Marco Gallo et al. (2007) International Worm Meeting "Effects of Daumone on C. elegans Physiology and Signal Transduction Pathways."

Marco Gallo, Donald L. Riddle

[International Worm Meeting, 2007]

Daumone is a pyranose sugar attached to a fatty acid & has dauer-inducing activity at sub-millimolar concentrations. We exposed mutants in different steps of the daf-2/IGF-1-like & TGF-<font face=symbol>b</font> dauer signal transduction pathways to synthetic daumone in order to understand which molecules are important to relay the daumone signal. Daumone treatment of strains carrying reporter constructs indicated that daumone induced DAF-16 nuclear localization in embryos & L1 animals. Furthermore, daumone partially suppressed daf-7 transcription in the ASI amphidial neurons. Daumone has toxic effects on the physiology of mutants with cuticular defects. At the concentrations needed for dauer induction, toxic affects are manifested. We propose a model of action for daumone in the process of dauer induction.

Marco Gallo et al. (2005) International Worm Meeting "The DPY-14 Paradox: an Essential Collagen Expressed by Both Hypoderm and Ciliated Neurons"

Marco Gallo, Robert C Johnsen, Allan K Mah, David L Baillie

[International Worm Meeting, 2005]

C. elegans dpy-14 encodes an essential collagen required for proper L1 cuticle formation. Study of dpy-14 promoter::GFP reporter constructs indicates that the gene is predominantly expressed during embryogenesis and supports SAGE data in revealing high levels of gene expression in both the hypodermis and ciliated neurons. Specifically, expression was observed in cells of AB lineage that have the potential to differentiate into amphids and their support cells. Furthermore, dpy-14 possesses the earliest known expression for a collagen gene.Animals carrying the dpy-14(e188) temperature sensitive allele are prone to conspicuous water leakage when placed in water at the 25C non-permissive temperature. Prominent water vesicles can be localised to the region of the amphid channels, suggesting that defects in these structures allow water entry into the animals in abnormal amounts.Structural integrity of the amphids was assessed by DiI staining. The results indicate that these ciliated neurons in Dpy-14 animals are morphologically indistinguishable from wild-type.Since mutations in DPY-14 result in structurally normal amphids, we went on to test their functionality. Promoter fusion constructs showed that dpy-14 is expressed in the progenitor cells of the ASER, ASEL, ASGR, ASIR and ASKR amphids, all of which are involved in positive chemotaxis to lysine. Chemoattraction assays indicate that dpy-14(e188) animals chemotax to Lys as well as N2 wild-type animals.Our experiments suggest that DPY-14 is a structural component of the amphid channels, but not of the amphids. We propose that ciliated neurons and their hypodermal support cells co-operate during embryo development in the synthesis and secretion of the DPY-14 collagen into the amphid channels.

Marco Gallo et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "The Novel Non-Essential Longevity Gene misc-1 Controls Mitochondrial Morphology and Apoptosis"

Donald Riddle, Marco Gallo

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

We identified misc-1 as a novel longevity gene. It is mainly expressed in the pharynx and putatively encodes mitochondrial 2-oxoglutarate carrier. MISC-1 and its human orthologue share 72% identity and 82% similarity at the amino acid level. RNA interference (RNAi) against misc-1 increases fer-15 average life-span by 20% and daf-2;fer-15 mean life-span by 42%. Animals carrying the knock-out allele, misc-1(tm2793), had an average life-span only 7% longer than wild-type. These effects on life-span do not seem to require daf-16 or skn-1. misc-1 seems to behave as most genes encoding mitochondrial proteins, for which levels of gene expression intermediate between wild-type and knock-out result in the largest extension in life-span. However, unlike most of these genes, misc-1 down-regulation results in increased life-span only if the RNAi treatment is applied after larval development. We employed a transgenic line carrying a mitochondrially-targeted form of Green Fluorescent Protein (GFP) to study the effects of this gene on mitochondrial morphology. misc-1 RNAi conveyed a mitochondrial fragmentation phenotype in adult N2. Mitochondrial fragmentation has been associated with apoptosis in C. elegans and mammalian systems. We therefore performed SYTO12 staining to detect apoptotic bodies in the germ-line of knock-out animals. Germ-line apoptosis in this strain was two-fold higher than in N2 control. These results were confirmed using a ced-1::gfp strain in an RNAi experiment. We then subjected the misc-1 knock-out worms to different stressors. Knock-out animals were more sensitive than N2 to juglone, which induces production of reactive oxygen species. However, they were not more sensitive than controls to thermal stress (both at 35degC and 37degC). Knock-out worms were more resistant than wild-type to 1 mM NaN, a mitochondrial poison. Interestingly, misc-1 is not an essential gene, although it is predicted to be the only C. elegans gene encoding a 2-oxoglutarate carrier. misc-1(tm2793) animals are phenotypically indistinguishable from N2, have the same developmental timing and rate of pharyngeal pumping (as assessed by feeding assays employing fluorescent microspheres). MISC-1 is therefore an excellent candidate drug target. One model to explain the results obtained thus far is that reduction of misc-1 activity positively affects life-span by stimulating a purifying selection in the germ line that conveys healthier mitochondria to the developing embryo.

