Rie Sakamoto et al. (2002) Worm Breeder's Gazette "A JNK/UNC-16 signaling complex regulates synaptic vesicle localization through a conventional kinesin in C. elegans"

Dana Thyra Byrd, Rie Sakamoto, Masato Kawasaki, Naoki Hisamoto, Yishi Jin, Kunihiro Matsumoto

[Worm Breeder's Gazette, 2002]

The c-Jun N-terminal kinase (JNK) of the MAP kinase (MAPK) superfamily is involved in various stress responses and apoptosis in mammal. The C. elegans JNK cascade is composed of JNK-1 (MAPK) and JKK-1 (MAPKK). The JNK-1 pathway functions in type-D GABAergic motor neuron and modulates coordinated locomotion. The C. elegans unc-16 gene encodes a protein homologous to mammalian JSAP1/JIP3, which acts as a scaffold protein in the JNK pathway by binding with MLK (MAPKKK), MKK7 (MAPKK) and JNK3 (MAPK). Like JSAP1/JIP3, UNC-16 physically interacts with JNK-1 and JKK-1, forming a JNK signaling module. unc-16(ju79) , jnk-1(gk7) , and jkk-1(km2) mutant animals exhibit mislocalization of synaptic vesicle marker SNB-1::GFP in L1 DD motor neurons. This suggests that the JNK-1 pathway containing UNC-16 regulates synaptic vesicle localization. To understand the mechanism regulated by UNC-16, we screened for UNC-16-binding proteins using yeast two-hybrid system. One of the isolated genes is klc-2 encoding a kinesin light chain. Co-immunoprecipitation experiments reveal that KLC-2 associates with UNC-16 and UNC-116 kinesin heavy chain when they are co-expressed in mammalian cells. We constructed the klc-2(km11) mutation that produces a truncated form of the KLC-2 protein lacking its C-terminal portion. The mutant animals exhibit Unc and weak Dpy phenotypes. Similar to unc-16(ju79) , jnk-1(gk7) , and jkk-1(km2) mutants, the SNB-1::GFP marker is mislocalized along the dorsal DD processes in the klc-2(km11) L1 larvae. These results raise the possibility that the UNC-16-JNK-1 pathway regulates synaptic vesicle localization through phosphorylation of KLC-2 in C. elegans .

Greenwald IS et al. (1986) Worm Breeder's Gazette "the lin-12 contig."

Greenwald IS, Sulston JE, Coulson AR, Priess JR

[Worm Breeder's Gazette, 1986]

[See Figure 1]

Villeneuve AM et al. (1985) Worm Breeder's Gazette "egl-16: a gene which unites the sex determination and dosage compensation pathways."

Meyer BJ, Villeneuve AM

[Worm Breeder's Gazette, 1985]

At the recent CSH worm meeting we reported that mutations in the gene egl-16 1) can promote male development in XX animals, and 2) have phenotypes which are sensitive to X-chromosome dosage, affecting XX but not XO animals. The former assertion was based on the observations that a) egl-16(n485.Y4)/Df XX animals display an incompletely penetrant Tra phenotype, and b) in double mutant strains, egl-16(n485) enhances the sexual transformation phenotypes of weakly masculinizing mutations in the genes tra-2 and egl-41. The latter assertion was based on the fact that her-1(e1520);egl-16(n485) XX hermaphrodites are Egl and slightly short, while XO hermaphrodites are essentially wild-type. Likewise, tra-1(e1099.e1488); 85) XX animals are short pseudomales with abnormal bursae, while XO males are wild-type in length and have normal bursae. Since the meeting, we have extended our understanding of egl-16 in several ways: The sexual transformation phenotype of egl-16 is effected through the major sex determination pathway. XX animals homozygous for the weak allele tra-2(n1106) are not visibly transformed but are merely HSN Egl hermaphrodites. The vast majority of animals of the genotype tra-2(n1106)unc-4(e120);egl-16( n485), however, are either visibly sexually transformed or sterile; a homozygous strain of this genotype cannot be maintained. To test whether this enhancement of sexual transformation caused by the egl-16 mutation occurs through the major sex determination pathway, we constructed the triple mutant her-1(e1520);tra-2(n1106)unc-4(e120); 85). The her-1 mutation blocks sexual transformation in this self-fertile Egl hermaphrodite strain, indicating that the egl-16 mutation promotes masculinization directly through the major sex determination pathway rather than via some peripheral mechanism. Arguing from epistasis, egl-16 acts upstream of her-1, perhaps by modulating her-1 activity. egl-16(n485) disrupts dosage compensation in XX animals. The fact that egl-16(n485) affects XX but not XO animals regardless of sexual phenotype suggested that this mutation may disrupt dosage compensation, producing an elevated level of X-linked gene expression in XX animals. This hypothesis was tested using both the biochemical assay for X- specific mRNA levels (Meyer and Casson) and the morphological assay for lin-14 expression (Plenefisch and De Long) described in this issue.

