Yamakawa, A. et al. (2011) International Worm Meeting "Analysis of immunogenic proteins in C. elegans (IV)."

Yamakawa, A., Ushiro, M., Tamura, A., Tanaka, T., Fukumoto, J.

[International Worm Meeting, 2011]

To construct monoclonal antibodies covering C. elegans immunogenic proteins, we immunized Balb/c mice with crude extracts from heterogenous population of C. elegans. The antibody producible spleen cells from these immunized mice were fused with P3U1 myeloma cells. Up to this day, the 21 kinds monoclonal anti-C. elegans antibodies were selected based on the strong staining in the immunoblotting for total C. elegans protein and were classified into four types (I, II, III and IV) based on the immunostained-band sizes detected on the Western blots. On the other hand, the 19 kinds monoclonal anti-C. elegans antibodies were additionally selected based on the weak staining in the immunoblotting for total C. elegans protein. These antibodies were analyzed by Western blotting and were classified into the following eleven types. Antibodies (1 kind) belonging to type A detected the 80 kDa-band. Antibody (2 kinds) belonging to type B detected several bands in addition to the 100 kDa-band. Antibody (3 kinds) belonging to type C detected the 180 kDa-band. Antibody (1 kind) belonging to type D detected the 150 kDa-band. Antibody (4 kinds) belonging to type E detected 40 kDa-band and 150 kDa-band. Antibody (3 kinds) belonging to type F detected several bands in addition to the 150 kDa-band. Antibody (1 kind) belonging to type G detected the 175 kDa-band. Antibody (1 kind) belonging to type H detected the 35 kDa-band. Antibody (2 kinds) belonging to type I detected the 20 kDa-band. Antibody (1 kind) belonging to type J detected the 110 kDa-band. Antibody (1 kind) belonging to type K detected several bands in addition to the 175 kDa-band.

Perez de la Cruz I et al. (1996) East Coast Worm Meeting "sup-9, A GENE INVOLVED IN THE REGULATION/COORDINATION OF MUSCLE CONTRACTION"

Perez de la Cruz I, Horvitz HR

[East Coast Worm Meeting, 1996]

Rare altered-function mutations in any of the interacting genes unc-93, sup-9, or sup-10 result in the abnormal regulation or coordination of muscle contraction. These mutants display sluggish movement, are unable to lay eggs, and exhibit a characteristic rubber-band phenotype (when worms are prodded on the head, they contract and relax without moving backwards) (1,2,3). Loss-of-function mutations in any of these three genes completely suppress a rubber-band mutation in any of the three genes, suggesting that all three act at the same step, possibly as subunits of a protein complex(1). Two additional genes have been identified through reversion screens of rubber-band mutants: loss-of-function sup-18 alleles completely suppress the rubber-band phenotype caused by sup-10(n983) while only partially suppressing the effects of unc-93(e1500) and sup-9(n1550) rubber-band alleles(2); rare sup-11 alleles were isolated as dominant suppressors of unc-93(e1500) and act as recessive, partial suppressors of sup-9 and sup-10 rubber-band alleles. Both unc-93 and sup-10 have been cloned. unc-93 encodes a novel, putative transmembrane protein(4). sup-10 also encodes a novel protein (C. Cummins, J. Levin, H. R. Horvitz and P. Anderson, unpublished results). Cloning sup-9 may provide new insights into the function of these interacting genes, especially since extensive genetic analysis of rubber-band mutants has yielded over 70 alleles of this gene, including loss-of-function, partial loss-of-function, and dominant-negative alleles. We are using transposon-tagged alleles of sup-9 isolated as suppressors of unc-93(e1500) to clone this gene. 1. Greenwald, I. and H.R. Horvitz (1980). Genetics 96, 147-164. 2. Greenwald, I. and H.R. Horvitz (1986). Genetics 113, 63-72. 3. Levin, J. and H. R. Horvitz (1992). Genetics 135, 53-70. 4. Levin, J. and H. R. Horvitz (1992). Journal of Cell Biology 117, 143-155.

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.

SJ Kwon et al. (1999) International C. elegans Meeting "Characterization of a C. elegans homologue of band 4.1 protein"

SJ Kwon, JH Ahnn, SK Jung

[International C. elegans Meeting, 1999]

Band 4.1 is a cytoskeleton protein found in human erythrocyte. It has been reported that Band 4.1 regulates membrane mechanical properties through modulation of interaction between spectrin and actin, and also regulates band 3-ankyrin interaction. However, the exact biochemical and pyshiological function of this protein has not been elucidated. When we searched the Yuji Kohara 's in situ database for genes specially expressed in body-wall muscle, we came across a clone, yk21h2 . This clone appeared to be expressed only in body-wall muscle during embryogenesis. We found a gene, F43d9.1 , which shows high homology with band 4.1 protein. This protein is highly conserved in the other organisms. It has 83% identity with band 4.1 protein. We are interested in the facts that C. elegans does not have erythrocytes but this protein is so highly conserved that we can suggest it in an evolutional aspect. According to In situ data( Yuji Kohara ), it is transcripted along the body-wall muscle during embryogenesis. So we expect that this protein may be associated with muscle development. Now we are trying to characterize it in C. elegans . To investigate the pyshiological role of this protein, we are trying to see the expression pattern in the C. elegans , and to get the deletion mutant using EMS mutagenesis.

