-
[
International Worm Meeting,
2003]
Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial parkinsons disease characterized by selective loss of dopaminergic neurons in substantia nigra and the locus coeruleus. parkin is the causative gene of AR-JP. The human parkin gene encodes 465 amino acids with a ubiquitin-like domain in the amino-terminus and two RING finger motifs in the carboxy terminus. So far, various deletion mutations and point mutations have been discovered in patients of AR-JP, suggesting that the loss of function of Parkin is the cause of AR-JP. Recently we and others showed that Parkin has a ubiquitin-protein ligase activity and therefore suggested that the defect of protein degradation in the neurons of AR-JP patients (Shimura H. et al. Nat. Genet. 25, 302-5, 2000). To investigate the function of Parkin in vivo, we began to analyze the Ce-PARKIN of C. elegans. Two deletion mutations in parkin genes show no defect in their viabilities. The expression of Ce-PARKIN seems to be specific to subset of neurons. Therefore, we focused on the function of Ce-PARKIN in the neurons and the analysis is underway.
-
[
International C. elegans Meeting,
1995]
We have been studying the genetic interactions between the genes
daf-2,
daf-23 and
daf-16 (Gottlieb and Ruvkun 1994). These genes either act in a branch separate from the
daf-11/daf-21 and
daf-7 group of the genetic epistasis pathway for dauer formation or further downstream in the pathway ( Riddle et al. 1981; Vowels and Thomas 1992; Thomas et al. 1993; Gottlieb and Ruvkun 1994). Molecular analysis of
daf-2,
daf-16 as well as
daf-23 (see abstract by Morris et al. this meeting) will help to elucidate function (e.g. neurons vs. target tissues) as well as shed light on the complex genetic interactions of these genes.
-
[
International C. elegans Meeting,
2001]
Mutations are the ultimate source of genetic disorders and offer important data for phylogenetic, forensic and population genetic studies. We directly assayed the rates and patterns of mutation in a set of 74 Caenorhabditis elegans mutation accumulation (MA) lines, where mutations accumulate over time in an effectively neutral manner as each MA line is propagated across generations as a single, random hermaphrodite (Vassilieva and Lynch, 1999). We performed a uniquely direct assay for the underlying rates and patterns of mutation in the MA lines with large-scale DNA sequence collection and analysis from both the nuclear and mitochondrial genomes. The mitochondrial DNA assays revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function and mutational hotspots at homopolymeric nucleotide runs (Denver et.al ., 2000). Mutational hotspots have also been identified in the nuclear DNA sequence assays. Furthermore, we have analyzed the same mitochondrial and nuclear loci in C. elegans natural isolates to evaluate the relative roles of mutation and natural selection in shaping molecular variation observed in natural populations. References: L.L. Vassilieva and M. Lynch , Genetics 151 , 119 (1999). D.R. Denver, K. Morris, M. Lynch, L.L. Vassilieva and W.K. Thomas, Science 289 , 2342 (2000).
-
[
International C. elegans Meeting,
1997]
Phosphoinositide 3-kinases (PI3Ks) have been found in mammals, yeasts, plants, Drosophila and Dictyostelium. They are involved in signal transduction and play a role in cellular processes like mitogenesis, cell growth, differentiation, actin-filament rearrangement, vesicular trafficking, glucose uptake and more. According to their in vitro substrate specificity and primary sequence, PI3Ks can be divided into three classes. In addition to them, there are PI3K-like proteins: ATM (ataxia telangiectasia mutated), DNA-dependent protein kinase (DNA-PKcs) and related genes contain a C-terminal catalytic domain similar to PIKs, but lipid phosphorylation activity has not been shown yet. The latter proteins seem to be involved in DNA repair processes and control of the cell-cycle. For some an intrinsic serine/threonine protein kinase activity has been demonstrated. Two PI3K family members have been identified so far in C.elegans. AGE-1 which controls dauer formation and lifespan (Morris et al., Nature 382, 536-539 (1996)) and a Vps34p homolog (our lab), which is needed for protein sorting to the yeast vacuole. We have recently initiated a search for more genes encoding PI3Ks and PI3K-like proteins in C.elegans. C.elegans database searches have identified promising candidates that are currently being analysed. Antisense-RNA injections shall be used to predict the phenotypes of defective mutants of the respective genes. Further studies will provide us with more information about their role in the development of a multicellular organism.
-
[
International C. elegans Meeting,
2001]
Homopolymeric nucleotide runs are a ubiquitous feature of eukaryotic genomes. Although their dominance in nuclear genomes is clear, homopolymer origins and mechanisms of maintenance and mutation are less obvious. To address these questions, we comprehensively examined the abundance and distribution of homopolymer loci ≥8 nucleotides in length in the genome of C. elegans . There are 148,625 homopolymer loci in the C. elegans genome. The four specific homopolymers are evenly distributed with respect to (+/-) strands of each chromosome. Homopolymer loci are under-represented in exons, show significant clustering in the arms of autosomes, and have significantly different densities between chromosomes. Homopolymers are over-represented in C. elegans genome compared to expectations based on individual nucleotide frequencies. A/T homopolymers vastly outnumber C/G homopolymers, and the size distributions of AT and CG homopolymers differ significantly, particularly for homopolymer loci less than 20 nucleotides in length. A direct investigation of mutation rates at numerous homopolymer loci in a set of C. elegans mutation accumulation lines (Denver, et al. 2000) revealed a significantly higher rate of mutation at C/G loci than at A/T loci. The abundance and distribution of homopolymer loci taken together with direct measures of their mutation rate suggest that differential stability may play a significant role in the maintenance of homopolymer loci. Literature cited: Denver, D.R., Morris, K., Lynch, M., Vassilieva, L., Thomas, W.K. "High direct estimate of the mutation rate in the mitochondrial genome of Caenorhabditis elegans " Science 289:2342-2344.
