Arturo Gutierrez et al. (2001) International C. elegans Meeting "Evolution of homeotic gene function in nematodes"

Arturo Gutierrez, Ralf J Sommer

[International C. elegans Meeting, 2001]

We are studying the evolution of vulval cell fate specification in nematodes by comparing Pristionchus pacificus with Caenorhabditis elegans . Many features of the posterior body region of nematodes are determined by the homeotic gene mab-5 . In C. elegans , the MAB-5 protein regulates the expression of different target genes in response to upstream signals like PAL-1 or the Wnt pathway. Cel-mab-5 plays an important role during male ray formation, M lineage specification and also during hermaphrodite vulva formation. P. pacificus mab-5 mutants have a similar ray phenotype, whereas the phenotype in the M lineage and the vulva differs strongly from Cel-mab-5 . For instance, the "multivulva phenotype" caused by the ectopic differentiation of P8.p in Ppa-mab-5 mutants depends on interactions between P8.p and the misspecified M lineage. Given the pattern of conservation and change of MAB-5 function, we want to study if regulatory and/or coding differences are responsible for these evolutionary alterations. In the present work, we expressed Ppa-mab-5 in a Cel-mab-5 mutant background under the control of the Cel-mab-5 gene and studied the rescue of the posterior rays. We cloned the Ppa-mab-5 ORF in a construct containing the complete Cel-mab-5 gene including the 6 kb upstream region. This basic construct has a frameshift mutation in the hexapeptide (HD-) to eliminate the function of the Cel-mab-5 gene but conserving its regulatory regions. Preliminary results show that Ppa-mab-5 ORF can rescue Cel-mab-5 during ray formation as efficient as Cel-mab-5 (HD+) or Cel-mab-5 ORF cloned in the same way. Further experiments are under way to study MAB-5 function in other tissues and to compare various Hox genes in the ray rescue assay.

Sequeira-Mendes J et al. (2016) Chromosoma "Genome architecture: from linear organisation of chromatin to the 3D assembly in the nucleus."

Gutierrez C, Sequeira-Mendes J

[Chromosoma, 2016]

The genetic information is stored in the eukaryotic nucleus in the form of chromatin. This is a macromolecular entity that includes genomic DNA and histone proteins that form nucleosomes, plus a large variety of chromatin-associated non-histone proteins. Chromatin is structurally and functionally organised at various levels. One reveals the linear topography of DNA, histones and their post-translational modifications and non-histone proteins along each chromosome. This level provides regulatory information about the association of genomic elements with particular signatures that have been used to define chromatin states. Importantly, these chromatin states correlate with structural and functional genomic features. Another regulatory layer is established at the level of the 3D organisation of chromatin within the nucleus, which has been revealed clearly as non-random. Instead, a variety of intra- and inter-chromosomal genomic domains with specific epigenetic and functional properties has been identified. In this review, we discuss how the recent advances in genomic approaches have contributed to our understanding of these two levels of genome architecture. We have emphasised our analysis with the aim of integrating information available for yeast, Arabidopsis, Drosophila, and mammalian cells. We consider that this comparative study helps define common and unique features in each system, providing a basis to better understand the complexity of genome organisation.

Knoch Lars et al. (2003) International Worm Meeting "Functional comparison of the EGF-like gene let-23 between Caenorhabditis elegans and Pristionchus pacificus"

Ralf Sommer, Knoch Lars

[International Worm Meeting, 2003]

