Hill, Andrew et al. (2015) International Worm Meeting "ADAM17/TACE homolog ADM-4 mediates stress-induced quiescence in C. elegans."

Gal, Danna, Van Buskirk, Cheryl, Hill, Andrew

[International Worm Meeting, 2015]

In C. elegans, intercellular signaling via the epidermal growth factor receptor (EGFR) specifies various cell fates, and recently our lab has discovered a role for EGFR signaling in a stress-induced sleep-like state. In response to various cellular stressors, C. elegans will enter a period of behavioral quiescence that is dependent on the sole EGFR, LET-23, and sole EGFR ligand, LIN-3. This finding contributes to a growing body of evidence that EGFR signaling contributes to the regulation of sleep states across species. EGFR ligands are produced as membrane-bound precursors that, upon proteolytic processing, release soluble EGF domain to bind target receptors on distant cells. EGFR receptor activation within a single neuron, ALA, is sufficient to induce sleep; however, the relevant source of LIN-3 and the protease responsible for its 'shedding' in stress-induced sleep remains unknown.In an RNAi screen for defective sleep, we identified the adm-4 metalloproteinase. We confirmed that adm-4(ok265) mutants are defective in sleep following exposure to several stressors, including ethanol, hyperosmotic solution, and pore-forming toxin, and are impaired for survival compared to wild type after extreme heat shock. We find that adm-4 mutants show wild-type viability, vulval development, and p11/p12 differentiation, suggesting that ADM-4 does not participate in other EGF signaling events.GFP reporter analysis indicates that adm-4 is predominantly expressed in pharyngeal tissue, where it overlaps with lin-3 expression. The function of pharyngeal lin-3 has yet to be ascertained. To assess whether pharyngeal adm-4/lin-3 mediates stress-induced quiescence, we are expressing adm-4 specifically in the pharyngeal tissue of otherwise adm-4(-) animals. The lin-3 gene is known to undergo alternative splicing, and we wish to determine whether a specific isoform is processed by ADM-4. We are conducting shedding assays in mammalian cell culture, expressing the C. elegans adm-4 cDNA and HA-tagged cDNAs of various lin-3 isoforms to determine if one isoform is preferentially or exclusively shed by ADM-4.

Kemphues KJ et al. (1991) International C. elegans Meeting "par MUTANT EMBRYOS SHOW ALTERED EARLY EXPRESSION OF CeMyoD."

Dickens S, Kemphues KJ, Kirby CM, MacCaulay A, Shakes DC

[International C. elegans Meeting, 1991]

Mutations in the par genes lead to a maternal effect lethal phenotype characterized by alterations in cleavage pattern and timing, aberrant partitioning of P granules, and extensive alterations in cell fates. We have proposed that the par genes encode components of a maternal system for asymmetric intracellular localization in early embryos and that proper functioning of this system is required for specification of early embryonic cell fates. Here we provide evidence that the par gene functions are necessary to establish proper lineage- specific transcription patterns in early cleavage stage embryos. Recently, the CeMyoD gene was shown to be an early marker of lineage specific transcription, with its earliest expression limited to the 2- 4 cells of the MS lineages at the 24-28 cell stages of embryogenesis. ( Krause et al., Cell 63,907). Andy Fire and Mike Krause anticipated our interest in this marker and generously gave us prepublication access to their data and a transgenic strain carrying a CeMyoD- -gal transcription reporter construct. We have constructed strains that segregate par homozygous worms carrying the reporter gene and are presently assaying -gal activity in the mutant embryos. Conclusions thus far: 1 ) All pars thus far examined (par-2,3,5) show ectopic expression of the CeMyoD reporter at the 32-cell stage (equivalent chronologically to wild type 24-28 cell stage). 2) No expression is seen prior to the 32-cell stage. 3) At least part of the early ectopic expression in par-3 and par-5 is transient; while many 32-and 64-cell-stage embryos are -gal positive in all nuclei, all embryos past the 64-cell stage have significant numbers of -gal negative nuclei. 4) ectopic 13-gal expression in par-2 embryos is limited to the posterior half of most embryos.