Marco Gallo et al. (2004) West Coast Worm Meeting "Molecular Characterization of C. elegans dpy-14, a Collagen Gene with Homology to the Human Collagen III Gene COL3A1"

Robert C Johnsen, Marco Gallo, David L Baillie, Allan K Mah

[West Coast Worm Meeting, 2004]

The purpose of this study is to characterize dpy-14 , a gene that codes for a collagen type III(alpha1). This gene is presently annotated on the WormBase database as col-59 . DNA sequencing analysis of the dpy-14 canonical allele, e188 , has revealed a mutation in the third exon, at position 747. Specifically, the codon GGA, coding for Gly, is changed to AGA, a codon for Arg. The severity of the Dpy phenotype in e188 mutants increases with higher temperatures, ranging from a mild dumpy appearance at 15 C, to a Dpy phenotype accompanied by vulval protrusions at 20 C, and to arrested development at 25 C. This gradient of temperature sensitivity might be explained by an interplay of steric hindrance and thermal motion involving the Arg side chain and water molecules. In fact, both of them are present in the channel formed in the centre of the collagen triple helix, and are thought to be involved in trimerization of the collagen monomers. The expression pattern of the gene was determined by analyzing the Long-SAGE data ( C. elegans II Project), and by the creation of promoter GFP fusion constructs. Both methods indicated that dpy-14 is highly expressed in embryonic neurons. In order to test whether the mutant DPY-14 protein affects the morphology of the amphids, a fluorescent dye filling experiment was performed on e188 animals. The results indicate that this class of ciliated neurons is affected by the mutation. DPY-14 shares 72% homology in its amino acid sequence with the human collagen type III(alpha1) COL3A1. Intriguingly, a mutation in COL3A1 similar to that observed in the e188 mutants, that is a Gly to Arg mutation within a collagen-like GXY repeat, is deemed responsible for the occurrence of a type of familial aortic aneurysm. It might be possible that the study of the C. elegans dpy-14 gene may help better understand this class of genetic diseases.

Yamasaki T et al. (2002) J Nat Toxins "Toxicity of tannins towards the free-living nematode Caenorhabditis elegans and the brine shrimp Artemia salina."

Tsukioka J, Mohamed ASA, Mori T, Fujii K, Yamasaki T, Sato M

[J Nat Toxins, 2002]

Toxicities of gallo- and condensed tannins towards the free-living nematode Caenorhabditis elegans is dependent on the tannins' molecular sizes. In the present paper we investigate the toxicity of ellagitannins to C. elegans and the toxicity of ellagi-, gallo-, and condensed tannins to the brine shrimpArtemia salina. Ellagitannins 1 and 2 were isolated from Euphorbia supina and identified as tellimagrandin I and rugosin A methyl ester, respectively. An ellagitannin preparation from Cornus officinalis was chromatographically fractionated into ellagitannins A through H, having different molecular weights and specific rotations. Three of the ten ellagitannins, 2, G, and H produced significant toxicity towards C. elegans, showing the presence of an activity-structure relationship, as opposed to the results from tests of gallo- and condensed tannins. Ellagi-, gallo-, and condensed tannins also produced toxicity in A. salina.

