Mingi Hong et al. (2001) International C. elegans Meeting "Ethanol Sensitivity Genes in the nematode Caenorhabditis elegans"

Min Sung Choi, Mingi Hong, Junho Lee

[International C. elegans Meeting, 2001]

Alcohol has many effects in higher organisms, especially in the nervous system. The mechanisms and sites of action of ethanol are not fully understood. In the hope of understanding the mechanisms of ethanol, we identified genes that control sensitivity to ethanol and anesthetics in the invertebrate system Caenorhabditis elegans. 15 mutations that confer resistance to the anesthetic effect of ethanol were identified, either by chemical mutagenesis or transposon insertion mutagenesis. We have cloned two of the genes responsible for the resistant phenotypes by mapping and rescue, and by transposon tagging. One of these, jud-1, encodes a protein similar to contactin. The cDNA analysis of the jud-1 gene shows that it is alternatively spliced. The shorter transcript has a single Ig domain, while the longer transcript has three Ig domains and one fibronectin type III domain. The functions of the two alternatively spliced transcripts are not known at this moment. In the mammalian system, contactin is known to be a cell adhesion/recognition molecule in the nervous system and has roles in the formation of axon projections in the developing nervous system. In C. elegans, jud-1 is also expressed in some neurons as shown by GFP expression analysis. jud-5, an ethanol sensitivity gene cloned by transposon tagging, encodes a novel protein. From the expression pattern of its GFP fusion protein, it could be seen that jud-5 is expressed in most muscles including pharyngeal, body wall, tail and vulva muscles. Further studies of these mutants are under way.

M Hong et al. (1999) International C. elegans Meeting "Isolation and Characterization of Genes Responsible for Ethanol Sensitivity in Caenorhabditis elegans"

M Hong, J Lee

[International C. elegans Meeting, 1999]

The mechanisms and sites of action of volatile anesthetics and ethanol are not fully understood. In the hope of understanding the mechanisms of general anesthetics, we identified genes that control sensitivity to ethanol and anesthetics in the invertebrate system Caenorhabditis elegans. Because the response of C. elegans to ethanol and anesthetics is similar to that of mammals, elucidating the action mechanism of ethanol and anesthetics in the nematode will help understanding their mechanisms in higher organisms including humans. As a start, we wanted to identify genes responsible for ethanol sensitivity by isolating mutations that confer ethanol resistance. About ten thousand wild-type nematode individuals (N2) were subject to mutagenesis, and the offspring were screened in the second generation for resistance to ethanol exposure. We found 9 independent mutations so far. ys9, the first ethanol-resistant mutant isolated in our screen, was not different from N2 in shape and behavior, but showed resistance to 7 vol% ethanol treatment. ys10, a second mutation, was also wild type in shape and behavior. ys11 animals displayed other phenotypes as well as ethanol resistance. The ys11 animals are medium dumpy (Dpy). In contrast to the results of ethanol test, ys9, ys10, and ys11 were more sensitive than N2 in halothane test. Interestingly, none of the ys9, ys10, and ys11 mutant animals survived after freezing and thawing, indicating that there may be alteration in membrane properties. From the results of thin layer chromatography and gas chromatography, composition of membrane phospholipids and steroids of ys9 was different from that of N2. Other mutations did survive freezing and thawing. We are currently performing genetic and molecular characterization of the genes for those mutations, and will present up-to-date results.

Hong Y et al. (1995) International C. elegans Meeting "RECONSTITUTION OF lin-4/lin-14 TRANSLATIONAL REGULATION IN VIVO AND IN VITRO"

Ambros VR, Hong Y

[International C. elegans Meeting, 1995]