J Hujova et al. (2004) European Worm Meeting "CAENORHABDITIS ELEGANS HAS ONLY ONE alpha-GALACTOSIDASE AND alpha-N-ACETYLGALACTOSAMINIDASE ORTHOLOG"

J Ledvinova, M Kostrouchova, J Sikora, D Holanova, B Asfaw, M Hrebicek, J Hujova, H Poupetova, R Dobrovolny

[European Worm Meeting, 2004]

Human alpha-galactosidase A (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGA) are true paralogs sharing a common ancestral gene. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders - Fabry (alpha-GAL deficiency) and Schindler (alpha-NAGA deficiency) diseases. BlastP searches for orthologs alphaof humanalpha alpha-GAL and alpha-NAGA (both from melibiase protein family) revealed a single C.elegans gene (CeGAL/NAGA) with significant homology to both human genes. We elucidated gene organization of CeGAL/NAGA by cDNA sequencing including 3 and 5 UTR. We performed phylogenetic analyses and homology modeling of CeGAL/NAGA based on 3D structure of chicken alpha-NAGA. alpha-GAL and alpha-NAGA activity measurements in C.elegans mixed culture homogenate revealed presence of both enzyme activities. However, the alpha-GAL activity was completely inhibited by N-acetylgalactosamine - alpha-NAGA inhibitor. To study the expression of CeGAL/NAGA in C.elegans, we created C-terminal GFP fusion of the whole CeGAL/NAGA ORF under the control of its natural promoter. Presence of CeGAL/NAGA :GFP transgene was confirmed at the level of gDNA, cDNA and protein. No GFP signal was observed under standard laboratory conditions. We have employed CON A and NH4Cl treatment in order to raise lysosomal pH. Both methods resulted in distinct GFP signal in membrane bound compartment of coelomocytes. Immunofluorescence detection of GFP fusion protein showed transgene expression in muscle and in some cases in gut and vesicular compartments of ceolomocytes suggesting differential accessibility of tissues to alkalization treatment. RNA interference assays directed against the CeGAL/NAGA employing combination of microinjection and feeding approaches have not revealed any abnormal phenotype. We found single C.elegans gene with high degree of similarity to both human alpha-GAL and alpha-NAGA. The hypothesis of single ortholog in C.elegans was further supported by phylogenetic, homology modeling and biochemical analyses. We determined tissue expression and examined the effect of RNAi with the aim to evaluate C.elegans as a model organism for Schindler and Fabry diseases. This work was supported by grant 303/02/1324 from the Grant Agency of the Czech Republic

Gallegos ME et al. (1995) International C. elegans Meeting "IN SEARCH OF A TRANS-ACTING TRANSLATIONAL REGULATOR FOR THE fem-3 3'UTR."

Kimble JE, Gallegos ME

[International C. elegans Meeting, 1995]

fem-3 specifies male fates in C. elegans. In hermaphrodites, gain-of-function (gf) fem-3 mutants fail to switch from spermatogenesis to oogenesis. The molecular defects found in fem-3(gf) mutations affect a 5 bp region within the fem-3 3'UTR and several lines of evidence suggest that this regulation acts at the level of translation. Our working hypothesis is that the fem-3 3'UTR possesses a negative cis-acting translational control element, which may include the 5 bp region altered in fem-3 (gf) mutants. We have developed a reporter assay to examine this 3'UTR-mediated translational control of fem-3. Two types of constructs have been made: lacZ::fem-3(+) 3'UTR has lacZ coding sequences fused to the wildtype fem-3 3'UTR, while lacZ::fem-3(gf) 3'UTR has lacZ coding sequences fused to a fem-3 (gf) 3'UTR. Both reporter transgenes are under the control of a heat shock promoter. Hermaphrodites containing the lacZ::fem-3(+) 3'UTR transgene show undetectable levels of B-gal activity (assayed by x-gal staining) whereas worms that contain the lacZ::fem-3(gf) 3'UTR show high levels of B-gal activity. Despite these dramatic differences in B-gal activity, both transgenic strains possess equal levels of reporter mRNA, as determined by RNAse protection. These results strongly support the idea that the fem-3 3'UTR carries a cis-acting translational regulatory element. Furthermore, this fem-3 3'UTR regulation is not limited to the germline: expression of the reporter transgene is observed in multiple somatic tissues. Somatic regulation of the fem-3 3'UTR was predicted due to somatic affects observed in tra-1(gf);fem-3(gf) double mutants (Schedl and Kimble, 1988). We are now using this assay to identify genes that when mutant, activate the lacZ::fem-3 (+) 3'UTR reporter transgene. Our results will be reported at the meeting.

