Jessica L Goldstein et al. (2003) International Worm Meeting "Characterization of a New Gene Involved in Ras-mediated Vulval Induction"

Kerry Kornfeld, Jessica L Goldstein

[International Worm Meeting, 2003]

The development of the C. elegans vulva requires the activity of the Ras signaling pathway. To identify components of this pathway, we screened for mutations that suppress the multivulva phenotype caused by let-60(n1046gf), a mutation that constitutively activates the ras gene. Forty-three alleles comprising 20 complementation groups were identified. These genes include conserved members of the Ras signaling cascade, such as Raf (lin-45), KSR (ksr-1), MEK (mek-2), MAPK (mpk-1), and an ETS transcription factor (lin-1). We are now analyzing n2528, an allele that defines a new complementation group. The n2528 mutation strongly suppresses the let-60(n1046) Muv phenotype, but does not suppress the lin-1(n383) Muv phenotype, suggesting that the affected gene may act downstream of let-60, but upstream of the transcription factor lin-1. In a wild-type genetic background, the n2528 mutation does not dramatically affect vulval development. n2528 mutants also display a cold-sensitive larval lethal phenotype that can be partially rescued by let-60(n1046). Thus, the gene affected by n2528 may contribute to the function of the Ras pathway at multiple times during development. We used single nucleotide polymorphisms in the Hawaiian strain, CB4856, to map the n2528 mutation to a 17.5 kilobase interval on LGX. We sequenced this interval to identify the molecular lesion, and confirmed the identity of the gene by transformation rescue. We then screened a deletion library to identify a second allele, am114, which appears to be a stronger loss of function allele than n2528. The gene affected by these mutations shares approximately 23% similarity with sec14, the yeast phosphatidylinositol transfer protein, mammalian alpha-tocopherol associated proteins, and squid cytosolic retinal binding protein. These findings suggest a protein that binds small hydrophobic molecules may play an important role in Ras signaling.

Jessica L Goldstein et al. (2002) Mid-west Worm Meeting "Characterization of a New Gene Involved in Ras-mediated Vulval Induction"

Kerry Kornfeld, Jessica L Goldstein

[Mid-west Worm Meeting, 2002]

The development of the C. elegans vulva requires the activity of the Ras signaling pathway. To identify components of this pathway, we screened for mutations that suppress the multivulva phenotype caused by let-60(n1046gf), a mutation that constitutively activates the ras gene. Forty-three alleles comprising 20 complementation groups were identified. These genes include conserved members of the Ras signaling cascade, such as Raf (lin-45), KSR (ksr-1), MEK (mek-2), MAPK (mpk-1), and an ETS transcription factor (lin-1). We are now analyzing n2528, an allele that defines a new complementation group. The n2528 mutation strongly suppresses the let-60(n1046gf) Muv phenotype and partially suppresses the lin-1(e1275) Muv phenotype, suggesting that the affected gene may act downstream of or in parallel to the transcription factor lin-1. In a wild-type genetic background, the n2528 mutation does not dramatically affect vulval development. n2528 mutants also display a cold-sensitive larval lethal phenotype that can be partially rescued by let-60(n1046gf). Thus, the gene affected by n2528 may contribute to the function of the Ras pathway at multiple times during development. We used single nucleotide polymorphisms in the Hawaiian strain, CB4856, to map the n2528 mutation to a 17.5 kilobase interval on LGX. We sequenced this interval to identify the molecular lesion and confirmed the identity of the gene by transformation rescue. The affected gene shares about 23% similarity with sec14, the yeast phosphatidylinositol transfer protein, mammalian alpha-tocopherol associated proteins, and squid cytosolic retinal binding protein, suggesting that proteins which function to bind small hydrophobic molecules may play an important role in Ras signaling.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Forrester S et al. (2007) Foodborne Pathog Dis "Evaluation of the pathogenicity of Listeria spp. in Caenorhabditis elegans."

