learning in Caenorhabditis elegans. Neuron 68, 1173-1186 (2010).3. Shtonda, B. B. & Avery, L. Dietary choice behavior in Caenorhabditis elegans. J. Exp. Biol. 209, 89-102 (2006).

Yi-Tzang Tsai et al. (2010) East Asia Worm Meeting "DAO-5 insufficiency leads to Histone H4 hypoacetylation on the rDNA promoter and oogenesis defect"

Yi-Tzang Tsai, Chi-Chang Lee, Chih-Wei Kao, Szecheng J. Lo, Huey-Jen Lai

[East Asia Worm Meeting, 2010]

Nucleolus is a nuclear compartment where ribosomal RNAs (rRNAs) are synthesized and processed, as well as assembled with ribosomal proteins to form the pre-ribosome. Nucleolar phosphorylated protein 140 (Nopp140), one of main nucleolar proteins, has been reported to be associated with rRNA gene (rDNA) promoter and able to maintain its proper nucleolar organization for efficient rRNA production in HeLa cell. Nevertheless, the significance of Nopp140 in organism is poorly documented. In C. elegans, we first identify that DAO-5 is the homolog of Nopp140 and thus designated DAO-5 as CeNopp140. A homozygous deletion mutant of dao-5 gene appears retarded growth, and gonad deformation accompanied with severely decreased brood size, as well as extra germ cell death during oogenesis. To elucidate the implication of CeNopp140 in rRNA synthesis, the RT-PCR analysis manifested that the null-mutant worms possessed a less quantity of pre-rRNAs as compared to that of wild type. RNA in situ run-on assay has also been established in embryonic cell culture of C. elegans. The results showed that cells from a CeNopp140-deletion mutant incorporated Br-UTP into newly-synthesized rRNA with a reduced efficiency, indicating the importance of CeNopp140 on rDNA transcription. The chromatin-immunoprecipitation assay with anti-acetyl-histone H4 revealed a significantly decreased amount of active rDNA repeats in CeNopp140 deficient embryos. In conclusion, the Nopp140 homolog of C. elegans may play an important role on facilitating rDNA transcription, as in consistence with its mammalian counterparts, which conveys the hypothesis that CeNopp140 renders gonad maturation favorably. (Y.-T. Tsai and C.-C. Lee contributed equally to this work) (Supported by CMRP grants from the Chang Gung Memorial Hospital)