L Saccomanno et al. (1999) International C. elegans Meeting "The role of the STAR family in tra-2 3'UTR translational regulation"

EB Goodwin, L Saccomanno

[International C. elegans Meeting, 1999]

The C. elegans sex determination gene, tra-2 , encodes a large transmembrane protein that is required for female sexual development. Repression of tra-2 translation is necessary for male cell fate development in both somatic and germ line tissues. Two elements located in the 3'UTR of the tra-2 mRNA, called TGEs, are required for tra-2 translational regulation. GLD-1 is a member of the STAR family of RNA binding proteins and regulates tra-2 translation in the hermaphrodite germline to permit spermatogenesis. However, translational regulation of tra-2 also occurs in the soma. The STAR family contains an approximately 200 amino acid domain (STAR domain) that is highly conserved, raising the possibility that another STAR protein represses tra-2 in the soma. The C. elegans sequencing project has identified at least five predicted open reading frames that have strong similarity to GLD-1. We have preliminary evidence that suggests at least one of these five STAR proteins, T21G5.5, can regulate translation through the tra-2 3'UTR. T21G5.5 is 67% identical to GLD-1 at the amino acid level within the STAR domain. I have performed RNA-mediated interference (RNAi) of T21G5.5 in transgenic C. elegans animals that carry a reporter transgene encoding the lacZ gene with the wild type tra-2 3'UTR. The F1 progeny following RNAi of T21G5.5 had an increased level of b -gal staining in the soma compared to wild type transgenic animals. 22% of the RNAi animals had b -gal activity in intestinal cells compared to 12.5% of non-injected. In addition, animals that stained for b -gal after injection with T21G5.5 also showed an increase in the number of b -gal stained intestinal cells (2-8 cells) compared to wild type (1-2 cells). Disruption of T21G5.5 activity also resulted in animals with interesting morphological phenotypes. F1 animals had a dumpy phenotype and an altered tail morphology. In addition, the gonads of these animals were sexually transformed. These preliminary data suggest that T21G5.5 is a likely candidate for a TGE-dependent translational repressor. However, these results do not eliminate the possibility that other STAR proteins are also 3'UTR-dependent translational regulators. We are currently testing this possibility.

Gallegos ME et al. (1996) Midwest Worm Meeting "mog-1 THROUGH mog-6 ARE REQUIRED FOR THE 3'UTR-MEDIATED POST-TRANSCRIPTIONAL CONTROL OF fem-3."

Gallegos ME, Kimble JE

[Midwest Worm Meeting, 1996]

fem-3 specifies male fates in C. elegans. In hermaphrodites, fem-3 gain-of-function (gf) alleles fail to switch from spermatogenesis to oogenesis. The molecular defects found in fem-3(gf) mutations affect a 5 bp region within the fem-3 3'UTR. Our working hypothesis is that the fem-3 3'UTR possesses a negative acting post-transcriptional control element, which requires the 5 bp region altered in fem-3 (gf) mutants. mog-1 through mog-6 mutant hermaphrodites, also fail to switch from spermatogenesis to oogenesis. Unlike the fem-3 gf alleles, however, the mog mutant alleles are recessive. In addition, epistasis analysis places mog-1 through mog-6 upstream of fem-3 in a negative regulatory pathway. This is consistent with a model where the mog genes act as post-transcriptional regulators of fem-3. To determine if the mog genes play a role in the post-transcriptional regulation of fem-3 I have assayed the expression of a heterologous reporter transgene, lacZ::fem-3(+)3'UTR, in the 6 mog mutant backgrounds. This transgene consists of lacZ coding sequences fused to the wildtype fem-3 3'UTR under the control of a heat shock promoter. Normally, lacZ::fem-3(+)3'UTR expresses B-gal to low levels (assayed by x-gal staining). However, lacZ::fem-3(+)3'UTR shows significantly higher levels of B-gal expression when placed in any of the six mog mutant backgrounds. These results strongly support the idea that the fem-3 3'UTR carries a negative-acting post-transcriptional control element and that the mog genes are required for proper negative regulation of fem-3 via its 3'UTR.

