Yuen-Yi Tseng et al. (2007) International Worm Meeting "Caenorhabditis elegans expresses a functional ArsA ATPase."

Vivian Liao, Chan-Wei Yu, Jui-Tung Liu, Yuen-Yi Tseng

[International Worm Meeting, 2007]

As arsenic is the most prevalent environmental toxin, it is imperative for us to understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Herein, we describe the identification of a Caenorhabditis elegans homologue (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We have evaluated the responses of wild-type and asna-1 mutant nematodes to various metal ions and found that asna-1 mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than that of wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans'' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)- ASNA-1 fusion protein was biochemically characterized, and its properties were compared with those of ArsAs. ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 ± 0.2 (SEM) fold, while Sb(III) stimulated it by 4.6 ± 0.15 (SEM) fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria but extend to animals by demonstrating that the asna-1 gene of the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.

Galagali, H. et al. (2019) International Worm Meeting "UNK-1 and CRI-1 modulate the activity of miRISC during dauer in C. elegans."

Yates III, J.R., Diedrich, J., Hill, M., Kim, J., Karp, X., Alessi, A., Galagali, H., Smith, I.T., Schmitter, A.

[International Worm Meeting, 2019]

C. elegans enters the stress-resistant dauer stage when exposed to unfavorable conditions like crowding and starvation early during development. The dauer stage is characterized by developmental arrest and negligible protein synthesis. The molecular pathways involved in dauer entry have been extensively studied. However, the mechanisms involved in maintaining developmental arrest and multipotency at the post transcriptional level remain unknown. MicroRNAs associate with the miRNA-Induced silencing complex (miRISC) and regulate gene expression programs during development inC. elegans. Here, we report that the zinc finger domain protein UNK-1 (C34D10.2) and the relatively uncharacterized protein CRI-1 modulate the efficiency of silencing of the targets of let-7 family microRNAs during dauer. Knockdown of unk-1and cri-1 led to the precocious expression of the adult collagen marker COL-19::GFP in dauer worms. The expression of UNK-1 is upregulated at the transcriptional level in a FOXO/DAF-16 dependent manner during dauer. UNK-1 is expressed in several tissues including neurons, hypodermis and body wall muscle. Immunoprecipitation followed by mass spectrometry revealed that UNK-1 and CRI-1 interact with each other and with the microRNA Argonautes ALG-1 and ALG-2 during dauer. Although microRNA levels are maintained, unk-1 mutants fail to downregulate target mRNAs during dauer, suggesting that UNK-1 is required for efficient microRNA mediated target degradation. However, when constitutive dauer forming daf-7(-); unk-1(-) animals are used, defective silencing is only observed about 20 hours after the dauer molt, indicating that UNK-1 may play a differential role during dauer entry and dauer maintenance. Future experiments in the lab aim to determine the mechanism by which UNK-1 and CRI-1 regulate miRISC to maintain multipotency during dauer.

Ikegami, Kohta et al. (2013) International Worm Meeting "Integral nuclear pore components associate with Pol III-transcribed genes and are required for Pol III transcript processing in C. elegans."

Lieb, Jason, Ikegami, Kohta

[International Worm Meeting, 2013]

Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of small non-coding RNAs transcribed by RNA Polymerase III (Pol III). Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral components of the nuclear pore, and importin-b IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and small nucleolar RNA (snoRNA) genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for its recruitment. Instead, NPP-13 are required for the cleavage of snoRNA precursor transcripts into mature snoRNAs, whereas processing of Pol II transcripts occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and Pol III transcript processing in C. elegans.

Chan-Wei Yu et al. (2007) International Worm Meeting "Characterization and regulation of the C. elegans GCS-1 by arsenite."

Chan-Wei Yu, Vivian Liao

[International Worm Meeting, 2007]

Arsenic is a potent toxin and carcinogen. We previously demonstrated that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress (Yu and Liao, 2005). Herein we investigate the regulation of GCS-1 in response to As(III). Our results showed that As(III) caused a concentration-dependent increase in the GSH levels up to a concentration of 10 <font face=symbol>m</font>M. Real-time RT-PCR analysis shows that gcs-1 mRNA transcript is most abundant in L1, L2 and adult stages. Whereas GCS-1 mRNA at L1 and dauer stages exhibited higher induction ratio after 1 h of As(III) exposure. Moreover, we observed that the increase in GCS-1 mRNA expression level caused by As(III) shows dose-dependence fashion, in parallel with the increase in the GSH level. RNAi feeding assay showed that worms that were fed GCS-1 dsRNA and were grown in the presence of 0.25 mM As(III) were obviously small and development arrest. Transcription of gcs-1 was observed in the pharynx, ASI chemosensory neurons, and intestine of post-embryonic C. elegans and upon exposure to As(III), As(V), or Sb(III), the intestinal expression increases dramatically; whereas Cd(II) induced significant expression in the pharynx. Together, our results indicated that GCS-1 is developmentally regulated and it plays a critical role for the protection of C. elegans against arsenic-induced oxidative stress.