Morgan PG et al. (2000) Worm Breeder's Gazette "Mutations in two C. elegans respiratory enzymes confer variable hypersensitivity to free radicals and volatile anestetics"

Kayser EB, Sedensky MM, Ishii N, Hartman PS, Morgan PG

[Worm Breeder's Gazette, 2000]

As with mev-1, a mutation in gas-1 resulted in hypersensitivity to the superoxide anion-generating chemical paraquat. gas-1 was more hypersenstive at 20C than 15C, which was not surprising given that gas-1 is difficult to even propagate at 25oC. As with mev-1, the gas-1 mutation also rendered animals hypersensitive to hyperoxia. In addition, the life spans of both gas-1 and mev-1 mutants were significantly shortened by elevated oxygen concentrations. It was determined previously that mev-1 animals were hypermutable to oxygen (Ponder, Hartman and Ishii, unpublished data). Conversely, gas-1 was only weakly hypermutable if at all. Finally, kn1 mutation in mev-1 did not confer hypersensitivity to the volatile anaestetic halothane.

Coulson AR et al. (1990) Worm Breeder's Gazette "GENOME MAP"

Coulson AR, Waterston RH, Sulston JE

[Worm Breeder's Gazette, 1990]

[See Figures 1-2]

Chan A et al. (2000) Worm Breeder's Gazette "Protocol for Antibody Staining Mitotic and Meiotic Chromosomes in the Gonad"

Chan A, Meyer BJ

[Worm Breeder's Gazette, 2000]

1. For any specific antibody try 1%, 1.5%, or 2% paraformaldehyde (PFA) with 5-minute fix:

Coulson AR et al. (1993) Worm Breeder's Gazette "The Genome Map of C. elegans"

Lutterbach B, Sulston JE, Waterston RH, Kozono Y, Coulson AR, Shownkeen R

[Worm Breeder's Gazette, 1993]

See Figures 1-6

Tanaka M et al. (1998) Worm Breeder's Gazette "Role of SEK-1 MAP Kinase Kinase in C.elegans"

Suzuki N, Kawasaki M, Matsumoto K, Tanaka M, Hisamoto N

[Worm Breeder's Gazette, 1998]

JNK and p38 belong to a subgroup of the mitogen-activated protein kinase (MAPK) superfamily and are activated in response to a variety of stresses in mammalian cells. SEK1/MKK4 is known to activate both JNK and p38 MAPKs as MAPK Kinase. We have identified a protein kinase, SEK-1, which has significant similarity with mammalian SEK1. SEK-1 can activate both of C. elegans JNK homolog JNK-1 and p38 homologs PMK-1 and PMK-2 in yeast Hog1 MAPK system. These results suggested that SEK-1 has substrate specificity similar to mammalian SEK1. To determine the SEK-1 expression pattern, we generated the animals carrying sek-1::gfp transcriptional fusion constructs. In L1 stage larvae, SEK-1::GFP was expressed in many cells including excretory/secretory cells, enteric muscles, procorpus in pharynx, AVA, AVD, AVE and unidentified neurons. SEK-1::GFP was also expressed in Uterine muscles and Vulval muscles in the adult. To investigate the function of SEK-1, we generated a sek-1 deletion mutant which lacks most of protein kinase catalytic domains. The sek-1 mutant animals did not show obvious developmental defects, but were defective in egg-laying and formed bag-of-worms (penetrancy>90%). Young sek-1 mutant adults responded partially to serotonin but were completely resistant to imipramine in egg-laying. These data suggest that the egl-d phenotype associated with sek-1 deletion is at least caused by defects in the sex muscles and possibily by defects in the secretion of Serotonin from neurons. In order to identify proteins directly involved in SEK-1 function, we screened for SEK-1 binding proteins using an yeast two-hybrid system. We isolated a new cDNA, termed seb-1 (sek-1 binding protein-1), whose product belongs to synaptotagmin family. SEB-1 also interacted JNK-1 by two-hybrid system. Synaptotagmin is an integral membrane protein and important for vesicle transporting including synaptic exocytosis. One possibility is that a SEB-1 may function to anchor both of SEK-1 and JNK-1 to the membrane.