Ayanori Yamakawa et al. (2005) International Worm Meeting "A preliminary work for construction of monoclonal antibody libraries against immunogenic proteins in C. elegans"

Ayanori Yamakawa, Yoshie Miyawaki

[International Worm Meeting, 2005]

Toward construction of monoclonal antibody (MAb) 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. We isolated the 49 kinds of MAbs being classified into 10 groups based on response to antigen and on size of antigen as following method. At first, the 35 of 49 kinds of MAbs were selected, based on the strong staining in the immunoblotting for total C. elegans protein and analyzed by Western blotting. Tentatively the 18 of 35 kinds of MAb were classified into five types (I, II, III, IV and V) based on band sizes detected on the blots. At the second, the 14 of 49 kinds of MAbs were selected, based on the weak staining in the immunoblotting for total C. elegans protein. These MAbs were analyzed by Western blotting and the 6 of 14 kinds of MAbs were classified into the new five types (VI, VII, VIII, IX and X). MAbs (2 kinds) belonging to type VI detected the 20kDa-band. MAb (1 kind) belonging to type VII detected the 37kDa-band. MAb (1 kinds) belonging to type VIII detected the 116kDa-band. MAb (1 kind) belonging to type IX detected the high MW (>205kDa)- band. MAb (1 kind) belonging to type X detected several bands in addition to the 116kDa-band. By the immunohistochemical methods, we are identifying cells showing response to the MAbs. In addition, we are isolating antigens responding to the MAbs and detecting peptide sequences of the antigens.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Perez de la Cruz I et al. (2000) East Coast Worm Meeting "Characterization of sup-11 and sup-18, two regulators of the SUP-9/SUP10/UNC-93 two-pore potassium channel complex"

Perez de la Cruz I, Horvitz HR

[East Coast Worm Meeting, 2000]

Rare altered-function mutations in the genes unc-93, sup-9, and sup-10 result in the abnormal regulation of muscle contraction. These mutants move sluggishly, are unable to lay eggs, and exhibit the rubber-band phenotype: when worms are prodded on the head, they contract and relax along their entire bodies without moving backwards. Genetic studies suggest that these three genes act at the same step and likely encode subunits of a protein complex. We have shown that sup-9 encodes a two-pore K+ channel subunit with similarity to mammalian TASK. unc-93 and sup-10 encode novel putative transmembrane proteins. Loss-of-function mutations in a fourth gene, sup-18, completely suppress the rubber-band phenotype caused by a sup-10(gf) mutation and partially suppress the unc-93(gf) and sup-9(gf) rubber-band phenotypes. We have shown that sup-18 encodes a single-pass transmembrane protein. Its intracellular domain shares sequence similarity with a family of bacterial NADH oxidases. A sup-18::gfp fusion is expressed in muscle and is localized to dense bodies, along with SUP-9 and UNC-93::GFP. We are making deletions in the sup-18::gfp reporter to identify regions of SUP-18 required for colocalization with SUP-9. In addition, we are assaying recombinant SUP-18 for nucleotide binding and dehydrogenase activities in vitro. Rare gain-of-function mutations in sup-11 cause a small scrawny phenotype, completely suppress the unc-93(gf) rubber-band defects and act as partial suppressors of the sup-10(gf) and sup-9(gf) rubber-band phenotypes. Loss-of-functions mutations in sup-11 cause embryonic lethality. While sup-11(gf) and sup-18(lf) mutations each only partially suppress the sup-9(gf) rubber-band phenotype, sup-11(gf); sup-9(gf); sup-18(lf)animals are completely suppressed for the rubber-band phenotype, suggesting that sup-11 and sup-18 act in parallel. Since sup-11 is an essential gene, SUP-11 may interact with channels in addition to SUP-9, with the gain-of-function mutations affecting mainly its interaction with the SUP-9/SUP-10/UNC-93 complex but not with other channels. sup-11 has been mapped to a small interval on LGI. We are further mapping sup-11 against polymorphisms in the region and determining the sequences of candidate genes from sup-11 mutants.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Perez de la Cruz I et al. (1997) International C. elegans Meeting "THE sup-9 RUBBER-BAND GENE MAY ENCODE A POTASSIUM CHANNEL"