Our lab studies the evolution of cell fate specification by comparing P. pacificus and C. elegans using vulva development as a model system. The comparison of loss of function mutations in orthologous genes between P. pacificus and C. elegans indicated substantial differences in gene function. To complement the genetic approach, we have used in vivo assays to analyze functional similarities and dissimilarities of orthologous proteins in C. elegans mutant backgrounds. Initial work with the two Hox genes lin-39 and mab-5 indicated that the P. pacificus genes can substitute for the C. elegans orthologs despite their functional differences (Grandien & Sommer, 2000; Gutierrez et al, 2003). To extend this work, we started to investigate components of the EGF/RAS/MAPK signal transduction pathway that mediate vulva development in C. elegans. In particular, we want to concentrate on the P. pacificus let-23 gene, the receptor tyrosine kinase that responds to the inductive signal from the LIN-3 ligand. Besides its role in vulva development, LET-23 also affects viability, male spicule formation, posterior development of the epidermis, and ovulation (Aroian and Sternberg, 1991). We cloned Ppa-let-23 using degenerate PCR and showed that the functional domains of LET-23 evolved differently from one another. For example, the C-terminal part of Ppa-LET-23 lacks three of the eight tyrosine-motifs involved in autophosphorylation and transduction important for individual Cel-LET-23 functions (Lesa and Sternberg, 1997). While one of these tyrosine motifs does not play any major role in C. elegans, the other two motifs have a strong effect on viability and vulva differentiation in C. elegans. We are currently testing the Ppa-let-23 cDNA for rescue of a Cel-let-23 mutant and planning further experiments with hybrid molecules between the let-23 genes of P. pacificus and C. elegans. AROIAN and STERNBERG, Genetics 128;251-267 (1991) LESA and STERNBERG, Mol. Biol. Cell 8:779-793 (1997) GRANDIEN & SOMMER, Genes Dev. 15:2161 -2172 (2001) GUTIERREZ et al., Development 130:983-993 (2003)

Anbalagan C et al. (2013) Ecotoxicology "Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils."

Anbalagan C, de Pomerai DI, Chowdhuri DK, Gutierrez C, Lafayette I, Martin JR, Antoniou-Kourounioti M

[Ecotoxicology, 2013]

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.

Cinar H N et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Vibrio cholerae Hemolysin Modulates Autophagy Response in Caenorhabditis elegans"

Sahu S N, Odusami O, Cinar H N

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Autophagy is a regulated cellular process responsible for the degradation of organelles and long lived proteins. This intracellular process acts as a survival pathway during starvation, and stress, also involved in cellular response against bacterial infections (1). Vibrio cholerae infects and kills C. elegans via cholerae toxin and toxin-co-regulated pili independent manner. V. cholerae hemolysin (VCC), which is a pore forming toxin, is required for the lethality, growth retardation and intestinal cell vacuolation during the infection (2). VCC induced vacuoles found to be co-localized with LC3 (mammalian homolog of lgg-1) punctuate, a feature indicative of autophagosome formation, in cultured cells (3). To asses the role of VCC in autophagy regulation at the organismal level we have analyzed the lgg-1::GFP distribution in C. elegans exposed to WT and hlyA deletion mutant strains of V. cholerae. While exposure of C. elegans to WT V. cholerae induced the punctuate distribution of lgg-1::GFP in the intestinal cells, hemolysin deletion mutants did not induce punctuate distribution. Accumulation of autophagosomes may reflect the induction of autophagy or reduced turnover of autophagosomes in blocked autophagy conditions. To differentiate between these two outcomes we genetically blocked the autophagy pathway using unc-51/ ATG1 mutants. Inactivation of autophagy pathway using unc-51(e369) mutants led to a decrease in the formation of lgg-1::GFP punctuate, during WT V. cholerae infection, suggesting that V. cholerae induces autophagy in C. elegans intestinal cells. Altogether our data suggest that V. cholerae induces autophagy response via VCC during infection in C. elegans. 1) Jia K. et al PNAS, 106 (2009) 14564-14569 2) Cinar HN. et al 17th International C. elegans Meeting, 2009 3) Gutierrez MG. et al PNAS, 104 (2007) 1829-1834

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

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.

Antika TR et al. (2023) J Biol Chem "Sequence-specific targeting of C. elegans C-Ala to the D-loop of tRNAAla."

Horng JC, Hsu HL, Nazilah KR, Wang CC, Wang TL, Wang SC, Antika TR, Chuang TH, Chrestella DJ, Wang SW, Tseng YK, Pan HC

[J Biol Chem, 2023]

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala_c) robustly binds both ligands. How Ce-C-Ala_c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala_c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala_c specifically recognized the conserved invariant base G¹⁸ in the D-loop of tRNA^Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala (Ce-C-Ala_m) robustly bound both tRNA^Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA^Ala.

Blumenthal TE et al. (1994) Worm Breeder's Gazette "C. elegans U2AF 65."

Zorio DAR, Lea K, Blumenthal TE

[Worm Breeder's Gazette, 1994]

C. elegans U2AF65

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.