Maduro MF et al. (1996) West Coast Worm Meeting "THE LG III DEFICIENCY eDf2 AND ASSOCIATED FREE DUPLICATION eDp6 ARE NOT MUTUALLY COMPLEMENTARY"

Maduro MF, Pilgrim DB

[West Coast Worm Meeting, 1996]

The deficiency eDf2 and the free duplication eDp6 were obtained concomitantly following acetaldehyde mutagenesis (Hodgkin, 1980). These two aberrations have been useful in mapping many LG III loci, which have demonstrated that eDp6 contains the right third of LG III and eDf2 contains the left portion. We used the eDf2 and eDp6 breakpoints as physical markers to clone unc-119, and showed that the genomic region corresponding to the unc-119 cDNA is completely absent. The gene unc-119 is the only LG III locus to not map to either eDf2 or eDp6. Our interest in these aberrations was renewed when we ran across a report by Starich et al. on the isolation of dyf-2 which maps to the far end of LG III(R) and is complemented by both eDf2 and eDp6. Using a plasmid mini-library approach, we walked across the eDf2 breakpoint, and probed the YAC grid to find that eDf2 contains sequences from the far right end of III. What is strange is that these same sequences are also present on eDp6, suggesting that eDf2 and eDp6 do not result from a simple chromosomal break, and the use of these rearrangements for mapping genes on LG III(R) should be approached with caution. The mapping evidence will be summarized and a model for the creation of eDf2 and eDp6 will be given.

Yamakawa, A. et al. (2009) International Worm Meeting "Analysis of immunogenic proteins in C. elegans."

Suzuki, J., Takimoto, M., Yamakawa, A., Tani, K.

[International Worm Meeting, 2009]

Toward construction of monoclonal antibody libraries covering C. elegans immunogenic proteins, we immunized Balb/c mice with crude extracts from heterogenous population of C. elegans. The spleen cells from the immunized mice were fused with P3U1 myeloma cells and the hybridomas producing monoclonal antibodies were synthesized. Up to the present, the 35 kinds monoclonal anti-C. elegans antibodies were selected based on the strong response to C. elegans crude extracts. To examine the C. elegans immunogenic proteins detected by these monoclonal antibodies, following experiments were done. At first, as the result of Western blotting analysis, tentatively 16 antibodies were classified into 4 types ( I, II, III and IV ). In these 4 types of monoclonal antibodies, the type III monoclonal antibody reacted with several C. elegans proteins which were main immunogenic proteins detected by antiserum against C. elegans. The next, the antiserum and three types ( I, III and IV) of these monoclonal antibodies were applied to the immunohistochemical staining of C. elegans. Type I monoclonal antibody stained the both of body and mouth, type III monoclonal antibody stained the both of intestine and a surface of body, and type IV monoclonal antibody stained intestine. On the other hand, antiserum stained the both of intestine and a surface of body, as well as type III monoclonal antibody. These results derived from Western blotting and immunohistochemical analysis suggest that the type III monoclonal antibody is one of the major antibody in antiserum.

Vivian Hsiu-chuan Liao et al. (2005) International Worm Meeting "gcs-1 confers resistance to arsenic-induced oxidative stress in Caenorhabditis elegans"

Chan-Wei Yu, Vivian Hsiu-chuan Liao

[International Worm Meeting, 2005]

47;-Glutamylcysteine synthetase (47;-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type / As(V) > gcs-1 / As(V) > wild-type / As(III) > gcs-1 / As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Addition of GSH significantly raised the survival rate of gcs-1mutant worms compared to As(III)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.