Victor L Jensen et al. (2008) European Worm Meeting "The maternal-effect Daf-c gene daf-25 is expressed in ciliated neurons and functions in neurosecretion"

Victor L Jensen, Sharon Bishop-Hurley, Marco Gallo, Donald L Riddle

[European Worm Meeting, 2008]

We isolated four alleles of daf-25 in screens for new temperature-sensitive. Daf-c (dauer-constitutive) mutants. At restrictive temperature, daf-25. mutants form dauer larvae even when food is abundant. The Daf-c phenotype. can be rescued by maternally supplied daf-25(+). Compared to N2, daf-25. worms failed to avoid sucrose and high concentrations of NaCl, indicating. that they are defective in sensing osmotic gradients. There is also a. reduction of brood size when worms are shifted to the restrictive. temperature (25degreesC) at L4, despite normal levels of progeny at the. permissive temperature (15degreesC). Epistasis results put DAF-25 function. downstream of DAF-6, but upstream of DAF-10. This implies that DAF-25. function requires cilia because DAF-10 is necessary for proper cilia. production. DAF-6 is required for proper formation of the amphid channel,. which connects the ciliated neurons to the environment. Indeed, a daf-. 25p::GFP reporter is expressed in the ciliated neurons among other tissues.. Because daf-25 dauer larvae are defective in dauer recovery, similar to. unc-31 dauers, we analyzed dense core vesicle secretion. Using a paex-. 3::ANF::GFP (gift from Erik Jorgensen), we observed a secretion defect in a. deletion mutant that affects both isoforms of daf-25. By contrast, a. deletion mutant that only affects one of the isoforms, and has a weaker Daf-. c phenotype, has no defect in ANF::GFP secretion. A model for the role of. DAF-25 in dauer formation will be presented.

Mohamed ASA et al. (2000) Journal of Pesticide Science "Lethal activity of galo- and condensed tannins against the free-living soil-inhabiting nematode, Caenorhabditis elegans."

Mohamed ASA, Sato M, Islam SQ, Yamasaki T, Mori T

[Journal of Pesticide Science, 2000]

Great amounts of structurally diverse tannins are annually produced. Tannins have been well known as food phytochemicals (e.g. 3T-O-a-L-arabinopyranosyl-ent-epicatechin-(2a->O-7,4a->8)-epicat echin in cacao mass), medicinal properties (e.g. geraniin in Geranium thunbergii as tonic or antidiarrhoic) or tanning materials for leather manufacture (e.g. wattle tannins in Acacia mollissima). Recently, condensed tannin dimers (procyanidins B-1 and B-3) and its polymers were isolated from Pinus densiflora and characterized as members of the oviposition-stimulating multicomponent system in pine for the cerambycid beetle, Monochamus alternatus. Condensed tannins have also been regarded as antifeedants for phytophagous insects. A marine phlorotannin preparation, polymer of phloroglucinol, obtained from the brown alga Sargassum furvellum inhibits growth of the green algae Dunaliella and Enteromorpha spp. This paper describes isolation and identification of gallo- and condensed tannins and their nematicidal acitivity against the soil-inhabiting nematode, Caenorhabditis elegans.

Peter Ruzanov et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "SAGE Protocol Combined With Illumina Flow-cell Sequencing (Tag-Seq) Improves Detection Of Rare Transcripts Associated With Longevity In C. elegans"

Peter Ruzanov, Donald Riddle

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Serial Analysis of Gene Expression (SAGE) allows extraction of short sequences (tags) from polyadenylated transcripts, which are concatenated, cloned, sequenced and matched to the transcriptome for estimating gene expression levels. We used a new method (Tag-Seq) developed by Martin Hirst, Marco Marra and others at Canada''s Michael Smith Genome Sciences Centre to improve detection of rare transcripts in long-lived mutants of C. elegans nematode. Tags were generated using the enzyme Mme I and subjected to parallel sequencing of more than 2 million tags per library in an Illumina flow cell, increasing yield of tag sequences 10-fold, while reducing overall cost by 3-fold. Tag-Seq detected transcripts on average from 11,000 of the 20,000 C. elegans genes, as compared to our previous SAGE libraries (about 100,000 tags each), which detected about 5,000 transcript species in a library. Detection of abundant transcripts for ribosomal proteins increased from a mean of 129 tags per gene in SAGE libraries to over 4,700 in Tag-Seq libraries. Tags for the less abundant transcriptional regulators increased from a mean of 3 to a mean of 96 tags per gene. With construction of multiple replicate libraries, we were able to conduct a much more detailed analysis of transcription profiles for long-lived dauer larvae and adults. Similar to our earlier SAGE study, we compared the expression data for daf-2 (insulin/IGF-1 receptor) and daf-2(+) adults and dauer larvae. We identified genes for which the expression was consistently changed in long-lived worms. The lists appear to show a strong biological relevance as we observed several ''signature'' genes among those up-regulated in dauer larvae: cki-1, aak-2, daf-16 and sod-3 (a well known target of the daf-16 transcription factor) among the genes consistently up-regulated in daf-2 adults. Having analyzed data for animals grown in liquid culture or on agar plates, we were able to distinguish between changes in gene expression associated with altered genotype rather than the environment.