Schwab U, Wiedmann M, Hoose WA, Milillo SR, Forrester S

[Foodborne Pathog Dis, 2007]

Caenorhabditis has proven to be a useful model for studying host-pathogen interactions as well as the ability of nematodes to serve as vectors for the dispersal of foodborne pathogens. In this study, we evaluated whether C. elegans can serve as a host for Listeria spp. While there was an effect of growth media on C. elegans killing, C. elegans exposed to L. monocytogenes and L. innocua pregrown in Luria-Bertani medium showed reduced survival when compared to nonpathogenic E. coli OP50, while L. seeligeri showed survival similar to E. coli OP50. In a preference assay, C. elegans preferred E. coli over L. monocytogenes and L. innocua, but showed no preference between L. monocytogenes and L. innocua. A gentamicin assay indicated that L. monocytogenes did not persist within the C. elegans intestinal tract. Our findings that L. monocytogenes and L. innocua strains tested have equally deleterious effects on C. elegans and that L. monocytogenes did not establish intestinal infection conflict with other recently published results, which found intestinal infection and killing of C. elegans by L. monocytogenes. Further studies are thus needed to clarify the interactions between L. monocytogenes and C. elegans, including effects of environmental conditions and strain differences on killing and intestinal infection.

Kenyon CJ et al. (1994) Worm Breeder's Gazette "A polyray Suppressor"

Kenyon CJ, Maloof JN

[Worm Breeder's Gazette, 1994]

A polyray-l suppressor Julin Maloof and Cynthia Kenyon, Dept of Biochemistry, U.C.S.F, San Francisco, CA 94143 The polyray-l (pry-l) gene is required to keep homeotic selector (HOM-C) genes repressed where their expression has not been initiated (see accompanying abstract and WBG 12(5):39). pry-l mutant worms have numerous homeotic transformations and are very sick, so we were intrigued by a plate of pry-l mutant worms which appeared much healthier than normal. I outcrossed the strain and found F2s with the full pry-l mutant phenotype, suggesting that the "healthy" strain contained both pry-l and an extragenic suppressor. Does the suppressor have a phenotype on its own? Since pry-l causes homeotic transformations due to ectopic HOM-C expression, it seemed possible that the suppressor would cause transformations in the opposite direction, similar to those seen in HOM-C loss of function mutants. Indeed some F2s from the outcross had descendants of the QL neuroblast in the anterior of the worms, a phenotype also caused by loss-of function mutations in the HOM-C gene mab-5. We are calling this newly identified gene spy-l for suppressor of polyray-l. spy-l seems to suppress all of the pry-l mutant-phenotypes: pry-l; spy-l double mutant worms do not have extra rays, are no longer Muv, are not Unc, and have anteriorly placed QR descendants. spy-l could suppress pry-l either by preventing ectopic HOM-C expression, or by preventing ectopically expressed HOM-C genes from having any effect. mab-5-lacZ expression was examined and found to be normal in the p1y-l; spy-l double, so wild-type spy-l is required for the ectopic HOM-C expression seen in pry-l mutant animals. The fact that there is a QL migration defect in worms mutant for spy-l alone suggests that spy-l may normally be required to turn on mab-5 in QL. We compared expression of mab-5-lacZ in wild- type and spy-l mutant worms at 3.5-5 hours and at 8-10 hours after hatching. At 3.5-5 hours 100% (n=20) of the mutant and 95% (n=20) of the wild-type worms had expression in QL. At 8- 10 hours 0% (n=30) of the mutant worms has expression of mab- 5 in the descendants of QL, but 87% (n=30) of the wild-type worms showed expression. It appears that spy-l is not required to initiate mab-5 expression in QL, but that it is required to maintain expression once it is initiated. It is possible that spy-l is required to maintain the ON state of a number of HOM-C genes, in a manner analogous to the brahma and trithorax genes of Drosophila. spy-l is located on X, in a region well covered by cosmids. Hopefully a molecular analysis of spy-l along with the isolation and study of more spy-l alleles will enable us to determine how similar the C. elegans and Drosophila maintenance systems are, and how spy-l and other maintenance genes are used to maintain and specify pattern.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Abraham D et al. (1989) Am J Trop Med Hyg "Immunity to larval Brugia malayi in BALB/c mice: protective immunity and inhibition of larval development."

Grieve RB, Christensen BM, Abraham D, Holy JM

[Am J Trop Med Hyg, 1989]

The objective of this study was to analyze the immune response of mice to the larval stages of Brugia malayi. Male BALB/c mice were inoculated with 3 doses of irradiated third-stage larvae (L-3) of B. malayi and were subsequently challenged with L-3 implanted ip within diffusion chambers. After 3 weeks, larvae were recovered to determine their viability, length, and stage of development. A significant reduction in parasite survival was observed in immunized mice. Furthermore, larvae recovered from immunized mice were significantly shorter than larvae recovered from control mice. All larvae recovered from immunized mice were L-3, whereas 96% of larvae recovered from controls were fourth-stage larvae (L-4). Sera collected from control and immunized mice were tested for the presence of antibodies reactive with L-3 and L-4 antigens using an indirect fluorescent antibody assay employing frozen larval cross-sections as antigen. Sera recovered after challenge of control mice reacted with internal, but not surface, antigens of L-3 and L-4. Alternatively, sera from immunized mice reacted with both internal and external antigens of both L-3 and L-4.