Ayanori Yamakawa et al. (2003) International Worm Meeting "IMMUNOGENIC PROTEINS IN C. elegans"

Ryuji Hosono, Yumi Sasano, Ayanori Yamakawa, Kazumi Sugitani

[International Worm Meeting, 2003]

To construct monoclonal antibodies covering C. elegans immunogenic proteins, we immunized Balb/c mice with crude extracts from heterogenous population of C. elegans. The spleen cells from the immunized mice were fused with P3U1 myeloma cells. Derived 35 kinds monoclonal anti-C. elegans antibodies were analyzed by Western blotting. Tentatively 18 antibodies were classified into five types (I, II, III, IV and V) based on band sizes detected on the blots. Antibodies (5 kinds) belonging to type I detected the 45kDa-band. Antibodies (3 kinds) belonging to type II detected the 60kDa-band. Antibodies (7 kinds) belonging to type III detected several bands in addition to the 40kDa-band. Antibody (1 kind) belonging to type IV detected several bands in addition to the 55-kDa-band. And antibodies (2 kinds) belonging to type V detected the 55kDa-band. These results indicate that in C. elegans at least three kinds of antigens with strong immunogenicity are present. We are especially interested in antibodies belonging to type III and investigated antigens by the immunohistochemical technique. 3D11, one of type III monoclonal antibodies, stained the intestinal regions. This antibody stained intensely the lumen rather than intestinal cells. Therefore, type III monoclonal antibodies may react with intestinal secretion such as digestive enzymes. We are now studying more detailed immunohistochemical localization of the antigens.

Y Kim et al. (1999) International C. elegans Meeting "cDNA cloning and functional analysis of the C. elegans dna topoisomerase IIIa gene"

Y Kim, H Koo

[International C. elegans Meeting, 1999]

Eukaryotic DNA topoisomerase III which is classified as type I DNA topoisomerase catalyzes the partial removal of negative DNA supercoils. The enzyme is required to maintain genome stability, and its null mutation blocks yeast sporulation and the embryogenesis of mice. cDNA clones of C. elegans DNA topoisomerase III a were obtained by performing RT-PCR using degenerate deoxynucleotides encoding amino acid sequences conserved in the human and yeast enzymes and then screening a C. elegans cDNA library using the RT-PCR product as a probe. The exact 5'-end cDNA clone was obtained by carrying out RT-PCR using a SL1 primer, which produced a 2.4 kb long cDNA sequence in combination with the clone from the cDNA library. C. elegans DNA topoisomerase III a polypeptide overexpressed in E. coli showed a catalytic activity of relaxing negatively supercoieled DNA in vitro . When the DNA topoisomerase III a expression in C. elegans was inhibited by microinjecting the double-stranded RNA, blockages in the oocyte formation and early embryogenesis were observed. The strong mRNA expression in the gonad coincided with the RNA interference phenotype. These results suggest that the DNA topoisomerase III a plays an important role in the gametogenesis and also embryogenesis. The specific reaction which the enzyme catalyzes in these developmental progressions remains to be explained.

Ikegami, Kohta et al. (2011) International Worm Meeting "RNA polymerase III transcribes a defined subset of tRNAs and snoRNAs at the nuclear pore in C. elegans."

Lieb, Jason, Ikegami, Kohta

[International Worm Meeting, 2011]

The "gene gating" hypothesis proposes that highly transcribed genes are localized near nuclear pore complexes (NPCs) for efficient mRNA export and translation. While studies in yeast have widely supported this idea, the situation in metazoans is unclear. Using genome-wide chromatin immunoprecipitation (ChIP) with antibodies specific to Nup93/NPP-13, a core component of NPCs, we identified genomic regions associated with NPCs in C. elegans embryos. NPP-13 associates almost exclusively with genes encoding transfer RNAs (tRNAs) and small nucleolar RNAs (snoRNAs). NPP-13 is associated with 27% (163) of all annotated tRNA genes, and 38% (46) of annotated snoRNA genes. ChIP of the RNA polymerase III (Pol III) machinery revealed that when tRNA or snoRNA genes are associated with NPP-13, they are strongly bound by TFIIIB, TFIIIC and Pol III itself. RNA-seq data allowed us to assess transcript levels of individual snoRNA genes and confirmed that almost all (96%) NPP-13-associated snoRNA genes are transcribed. Of snoRNA genes that are not associated with NPP-13, only about half are transcribed, and they are transcribed by Pol II and not Pol III. Pol II-transcribed genes surrounding sites of NPP-13 association are not particularly highly transcribed. Our data suggest that metazoans utilize a gene gating system that involves Pol III-mediated transcription at the nuclear pore, possibly to facilitate export of tRNAs or snoRNAs, ultimately facilitating protein synthesis.