Berninsone P et al. (2000) East Coast Worm Meeting "SQV-7, a protein involved in vulval invagination and embryonic development, transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose"

Hwang H-Y, Horvitz R, Berninsone P, Hirschberg C

[East Coast Worm Meeting, 2000]

Mutations in eight sqv (squashed vulva) genes (sqv-1 to 8) cause defects in vulval invagination and early embryonic development (1). SQV-1 and SQV-4 are similar to enzymes involved in nucleotide sugar metabolism (2), while SQV-3 and SQV-8 are similar to glycosyltransferases (3). SQV-7 is a multi-transmembrane protein resembling Golgi membrane nucleotide sugar transporters (4); these proteins specifically translocate nucleotide sugars from the cytosol into the lumen of the endoplasmic reticulum and Golgi apparatus where they are used as sugar donors by glycosyltransferases (4). Translocation is essential for glycosylation: yeast, protozoa and mammalian cell mutants defective in this function have a severe deficiency of the corresponding sugar in their glycoconjugates (4). Because GDP-mannose and UDP-glucose are the only nucleotide sugars transported into S.cerevisiae Golgi and ER vesicles in vivo and in vitro (5), this organism was used for expression of SQV-7 to determine its substrate specificity. We found that SQV-7 transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose in vitro, the first protein known to transport the former two nucleotide sugars. All three nucleotide derivatives are competitive, alternate, non-cooperative substrates, which suggests there is a single active site for all three substrates on SQV-7. Expression of the two mutant sqv-7 missense alleles results in significant reduction of the three transport activities. We hypothetize that in these mutants the biosynthesis of chondroitin, the major glycosaminoglycan in C. elegans (6), is more likely to be impaired than that of heparan sulfate since chondroitin polymerization requires both UDP-glucuronic acid and UDP-N-acetylgalactosamine while heparan sulfate polymerization does not require UDP-N-acetylgalactosamine. (1)Herman T. et al (1999) PNAS 96:968 (2)Hwang H. et al.(2000) East Coast C.elegans Meeting (3)Herman T. and Horvitz H. R. (1999) PNAS 96: 974 (4)Hirschberg C. et al (1998) Annu. Rev. Biochem.67: 49 (5)Berninsone P. et al (1997) J. Biol. Chem. 272: 12616 (6)Toyoda H. et al (2000) J. Biol. Chem. 275: 2269

J Grabitzki et al. (2004) European Worm Meeting "THE PCOME OF CAENORHABDITIS ELEGANS IDENTIFICATION OF PHOSPHORYLCHOLINE-SUBSTITUTED PROTEINS AND COMPARISON OF DEVELOPMENTAL CHANGES IN THE EXPRESSION OF THIS EPITOPE"

R Geyer, G Lochnit, J Grabitzki

[European Worm Meeting, 2004]

Caenorhabditis elegans has been found to be good model system for parasitic nematodes, drug screening and developmental studies. Like the respective parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins, carrying, in part, phosphorylcholine (PC) substitutents, which might play important roles in nematode development, fertility and, at least in the case of parasites, the survival within the host (1). With the exception of a major secretory/ excretory product from Achanthocheilonema viteae (ES-62) (2) and the aspartyl-protease ASP-6 (3), no other proteins carrying this epitope has been identified and studied in detail yet. For C. elegans two N-linked PC-epitopes have been reported so far: (I) a pentamannosyl-core structure carrying three PC-residues (4) and (II) a trimannosyl-core species elongated by a N-acetylglucosamine substituted at C-6 with PC (5). Furthermore, in Dauer larvae of C. elegans there was evidence for the presence of glycans with the composition PC1Hex3HexNAc3 to PC2dHex2Hex4HexNAc7 (6). Here we present the 2D-electrophoretic separation of C. elegans proteins, the comparison of the PC-substitution pattern in distinct developmental stages and the mass spectrometric identification of PC-modified proteins. References: 1.Lochnit, G., Dennis, R. D., and Geyer, R. (2000) Biol Chem 381, 839-847 2.Harnett, W., Harnett, M. M., and Byron, O. (2003) Curr Protein Pept Sci 4, 59-71 3.Lochnit, G., Grabitzki, J., Henkel, B., and Geyer, R. (2003) Biochemical Journal submitted 4.Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002) J Biol Chem 277, 49143-49157 5.Haslam, S. M., Gems, D., Morris, H. R., and Dell, A. (2002) Biochem. Soc. Symp. 69, 117-134 6.Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004) Proc Natl Acad Sci U S A 101, 3404-3408

Berninsone P et al. (2000) East Coast Worm Meeting "Defects in glycosaminoglycan biosynthesis cause the C. elegans Sqv phenotype and human Ehlers-Danlos syndrome"