Grant BD et al. (1994) Worm Breeder's Gazette "sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein."

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1994]

sel-l, a negative regulator of lin-12 and glp-l activity, encodes a novel extracellular protein. Barth Grant and Iva Greenwald HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

Sese BT et al. (2009) J Toxicol Environ Health A "Toxicity of polycyclic aromatic hydrocarbons to the nematode Caenorhabditis elegans."

Reid BJ, Grant A, Sese BT

[J Toxicol Environ Health A, 2009]

The presence of polycyclic aromatic hydrocarbons (PAHs) in the environment has attracted much concern owing to their mutagenic and carcinogenic properties. Regulatory authorities have favored the use of biological indicators as an essential means of assessing potential toxicity of environmental pollutants. This study aimed to assess the toxicity of acenaphthene, phenanthrene, anthracene, fluoranthene, pyrene, and benzo[a]pyrene to Caenorhabditis elegans by measuring LC50 and EC50 values for growth and reproduction. The exposure to all chemicals was carried out in aqueous medium. All PAHs showed a low acute toxicity to C. elegans. There was no significant mortality in C. elegans after 24 h of exposure at PAH concentrations within (and indeed above) their respective solubility limits. Prolonged exposure (72 h) at high concentrations for acenaphthene (70,573 microg/L), phenanthrene (3758 microg/L), anthracene (1600 microg/L), fluoranthene (1955 microg/L), pyrene (1653 microg/L), and benzo[a]pyrene (80 microg/L) produced mortality. Results also showed that reproduction and growth were much more sensitive parameters of adverse response than lethality, and consequently may be more useful in assessing PAH toxicity using C. elegans. In comparison with previous studies, C. elegans was found to be approximately 2-fold less sensitive to acenaphthene, 5-fold less sensitive to phenanthrene, and 20-fold less sensitive to fluoranthene than Daphnia magna. However, the 48-h LC50 for benzo[a]pyrene (174 microg/L) reported in the present study with C. elegans was similar to that reported elsewhere for Daphnia magna (200 microg/L). Although C. elegans indicated greater sensitivity to benzo[a]pyrene than Artemia salina (174 microg/L vs. 10000 microg/L), the organism showed less sensitivity to pyrene (8 microg/L vs. 2418 microg/L), fluoranthene (40 microg/L vs. 2719 microg/L), and phenanthrene (677 microg/L vs. 4772 microg/L) than Artemia salina. Caenorhabditis elegans, while not the most sensitive of species for PAH toxicity assessment, may still hold applicability in screening of contaminated soils and sediments.

Lackner MR et al. (1995) International C. elegans Meeting "The role of mpk-l in vulval development"

Kim SK, Lackner MR

[International C. elegans Meeting, 1995]

The mpk-l gene encodes a MAP kinase protein that is approximately 75% identical to vertebrate MAP kinases. Previously, we have shown that mpk-l plays an important role in ras-mediated induction of vulval cell fates, as reduction-offunction mutations in mpk-l completely suppress excess vulval differentiation caused by a gain-of-function mutation in let-60 ras. We have now characterized a strong reduction-offunction allele of mpk-l that results in temperature-sensitive vulvaless phenotype (84% Vul at 25 degrees Celsius) and a completely penetrant sterile phenotype at all temperatures. Epistasis analyses suggest that mpk-l acts downstream of lin-15 and let-60 ras, but upstream of the putative transcription factors lin-l and lin-31. To determine whether mpk-l is absolutely essential for vulval induction, or whether some induction may occur in the absence of mpk-l activity, we are in the process of isolating a null allele of mpk-l. We used mutant activated forms of mpk-l and Drosophila MEK to address the question of whether activation of these two kinases is sufficient to promote vulval fates in the absence of upstream signaling. Coexpression of both activated mpk-l and activated D-MEK causes a strong Muv phenotype (~90% Muv), whereas coexpression of wild-type mpk-l and wild-type D-MEK results in a weak Muv phenotype (~10% Muv). The strong Muv phenotype is unaffected by ablation of the anchor cell or a reduction-offunction mutation in lin-45 raf, consistent with activation of mpk-l and MEK being at least partially independent of upstream signaling. Our future plans are to identify targets of mpk-l by isolating suppressors of this strong Muv phenotype.