Perez de la Cruz I, Horvitz HR

[International C. elegans Meeting, 1997]

Rare altered-function mutations in the genes unc-93, sup-9, or sup-10 result in the abnormal regulation or coordination of muscle contraction. These mutants move sluggishly, are unable to lay eggs, and exhibit a so-called rubber-band phenotype: when worms are prodded on the head, they contract and relax without moving backwards1,2,3. Loss-of-function mutations in any of these three genes completely suppress any rubber-band mutation, suggesting that all three genes act at the same step, possibly by encoding subunits of a protein complex. Loss-of-function mutations in a fourth gene, sup-18, completely suppress the rubber-band phenotype caused by sup-10(n983) and partially suppress the unc-93(e1500) and sup-9(n1550) rubber-band alleles2. unc-93 and sup-10 have been cloned and encode novel putative transmembrane proteins4. To understand better the role of sup-9 and sup-18 in the regulation of muscle contraction we cloned both genes. We identified a sup-9 candidate gene using a transposon-tagging strategy. A cosmid containing this candidate gene rescued the sup-9 mutant phenotype in transformation-rescue experiments. This putative sup-9 gene is predicted to encode a potassium channel containing four transmembrane domains and two P domains. We have sequenced the putative sup-9 gene in several mutants: sup-9(1914) animals have a deletion spanning five of the eight predicted exons, sup-9(n180) animals have G to C change in the splice acceptor site of the first intron, and sup-9(n241) animals carry a valine to isoleucine substitution in a predicted loop region. We mapped sup-18 to the interval between ncl-1 and unc-36. Transformation-rescue experiments showed that a 3.8 kb BstBI-SacI subclone of cosmid C02C2 rescued the sup-18 mutant phenotype. This subclone contains a single predicted gene encoding a novel 344 amino acid transmembrane protein. A spontaneous allele, sup-18(n463), contains a four base pair deletion predicted to result in a truncated protein of 200 amino acids. One possibility is that SUP-18 may act as an auxiliary subunit to regulate the channel conducting properties of SUP-9. 1. Greenwald, I. and H.R. Horvitz (1980). Genetics 96, 147-164. 2. Greenwald, I. and H.R. Horvitz (1986). Genetics 113, 63-72. 3. Levin, J. and H. R. Horvitz (1992). Genetics 135, 53-70. 4. Levin, J. and H. R. Horvitz (1992). Journal of Cell Biol. 117, 143-155

Lee RC et al. (1991) International C. elegans Meeting "CLONING OF THE HETEROCHRONIC GENE lin-4 BY POLYMORPHISM MAPPING AND TRANSFORMATION RESCUE."

Ambros VR, Lee RC, Feinbaum RL

[International C. elegans Meeting, 1991]

We genetically mapped the lin4 locus to a position on LGII closely linked and to the left of bristol/bergerac polymorphism rnaP13, detected by YAC Y42A4. Using YAC and cosmid clones in the region as probes to southern blots, we analysed DNA from the only lin4 allele available at the time, e912, (see Feinbaum and Ambros, these abstracts) and found that the YAC Y18Cl detected a polymorphism in a 5 kb EcoRI band. Cosmids drawn under Y18Cl on the physical map did not detect this band, so we suspected that lin-4 was at the right end of Y18Cl, which extended into a cosmid-free gap. Therefore, to clone the sequences corresponding to the 5 kb EcoRI band, we used Y18Cl, which detects the 5 kb band, and Y42A4, which almost completely overlaps Y18Cl but does not detect the band, in a differential screen of an EcoRl lambda library prepared by Ann Rougvie. In this way we isolated two different 5 kb EcoRI fragments that both turn out to be partially deleted in lin4(e912) DNA. These EcoRI clones lie not in the gap, but in the contig and to the left of the previous end of Y18Cl. We have therefore revised the extent of Y18Cl to include the cosmids containing these two 5 kb EcoRI fragments. One of the polymorphic 5 kb EcoRI clones, pRL2D, rescues the mutant phenotype of lin-4(e912J, as does an approximately 2.9 kb Cla-l subclone of pRL2d, pRL2DCla. (Two overlapping cosmids, C02B6 and C31Gll, also rescue lin4). Therefore, pRL2DCla contains a functional lin4 gene, although the lin-4(e912) lesion, originally generated by 32P decay, is somewhat complex, involving deletion of more than 5 kb of additional sequence in the region, and partial duplication of another sequence. We are currently sequencing pRL2DCla and testing smaller subclones for rescue. cDNAs that hybridize to the lin-4 rescuing fragments are also under analysis.