Wang, Jennifer T. et al. (2013) International Worm Meeting "Asymmetric segregation of P granules requires granule remodeling by two novel serine-rich proteins."

Seydoux, Geraldine, Wang, Jennifer T.

[International Worm Meeting, 2013]

P granules are RNA-rich granules that are found exclusively in the germline. In adult germ cells, P granules are mostly perinuclear and stable. In contrast, in early embryos, P granules are cytoplasmic and highly dynamic. Activation of P granule dynamics in embryos is required to segregate P granules asymmetrically to the nascent germline [1,2]. We have found that activation of P granule dynamics requires P granule remodeling by two serine-rich proteins. We used a GFP::PGL-1 fusion to analyze P granule dynamics during the oocyte-to-embryo transition. Oocyte P granules are disassembled during ovulation and reappear during meiosis throughout the cytoplasm of the newly fertilized zygote. A similar disassembly/reassembly cycle is repeated in the zygote at each mitotic division, except that reassembly occurs preferentially in the cytoplasm destined for the germline blastomere. We have found that depletion of GEI-12 and its paralog C36C9.1 blocks the disassembly/reassembly cycle. In gei-12+C36C9.1(RNAi), oocyte P granules persist through ovulation and the first mitotic division, and do not segregate asymmetrically. Formation of new P granules is also blocked. GEI-12 and C36C9.1 are 70% identical serine-rich proteins with no recognizable motifs. Sequence analyses indicate that these proteins have low-complexity sequence (LCS) throughout their length. LCS domains have recently been proposed to form dynamic fibers that hold RNA granules together [3,4]. Consistent with functioning as a P granule scaffold, GEI-12 associates with P granules after reassembly in embryos. Deconvolution confocal microscopy suggests that, in the reassembled P granules, GEI-12 localizes to a core that joins together several small PGL-1 granules. We conclude that 1) GEI-12 and C36C9.1 remodel P granules during the oocyte-to-embryo transition and 2) P granule remodeling is essential for the asymmetric segregation of P granules in embryos. 1. Brangwynne CP et al (2009). Science 324, 1729 2. Gallo C et al (2010). Science 330(6011) 1685 3. Kato M et al (2012). Cell 149(4), 753-767 4. Han TW et al (2012). Cell 149(4), 768-779.

Chissoe SL et al. (1997) International C. elegans Meeting "Characterization of a C. elegans Fosmid Library: Investigating Representation and Sequence Closure"

Chissoe SL, Wilson RK, Marra MA, Waterston RH, Shownkeen R, McDonald K, Coulson AR

[International C. elegans Meeting, 1997]

The physical map of the 100 Mb C. elegans genome consists of 17,600 mapped cosmids and 3,000 mapped YACs, which together contain in excess of 95% of the genome, and more than 99% of the genes. The central gene-rich portions of the chromosomes are well represented in cosmids, and comprise about 60% of the genome. In contrast, the gene-poor chromosomal arms are not well represented in cosmids, and comprise about 40% of the genome. Approximately half of the chromosomal arm regions is represented by cosmids and the other half (or approximately 20% of the genome) is covered only by YAC clones. As the systematic large scale sequencing effort progresses (65% of the genome sequence is finished and another 20% of the genome is in active sequencing), we increasingly have focused our attention on strategies to provide sequence-ready clones for the remainder of the C. elegans genome (see abstract by Alan Coulson). To examine the extent to which these cosmid-lacking regions of the C. elegans genome can be recovered in single copy vectors, we have constructed a genomic library in the fosmid vector pFOS1. Approximately 18,000 C. elegans fosmids have been picked and gridded onto high density filter arrays (courtesy of Genome Systems, Inc., St. Louis, MO). The analysis of radioactive fingerprint data from random C. elegans fosmids and from those identified by direct probing has indicated that we may expect to recover half of the cosmid-lacking regions in fosmid clones. Furthermore, the deletion rate observed in random C. elegans fosmids is much lower than for random cosmids, and we are investigating the stability of selected C. elegans fosmids specific for regions known to delete when cloned in cosmids. Recently, we have generated an in silico fingerprint database derived from finished genomic sequence data and are incorporating agarose gel-based restriction digest data from random C. elegans fosmid clones. This method (see abstract by Marco Marra) could enable rapid identification of potential gap closing fosmid clones using a non-radioactive, high-throughput approach. The current progress of the use of fosmid clones to complement the existing YAC and cosmid coverage of the C. elegans genome will be presented.