Crowell T et al. (1995) International C. elegans Meeting "SYNAPTOTAGMIN-DEFICIENT (SNT-1) MUTANTS OF C. ELEGANS: MOLECULAR AND PHENOTYPIC ANALYSIS"

Crowell T, Rand JB, McManus JR

[International C. elegans Meeting, 1995]

Synaptotagmin is a major synaptic vesicle protein; it contains 2 apparent calcium-binding (PKC-CII) domains and seems to be involved in both exocytosis and endocytosis. The cloning of snt-1 and the isolation of mutants were reported by Nonet et al. (1993). We have now analyzed 17 independent spontaneous snt-1 mutations isolated in our laboratory, as well as alleles isolated by Leon Avery and Erik Jorgensen. Molecular analysis indicates that 12 of the alleles are associated with DNA rearrangements (3 Tc1 insertions, 1 non- Tc1 insertion and 8 deletions), with the remaining alleles due to point mutations. Some of the deletions provide guaranteed molecular null alleles; null mutants are quite uncoordinated, but viable. Phenotypic analysis of snt-1 mutants included quantitative behavioral studies (pharyngeal pumping and head thrashing) and immunoblots. Several of the mutants clearly retain some gene function, and it was possible to establish an alleleic series of phenotypic severity. Partial gene function is retained not only by some of the point mutants, but also by a 66-bp (inframe) deletion which eliminates most of the first CII domain, and by a 9-bp deletion which disrupts the second CII domain. Supported by a grant from MDA

Crowell T et al. (1997) International C. elegans Meeting "ANALYSIS OF C. elegans SYNAPTOTAGMIN (snt-1) MUTANTS REVEALS A SITE REQUIRED FOR PROPER TARGETING"

Duke A, McManus JR, Rand JB, Crowell T, Duerr JS, Gaskin J

[International C. elegans Meeting, 1997]

Synaptotagmin is a major synaptic vesicle protein; it contains 2 apparent calcium-binding (PKC-C2) domains and may be involved in both exocytosis and endocytosis. A large number of mammalian synaptic proteins appear to have specific, high-affinity interactions with different domains of synaptotagmin. The cloningof snt-1 and the isolation of mutants were reported by Nonet et al. (1993). Because C. elegans snt-1 mutants are viable and resistant to cholinesterase inhibitors, it has been relatively easy to identify a large number of alleles. We have now analyzed the molecular, behavioral, and immunocytochemical properties of 17 independent snt-1 mutations. Some of the alleles provide guaranteed molecular null alleles; null mutants are quite uncoordinated, but viable. Analysis of all snt-1 mutants included quantitative behavioral studies (pharyngeal pumping, body thrashing in liquid, and expulsion failure) and immunostaining. Partial gene function was retained by some point mutants, and also by several deletions. Of particular interest was snt-1(md220), an (in-frame) 9-bp deletion which disrupts part of the second C2 domain. Immunocytochemical and Western analyses indicate that the mutant protein is present at approximately the wild-type level, but virtually all of the immunostaining is in the cell bodies, rather than at synapses. Since other synaptic vesicle markers (e.g., UNC-17) are properly localized in snt-1(md220) animals, it appears that this mutation disrupts a site on the synaptotagmin molecule necessary for proper targeting of the protein to synaptic vesicles. This mutant is of interest behaviorally because it has the null phenotype for pharyngeal pumping and body thrashing, but its expulsion failure rate is almost that of wild-type. Expulsion is different from the other behaviors because: a) it is 100-fold less frequent; b) it is mediated by excitatory GABAergic synapses; c) which seem to be located in the cell body of the DVB neuron. (Supported by grants from NINDS and MDA.)

Samuel ADT et al. (2003) J Neurosci "Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory."

Murthy VN, Samuel ADT, Silva RA

[J Neurosci, 2003]

Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm's ambient temperature (T-amb) and its memory of the temperature of its cultivation (T-cult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25degreesC, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either T-amb > T-cult or T-amb > T-cult, but AFD synaptic release is low if T-amb congruent to T-cult. This suggests that AFD encodes a direct comparison between T-amb and T-cult.

Rand JB et al. (1997) International C. elegans Meeting "ANALYSIS OF C. elegans CHOLINE ACETYLTRANSFERASE (CHA-1) MISSENSE MUTANTS SUGGESTS A TEMPERATURE-DEPENDENT REQUIREMENT FOR CHOLINERGIC FUNCTION"

Rand JB, McManus JR, Crowell T, Duerr JS

[International C. elegans Meeting, 1997]

The cha-1 and unc-17 genes encode choline acetyltransferase and a vesicular acetylcholine transporter respectively. Both of these genes are required for cholinergic transmission; and null alleles of both genes lead to L1 arrest and death. In addition, many viable cha-1 and unc-17 mutants have been isolated that have low levels of residual function. These mutants are small, slow-growing, uncoordinated, and resistant to inhibitors of cholinesterase. Some of the cha-1 mutants were reported to be temperature-sensitive, i.e., they were behaviorally normal at 16! but uncoordinated at 25!, or else the mutant phenotype was considerably stronger at the higher temperature. We sequenced and performed quantitative behavioral analysis on 16 viable cha-1 mutants with some residual gene function and thus some residual ChAT activity. Most of these mutations result in dramatic decreases in ChAT immunoreactivity in neurons, suggesting that the mutant proteins are preferentially degraded. In addition, most of these mutations confer temperature-sensitive development and behavior. While a few of the alleles have dramatic temperature effects, most cha-1 alleles tested exhibited at least mild heat-sensitivity of growth and behavior. (One allele showed both cold sensitivity and heat sensitivity; this mutation is due to a Tc1 insertion at a splice junction.) In contrast, neither unc-17 mutants nor snt-1 mutants displayed ts-growth or behavior. It thus appears that the requirement for acetylcholine itself may be temperature sensitive. (Supported by grants from NIGMS and OCAST.)