Herman T, Hirschberg C, Hwang H-Y, Horvitz R, Berninsone P

[East Coast Worm Meeting, 2000]

Mutations in eight sqv (squashed vulva) genes result in several developmental abnormalities, including defective vulval invagination and maternal-effect lethality. The molecular identities of the six sqv genes cloned to date now suggest that the molecular basis for these defects lies in the disruption of the biosynthesis of glycosaminoglycans (GAG) of the structure (serine residue in the protein core)-xylose-galactose-galactose-glucuronic acid-(X-glucuronic acid)n, where X is either N-acetylgalactosamine or N-acetylglucosamine. Biosynthesis of GAGs requires the synthesis of nucleotide sugars in the cytoplasm and translocation of nucleotide sugars into the endoplasmic reticulum (ER) and/or Golgi, where polymerization of sugars is catalyzed by glycosyltransferases. SQV-4 is a UDP-glucose dehydrogenase, a key enzyme in UDP-glucuronic acid synthesis. SQV-7 is a multi-pass transmembrane protein that transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose from the cytoplasm into the ER and/or Golgi (see abstract by Berninsone et al.). Recently cloned mammalian homologs of sqv-3 and sqv-8 encode glycosyltransferases necessary for the biosynthesis of the GAG-protein linkage region of proteoglycans. SQV-3 is similar to galactosyltransferase I, and SQV-8 is similar to glucuronyltransferase I. SQV-1 is a cytoplasmic protein with weak similarities to nucleotide-sugar modifying enzymes, and SQV-5 is a novel protein with a single predicted transmembrane domain. We postulate that sqv-1 and sqv-5 are components of the same GAG biosynthesis pathway and that the GAGs are important for cell-cell or cell-matrix interactions in embryonic and vulval development. Mutations in the human homolog of sqv-3 are implicated as the cause of a progeroid variant of the connective-tissue disorder Ehlers-Danlos syndrome. The other five sqv genes also have close human counterparts, which suggest that a common pathway for modifying important cell surface and/or extracellular GAGs is present in humans and in C. elegans. Defects in the human counterparts of other sqv genes therefore may be responsible for aging disorders and connective tissue diseases such as Ehlers-Danlos syndrome.

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and seven from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last few years. There is no indication that we are even close to knowing all species in this genus.

Xiaodong Wang et al. (2003) International Worm Meeting "Evolution of a novel gene expression pattern in C. elegans"

Xiaodong Wang, Helen M Chamberlin

[International Worm Meeting, 2003]

Previous studies have shown that C. elegans ovo-related gene lin-48 expresses in a small number of cells including the excretory duct cell. In the related species C. briggsae, the expression is conserved in all cells except the excretory duct. This lin-48 expression difference affects excretory duct morphogenesis. In C. briggsae, as well as in C. elegans lin-48(sa496) mutants, the excretory duct is more anterior than in C. elegans wild type. This indicates that C. elegans lin-48 (Ce-lin-48) is involved in duct morphogenesis and positioning, but this gene function is absent in C. briggsae (1). We have made reporter transgenes composed of the lin-48 regulatory sequences from C. elegans or C. briggsae driving expression of green fluorescent protein (GFP). Tests of these clones in each species showed that only the Ce-lin-48 is expressed in excretory duct cell in C. elegans animal. These results indicate that there are differences in both cis-regulatory sequences and trans-acting proteins between the two species. By creating chimeric reporter transgenes including C. elegans and C. briggsae regulatory sequences, we have found that one difference between the two species is the presence of regulatory sequences in Ce-lin-48 that respond to the bZip protein CES-2 (1). The lin-48 gene expression differences between C. elegans and C. briggsae could result from loss of excretory duct expression in the C.briggsae lineage or acquired expression in the C. elegans lineage. To distinguish between these possibilities, we have analyzed three additional Caenorhabditis species (C. remanei, C. sp. CB5161 and C. sp. PS1010). We found these species have a duct morphology similar to C. briggsae indicating the C. elegans morphology is unique to this species. For comparison to C. elegans and C. briggsae, we have isolated the lin-48 gene from C. remanei and C. sp. CB5161. Alignment of the lin-48 regulatory sequences reveals that the sequences are more conserved among C. briggsae, C. remanei and C. sp. 5161. Several conserved domains are absent from C. elegans, whereas the previously identified CES-2 binding sites are absent from the other species. Currently, we are creating lin-48::gfp reporter transgenes for each species to observe the gene expression patterns. Further experiments with these transgenes will allow us to test whether the differences between C. elegans and the other species result from a loss of repressor elements or gain of activator elements in the C. elegans gene. (1)X. Wang and H. M. Chamberlin (2002) Genes & Development 16: 2345-2349.