lin-4 encodes an abundant 22 nt RNA (lin-4S) that is complementary to an element in the 3'UTR of lin-14 mRNA. lin-4S represses the translation of lin-14 mRNA, probably by base-pairing with this lin-14 3'UTR sequence. We are attempting to reconstruct this translational control in yeast and in an in vitro translation system. In yeast S. cerevisiae, our most difficult task has been to efficiently express lin-4S RNA. A yeast U6 promoter was used to drive expression of lin-4L (altered by a 2 nt change to create a A-box consensus in the transcribed sequence), but the lin-4L transcript, the presumed precursor to lin-4S, was poorly processed to lin-4S. We also attempted to express lin-4S in yeast as an intron of a suppressor tRNA (sup70). The lin-4S intron was spliced out (as judged by the suppressor tRNA phenotype displayed by the cells), but we did not detect inhibition of a lacZ-lin-14 3'UTR reporter gene. We are currently exploring other methods for expressing high levels of lin-4S in yeast. In a reticulocyte lysate translation system (nuclease treated), a luciferase (lux) mRNA was used as a reporter for lin-4 activity. Synthetic lin-4S RNA at up to 10,000x molar ratio to lux mRNA shows only a moderate non-specific repression of luciferase activity that occurs independently of the presence of lin-14 3'UTR sequences. We are currently testing whether alternative assay conditions may permit a specific translational repression by lin-4. Surprisingly, an in vitro transcribed 50nt RNA (Y26) that mimics the predicted structure of lin-4/lin-14 hybrid stimulated the translation of lux reporter RNAs. The Y26 RNA may titrate some general translation repressor(s) in the in vitro translation system.

Hong, Ray et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The Characterization of Insect Pheromone Insensitive Mutants in Pristionchus pacificus"

Cinkornpumin, Jessica, Yaghoobian, Jonathan, Hong, Ray

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

A systematic effort to identify the natural ecology of Pristionchus nematodes revealed species-specific host preferences for several beetle species, including the oriental beetle found in Japan and northeastern U.S. In Pristionchus pacificus, the first and only molecular component identified so far involved in odor signaling is the cGMP dependent protein kinase, Ppa-EGL-4. To obtain additional factors involved in insect pheromone attraction, we performed a non-saturating X-ray mutagenesis on P. pacificus for mutants that do not show attraction toward their host oriental beetle pheromone (ZTDO). We selected two promising candidates named obi-1 and obi-3 (Oriental Beetle pheromone Insensitive mutants). The chemosensory phenotype of obi-1 and obi-3 is specific for ZTDO attraction and neither obi-1 nor obi-3 show dauer formation defect (daf-c). However, both obi-1 and obi-3 animals show higher amplitude turns than wildtype, as well as differences in body morphology. Adult obi-1 hermaphrodites are ~20% longer than wildtype. In contrast, the Ppa-egl-4 loss-of-function is short and does not have an obvious egg-laying defect. We were able to confine obi-1 to a <153 kb region by positional mapping of 550 F2 progeny. Other morphological and behavioral characteristics of obi-1 will also be discussed.

Hong K et al. (1993) International C. elegans Meeting "Structure/function analysis of MEC-4 -- A protein involved in mechanosensory neuron function and neurodegeneration."

Driscoll MA, Hong K

[International C. elegans Meeting, 1993]

Hong Y et al. (1997) International C. elegans Meeting "Identification of protein domains associated with LIN-14 function, nuclear localization and chromosome association"

Ambros VR, Hong Y

[International C. elegans Meeting, 1997]

Heterochronic genelin-14 has a complex genomic DNA structure with 13 exons spanning over 20kb, and produces at least three different transcripts by alternative splicing of 5' exons. LIN-14 is a novel protein whose biochemical activity remains unknown. Based on a fully functional LIN-14::GFP fusion protein construct we made, a series of deletions of the LIN-14::GFP were then constructed and expressed under the control of the hypodermal-specific col-10 promoter for the in-vivo assay. Judged by its rescuing activity in hypodermal cell lineages, an exon9-13 construct (aa292-535) appears to be the minimum rescuing contstruct that we have tested so far. An exon10-13 construct does not rescue, arguing that exon9 is indispensable for executing LIN-14 function. In fact, the exon10-13 construct shows dominant negative activity, suggesting that this truncated LIN-14 polypeptide can interfere with endogenous lin-14 activity. The data suggests that LIN-14 exons 1-8 are not strictly required for lin-14 function and at least in the hypodermis, the alternative LIN-14 products predicted are also not essential. The results also shown that LIN-14 has an unconventional, extended NLS domain, rather than a more typical discrete NLS, as the whole region from aa284-466 is required for efficient nuclear localization. In addition, we observed that deletion constructs that display lin-14 activity like exon9-13 etc. show strong association with chromosomes during mitosis. Chromosomes show strong LIN-14::GFP fluorescence from metaphase to anaphase. In contrast, non-rescuing constructs like exon10-13 and exon11-13 shows no chromosomal association, suggesting the ability to associating with chromosomes is closely related to LIN-14 function. One model consistent with our data so far is that lin-14 encodes a transcription factor with DNA binding activity in its more carboxy-terminal exons.