Bommireddy R et al. (2007) Trends Mol Med "TGFbeta1 and Treg cells: alliance for tolerance."

Bommireddy R, Doetschman T

[Trends Mol Med, 2007]

Transforming growth factor beta1 (TGFbeta1), an important pleiotropic, immunoregulatory cytokine, uses distinct signaling mechanisms in lymphocytes to affect T-cell homeostasis, regulatory T (T(reg))-cell and effector-cell function and tumorigenesis. Defects in TGFbeta1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. TGFbeta1 prevents abnormal T-cell activation through the modulation of Ca(2+)-calcineurin signaling in a Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)3 and SMAD4-independent manner; however, in T(reg) cells, its effects are mediated, at least in part, through SMAD signaling. TGFbeta1 also acts as a pro-inflammatory cytokine and induces interleukin (IL)-17-producing pathogenic T-helper cells (T(h) IL-17 cells) synergistically during an inflammatory response in which IL-6 is produced. Here, we will review TGFbeta1 and its signaling in T cells with an emphasis on the regulatory arm of immune tolerance.

Grundahl K et al. (1997) International C. elegans Meeting "unc-41 ENCODES A PRESYNAPTIC PROTEIN"

Rand JB, Grundahl K, Eustance RJ, Crowell T

[International C. elegans Meeting, 1997]

unc-41 mutants display a coiling, uncoordinated behavior, resistance to inhibitors of cholinesterase, elevated levels of acetylcholine, slow growth, and small adult size. This set of phenotypes is associated with defects in acetylcholine release and/or synaptic vesicle function. In addition, unc-41 animals display a defecation-expulsion defect associated with defective GABA function. It is therefore likely that unc-41 encodes a protein important for the release of most or all neurotransmitters. We cloned the gene by transposon tagging, and we have isolated and sequenced unc-41 genomic and cDNA clones. Northern and cDNA analysis reveals that there are two unc-41 transcripts. These are 5.3 and 4.5 kb long; they are both rare and appear to be transcribed from different promoters. The structural difference between the two transcripts arises from the presence of additional exons upstream of the 4.5 kb transcript and its presumed promoter. The gene encodes a pair of nested hydrophilic proteins (1650- and 1400-amino acids). These have slight similarity to the AP50 clathrin adaptor protein family (which functions in membrane trafficking and endocytosis) and a strong similarity to the 1260-amino acid stonedB protein of Drosophila (Andrews et al., 1996). The stoned locus is a complex locus which encodes two structurally unrelated proteins. There are genetic interactions between the stoned and shibire loci (shibire encodes the endocytosis protein dynamin); it is therefore hypothesized that the stoned protein(s) also function in endocytosis. The sequence similarity is limited to the 500-amino acid C-terminal domain of the UNC-41 and stonedB proteins. Immunolocalization studiesindicate that the UNC-41 proteins are located at synapses in most or all neurons, and that they are not associated with synaptic vesicles. We have analyzed a large collection of unc-41 alleles - this includes 27 alleles isolated in our lab, 9 MRC alleles, and two MIT alleles. Many of them have residual immunostaining, and some alleles have only partial behavioral impairment. (Supported by a grant from NINDS.)

Agulnik SI et al. (1995) Genomics "Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans."

Bollag RJ, Silver LM, Agulnik SI

[Genomics, 1995]

Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

Ju T et al. (2006) Glycobiology "Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans."

Ju T, Zheng Q, Cummings RD

[Glycobiology, 2006]

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-Gal:GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122)(2). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by PCR using a C. elegans cDNA library as the template, contains 1,170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type-II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has 7 Cys residues in the lumenal domain including 6 conserved Cys residues in all of the orthologs. The Ce-T-synthase has 4 potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search and it contains 8 exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein constructs show that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc, and might require invertebrate-specific factors for formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and opens new avenues to explore O-glycan function in this model organism.

Muir RE et al. (2007) Int J Syst Evol Microbiol "Leucobacter chromiireducens subsp. solipictus subsp. nov., a pigmented bacterium isolated from the nematode Caenorhabditis elegans, and emended description of L. chromiireducens."

Muir RE, Tan MW

[Int J Syst Evol Microbiol, 2007]

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504(T)) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504(T) was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504(T) and Leucobacter chromiireducens LMG 22506(T) indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1(T)=CIP 108389(T)=LMG 22506(T)) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504(T)=DSM 18340(T)=ATCC BAA-1336(T)).

Agulnik SI et al. (1997) Genome "Three novel T-box genes in Caenorhabditis elegans."

Ruvinsky I, Agulnik SI, Silver LM

[Genome, 1997]

The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Seven other recently described mouse T-box genes are also expressed during development. In the nematode Caenorhabditis elegans, four T-box genes have been characterized to date. In this study, we describe three new C. elegans T-box genes, named Ce-tbx-11, Ce-tbx-12, and Ce-tbx-17. Ce-tbx-11 and Ce-tbx-17 were uncovered through the sequencing efforts of the C. elegans Genome Project. Ce-tbx-12 was uncovered through degenerate PCR analysis of C. elegans genomic DNA. Ce-tbx-11 and Ce-tbx-17 are located in close proximity to the four other previously described T-box genes in the central region of chromosome III. In contrast, Ce-tbx-12 maps alone to chromosome II. Phylogenetic analysis of all known T-box domain sequences provides evidence of an ancient origin for this gene family.