Fire A et al. (1995) International C. elegans Meeting "EFFICIENT GENE-EXPRESSION: REQUIRES PUNCTUATION!"

Xu SQ, Seydoux GC, Fire A

[International C. elegans Meeting, 1995]

We find that transcripts produced by lacZ fusions and some other heterologous transgenes are predominantly nuclear, frequently in 1-2 spots which may be sites of transcription (Development 120, 2823; P.Okkema, unpublished). In contrast, most endogenous transcripts accumulate in the cytoplasm. One potential difference between lacZ transcripts and endogenous messages is the long intron-less lacZ coding region. Consistent with this idea, we found several years ago (Genetics 135, 385) that a single intron just 5' to lacZ stimulates expression from some promoters. To generate reporter genes which were closer in character to endogenous nematode genes, we have produced vectors with 1-12 artificial introns within the lacZ coding region. For weak promoters, these increase beta-gal expression (by 1-2 orders of magnitude). Stimulation has likewise been seen by making an intron-rich gfp coding region. We've used in situ hybridization to examine RNAs produced from intron-poor and intron-rich unc-54::lacZ fusions. Constructs without introns (or with just one intron) show predominantly nuclear RNA localization, while intronrich constructs accumulate mRNA at high levels in the cytoplasm. The intron-rich reporters provide the potential for more sensitive vectors to assay gene expression. Promoterless and basal-promoter (enhancer-trap) vectors have been constructed with multiple introns in the reporter coding region. These are indeed more sensitive for detecting weak promoter & enhancer activities (higher staining frequency and greater intensity). Zygotic beta-gal expression in very early embryos is dramatically improved, while the germline still refuses to express beta-gal. As the signal increases, background also increases. Injected alone, promoterless intron-rich vectors give some background staining in gut and pharynx. Introns also appear to enhance sporadic background staining in the enhancer assay vectors.

Jan E et al. (1996) Midwest Worm Meeting "THE TRANSLATIONAL REGULATION VIA THE tra-2 3'UTR MAY BE CONSERVED ACROSS SPECIES"

Goodwin EB, Jan E

[Midwest Worm Meeting, 1996]

tra-2 promotes female cell fates in C. elegans. tra-2 loss of function mutants transform XX animals into males whereas tra-2 gain of function (gf) mutants cause XX animals to develop as females (they do not make sperm). tra-2(gf) mutants map to two 28 nt direct repeat elements (DRE) separated by a 4 nt spacer located in the 3' untranslated region (3'UTR). Previous evidence suggests that the DREs regulate tra-2 activity by inhibiting the translation of tra-2. The DREs are a binding site for a factor (DRF) that is present in crude worm extracts, and which we hypothesize is a repressor of translation. Our working model is that the binding of DRF to the DREs represses translation of tra-2. To investigate the biological importance of the translational regulation of tra-2, we asked whether this 3'UTR control is evolutionarily conserved by assaying for the presence of the regulation in the nematode C. briggsae and mammalian tissue culture cells. Previously, we developed an assay in which the translation of a b-gal reporter transgene is controlled by the presence of the wild-type tra-2 3'UTR (both DREs are present) but not by mutant 3'UTRs (where one or both of the DREs have been deleted) (ref 1). In this assay, little b-gal activity is detected in the soma of animals carrying a transgene with the wild-type 3'UTR, but strong b-gal activity is detected in animals that carry a transgene with a mutant 3'UTR. RNAse protection analysis demonstrated that the different transgenes produce similar amounts of RNA, indicating that the differences in b-gal activity is likely due to translational control. We used the identical transgenes to address whether the 3'UTR control mechanism is present in C. briggsae. C. briggsae is estimated to have diverged from C. elegans approximately 50 million years ago. Similar to C. elegans, the wild-type tra-2 3'UTR was able to repress the translation of b-gal but mutant 3'UTRs could not, suggesting that the molecular mechanism that mediates the translation of tra-2 is present in C. briggsae. To ask whether the 3'UTR regulation is conserved in mammals, we tested whether the wild-type C. elegans 3'UTR can repress translation of a reporter transgene in Hela cells. In these experiments a reporter construct that contained the SV40 promoter and the luciferase gene was used. Preliminary experiments show that the presence of the wild-type 3'UTR can repress luciferase expression, but a mutant 3'UTR can not. Therefore, the fact that the C. elegans 3'UTR can control translation of reporter transgenes both in C. briggsae and Hela cells suggests that the molecular mechanism that governs the 3'UTR regulation is a fundamental process that is conserved across species. 1. Goodwin E.B., Okkema P.G., Evans T.C., and Kimble J. (1993) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans. Cell 75: 329-339