Hong K et al. (1995) International C. elegans Meeting "Structure/function/topology of MEC-4--A Channel Subunit Involved in Mechanosensory Neuron Function and Neurodegeneration"

Driscoll MA, Xue J, Hong K, Lai CC

[International C. elegans Meeting, 1995]

mec-4 encodes protein related to deg-1, mec-10 and subunits of the mammalian amiloride-sensitive epithelial Na+ channel. mec-4 and mec-10 are co-expressed in the six touch receptor neurons and are postulated to encode subunits of a mechanosensory ion channel. This working hypothesis is exciting because, unlike voltage-gated and ligand-gated channels, mechanically-gated channels (those opened by membrane stretch or physical deflection) have not been previously cloned from eukaryotes. mec-4 has a second interesting feature--specific dominant gain-of- function alleles induce degeneration of the touch receptor neurons.

Willett JD et al. (1993) International C. elegans Meeting "Age related changes in metabolites of tryptophan and tryrosine in Caenorhabditis elegans."

Hong C, Willett JD, Chandhoke V

[International C. elegans Meeting, 1993]

Willett JD et al. (1991) International C. elegans Meeting "SEROTONIN AND AGING IN CAENORHABDITIS ELEGANS."

Madan S, Oates KK, Willett JD, Hong C, Zuckerman BM

[International C. elegans Meeting, 1991]

Alterations in neuroendocrine function are fundamental to altered physiological responses to stimuli observed in aging organisms. In mammalian systems the neurotransmitter serotonin declines in the senescent central nervous system. Such changes are believed relevant to functional declines in learning, memory and behavior. Since C. eleqans is an ideal system in which to explore the molecular mechanisms of age-dependent alterations in serotonin levels, studies have been undertaken on serotonin levels throughout the nematode life cycle. While serotonin is present in C. eleqans and 'serotonergic' cells have been identified using immunofluorescent techniques, its functional role in these organisms remains obscure. As in mammalian systems, a decline in serotonin levels in the senescent nematode is observed. We have also found an indolealkylamine of unknown structure present in amounts greater than that of serotonin. This substance appears to be a 5-oxygenated indolealkylamine with a MW of 234. Work is underway to fully characterize this material.

Hong Xiao et al. (2005) International Worm Meeting "The C-terminal SER-1 PDZ motif is essential for optimal 5-HT stimulation of egg-laying"

Rob Hobson, Li Lin, Richard Komuniecki, Vera Hapiak, Katherine Smith, Hong Xiao

[International Worm Meeting, 2005]

Serotonin (5-HT) stimulates egg-laying in nematodes and multiple 5-HT receptors appear to be involved in the process. In the free-living nematode, Caenorhabditis elegans, the 5-HT stimulation of egg-laying is abolished in ser-1 null worms that lack a 5-HT2 like receptor and the phenotype is rescued by ser-1 expression in vulval muscle. A type I PDZ motif (ETFL) at the C-terminus of both the C.elegans (SER-1) and Ascaris suum (AS-1) 5-HT2-like receptors is not essential for rescue, but appears to optimize signaling. The C-termini of both SER-1 and AS-1 bind specifically to PDZ domain 10 of the multi-PDZ domain containing protein MPZ-1 (C52A11.4), based on yeast 2 hybrid screening, GST pulldown and co-immunoprecipitation. Most importantly, the overexpression of MPZ-1 PDZ domain 10 in vulval muscle, predicted to disrupt the SER-1:MPZ-1 interaction, also specifically inhibits 5-HT stimulated egg-laying. The expression of mpz-1 is complex and differentially driven by multiple promoters. Expression is confined to about 60 neurons and body wall and vulval muscles. However, only 3 pairs of these neurons also appear express ser-1 (RMH, RMF, RMD). Indeed, MPZ-1 also appears to interact with other GPCRs with acidic amino acids in the -3 position of their PDZ motifs. The expression of GFP-tagged MPZ-1 PDZ domains 6, 9 or 10 driven by specific mpz-1 promoters is uniform throughout neurons or muscles. In contrast, the expression of GFP-tagged MPZ-1 PDZ domain 8 is punctate. In neurons, PDZ domain 8 colocalizes with synaptobrevin, suggesting a synaptic localization and in body wall muscle is associated with the dense bodies, suggesting a role in tight junctions. These studies suggest that the SER-1:MPZ-1 interaction is essential for optimal signaling and that MPZ-1 may be involved in the assembly, trafficking or localization of multiple GPCRs.