Adachi H et al. (1995) Worm Breeder's Gazette "mev-1 Is a Mutant of Premature Aging."

Hosokawa H, Ishii N, Adachi H

[Worm Breeder's Gazette, 1995]

mev-1 is a mutant of premature aging Hiroshi Adachi (1 2), Hideaki Hosokawa (2) and Naoaki Ishii (2) (1) Research and Development Headquarters, Biological Science Research Center, Lion Corporation, Tajima, Odawara, Kanagawa, 100, (2) Department of Molecular Biology, Tokai University School of Medicine, Isehara, Kanagawa 259-1 1, Japan Mutation in mev-1 render animals hypersensitive to high oxygen concentration (1). The life span of this mutant is shorter than that of wild type under atmospheric conditions and various depending upon the oxygen concentration (2). To further our understanding of mev-1, we. measured the level of peroxylipid and oxidized protein as assessed by fluorescent materials and carbonyl residues, respectively.These are good markers of aging, as it accumulated with increasing aging. Fluorescent materials in methanol/water extracts of both wild type and mev -1 animals accumulated with increasing age. The fluorescent material in mev-1 accumulated more than in the wild type. The amount of the extracts of mev-1 on day 10 was approximately two times that observed in the wild type on the same day (3). We also found that the mev-1 mutant accumuiates more carbonyl residues at a greater rate than dose wild type. When incubated under 90percent oxygen, the fluorescent materials in the mev-1, but not wild-type animals accumulated more rapidly compared with when incubated under atmospheric condition. Conversely, the materials did not accumulated in either wild type or mev-1 animals under 2percent oxygen3. The rate of accumulation of fluorescent material was influenced by oxygen.The activity of superoxide dismutase was about half the wild-type level(1). These observation suggests that the mev-1 may be a mutant of premature aging and oxygen may be a determinant of aging in C. elegans. 1. N. Ishii et al, Mutation Research, 237, 16*171 2. S. Honda et al, J. Geront.48, B*7-B61 (1993) 3. H. Hosokawa et al, Mech. Ageing Develop. 74, 161- 1 70 (1994)

Bakaev VV (1996) Worm Breeder's Gazette "AN ATTEMPT TO SLOW AGING IN C. elegans. 5. THE EFFECT OF ROYAL JELLY EXTRACT WATER SOLUTIONS IN DIFFERENT CONCENTRATIONS"

Bakaev VV

[Worm Breeder's Gazette, 1996]

The purpose of this study was the effect of different concentrations of royal jelly extract on nematode life span. In this experiment royal jelly extract was used in the form of "Apilactosa" (produced by "Fertane", Moscow, Nagatinskaya str., 1) in following dilutions 1:10*3, 1:10*4, 1:10*5, 1:10*6 , 1:10*7 , 1:10*8 , 1:10*9 and 1:10*10. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0,75 ml of liquid medium (with E. coli and without royal jelly extract) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with royal jelly extract in any concentration) every day (one worm in one well). This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Concentration of royal Longevity jelly extract n mean +/- S.D. maximal 1:10*3 12 Lethal 1:10*4 11 18,2 +/- 1,8 28 1:10*5 12 19,2 +/- 2,2 32 1:10*6 12 19,3 +/- 1,7 28 1:10*7 12 15,8 +/- 2,2 28 1:10*8 11 20,6 +/- 3,1 32 1:10*9 8 12,3 +/- 3,5 34 1:10*10 12 17,5 +/- 2,3 31 Conclusion: If the extract from royal jelly was applied to C. elegans during the whole life span in above described conditions, the most effective concentration was 1:10*8. It seemed for me more reasonable to study the effect on longevity of any concentrations near 1:10*8. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (Bristol, N2) and E. coli OP50.