Papp A et al. (1997) International C. elegans Meeting "WORM ZAPPING II: C. ELEGANS ELECTROPORATION?"

Lee JH, Farris K, Papp A

[International C. elegans Meeting, 1997]

C. elegans can be transformed efficiently with exogenous genetic material using the technique of microinjection (Mello et. al, 1991). Although microinjection has become straightforward through the use of devices like our $425 microINJECTOR System, the procedure relies on a microscope system and accessories costing between $10,000 and $30,000, as well as some dexterity and knowledge of worm anatomy. We previously reported evidence suggesting that exogenous DNA can be introduced into worms using The Cloning Gun, an inexpensive, hand-held, cordless, rechargeable electroporation device designed for high-efficiency transformation of Stationary-Phase E. coli, developed here at Tritech Research. The data were preliminary, and B-gal staining suggested gene expression in some patches of somatic cells in worms that could have either been P0 or F1; however, data were not available as to whether this transformation was heritable. Here we present data that electroporation can produce heritable transformation: F3 worms staining completely for B-gal. Attempts to replicate this result with rol-6 have produced only non-heritable rollers and dumpies. Because electroporation seems to be a low frequency transformation procedure, we are investigating other genetic markers that may allow for the recovery of individual transformants from a large population of non-transformed worms. Experiments with dye show that electroporation can readily permeablize both adults and eggs. Mello, C. et al. (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J. 10(12):3959-70.

Broeks A et al. (1991) International C. elegans Meeting "DEVELOPMENT OF AN ENHANCER-TRAP USING THE Tc1 TRANSPOSABLE ELEMENT."

Plasterk RHA, Broeks A

[International C. elegans Meeting, 1991]

We want to develop an enhancer-trap system, by creating a mobile lacZ gene that can be used to probe the C. elegans genome for tissue specific expression. Recombinant Tc1 elements are used for this purpose. We constructed trangenic lines containing extrachbomosomal arrays consisting of a mixture of two plasmids; one with the rol 6 marker gene and one with a modified Tc1 transposable element. The modified Tc1 element contains, between its ends, the -galactosidase encoding gene lacZ under the control oLthe enhancerless glp-1 promoter (A.Fire et.al. 1990). Mutator positive roi6 /Tc1-1acZ transgenic lines were stained with X-gal for iacZ expression. No expression of the extrachromosomal -gal gene was found. To trap enhancers large amounts of transgenic nematodes were examined for iacZ expression: blue staining would suggest insertion of the introduced Tc1 element near an enhancer. The frequency of Tc1 transposition from the extrachromosomal DNA into the genome was low. so this strategy is not useful for trapping enhancers. Therefore we are now using an improved method: a second marker gene. the fem-1 gene. is cloned into the modified Tc1 element. The Tc1-1acZ/fem-1 construct is coinjected with an unc-22 antisense construct into a mut/fem-1 ts strain. In the progeny of transgenic lines Owe are currently searching for wild type segregants which are self fertile at 25 C. These are expected to contain a Tc1 element that has jumped into the chromosome; among these lacZ expression can be investigated.