-
[
International Worm Meeting,
2003]
Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991
-
[
European Worm Meeting,
2004]
Caenorhabditis elegans has been found to be good model system for parasitic nematodes, drug screening and developmental studies. Like the respective parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins, carrying, in part, phosphorylcholine (PC) substitutents, which might play important roles in nematode development, fertility and, at least in the case of parasites, the survival within the host (1). With the exception of a major secretory/ excretory product from Achanthocheilonema viteae (ES-62) (2) and the aspartyl-protease ASP-6 (3), no other proteins carrying this epitope has been identified and studied in detail yet. For C. elegans two N-linked PC-epitopes have been reported so far: (I) a pentamannosyl-core structure carrying three PC-residues (4) and (II) a trimannosyl-core species elongated by a N-acetylglucosamine substituted at C-6 with PC (5). Furthermore, in Dauer larvae of C. elegans there was evidence for the presence of glycans with the composition PC1Hex3HexNAc3 to PC2dHex2Hex4HexNAc7 (6). Here we present the 2D-electrophoretic separation of C. elegans proteins, the comparison of the PC-substitution pattern in distinct developmental stages and the mass spectrometric identification of PC-modified proteins. References: 1.Lochnit, G., Dennis, R. D., and Geyer, R. (2000) Biol Chem 381, 839-847 2.Harnett, W., Harnett, M. M., and Byron, O. (2003) Curr Protein Pept Sci 4, 59-71 3.Lochnit, G., Grabitzki, J., Henkel, B., and Geyer, R. (2003) Biochemical Journal submitted 4.Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002) J Biol Chem 277, 49143-49157 5.Haslam, S. M., Gems, D., Morris, H. R., and Dell, A. (2002) Biochem. Soc. Symp. 69, 117-134 6.Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004) Proc Natl Acad Sci U S A 101, 3404-3408
-
[
International C. elegans Meeting,
2001]
The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage in early C. elegans embryos. PIE-1 has at least two functions in the embryonic germ lineage: 1) inhibition of mRNA transcription, and 2) promotion of efficient NOS-2 expression, a maternal protein required for primordial germ cell development. Structure-function studies have indicated that these two functions require sequences in the C-terminal region of PIE-1 (Tenenhaus et al., 2001). The C-terminus of PIE-1 is also required for asymmetric segregation of PIE-1 in dividing germline blastomeres (Reese et al., 2000). We have used the C-terminus of PIE-1 as a bait in a yeast two hybrid screen in the hope of identifying proteins required for PIE-1 activity and/or localization. Screening of 900,000 transformants yielded 365 candidate interactors (thanks to Zheng Zhou and Bob Horvitz for their excellent library). To quickly identify functionally relevant candidates, we designed a simple protocol to test the in vivo function of each candidate by RNAi. We first isolate the "prey" plasmids from the positive yeast transformants by selection on an auxotrophic E. coli strain. The prey inserts are then PCR amplified and transferred by recombinational cloning into Lisa Timmons and Andy Fire’s feeding vector and transformed into E. coli strain HT115. Resulting transformants are used to "feed" three tester worm strains: 1) a MED-1:GFP line (thanks to Morris Maduro and Joel Rothman) to assay for defects in transcriptional repression, 2) a GFP:
nos-2 3’UTR line to assay for defects in NOS-2 expression, and 3) a PIE-1:GFP line to assay for defects in PIE-1 localization. The screen is ongoing and progress will be reported at the meeting.
-
[
East Coast Worm Meeting,
1996]
A number of dauer-constitutive (Daf-c) and dauer-defective (Daf-d) mutants have been isolated, and most of them are ordered into neuronal signal transduction pathways.
daf-2 and
daf-23 are the only two daf-c genes which are positioned either in a separate branch of the pathway or downstream of
daf-12. In addition, weak
daf-2 and
daf-23 mutants cause prolonged life span of the adult worms, which is not caused by any other daf-c mutants (ref. 1-3). These data suggest that
daf-2 and
daf-23 may play different role(s) from the other daf genes. Recently,
daf-23 has been cloned in our lab (see abstract of J. Morris et al., this meeting) and found to be a homologue of the catalytic subunit of mammalian PI 3-kinase. Because
daf-2 is positioned at the same place of the genetic epistatic pathway as
daf-23, it could be part of the PI 3-kinase signaling cascade. Cloning of
daf-2 could help to elucidate the function of PI 3-kinase signaling in dauer formation and aging. We have mapped
daf-2 gene by RFLP analysis to a region between mgP34 and mgP35 on the left arm of chromosome III. We have detected a
daf-2 allele-specific polymorphism associated with a uv-induced allele using YACs as a probe. We are currently cloning the polymorphic genomic fragment and screening cDNA library using the YAC probes. We are also doing microinjection of the YACs to rescue the
daf-2 phenotype. References: (1) Kenyon et al. (1993) Nature 366, 461-464, (2) Larsen et al. (1995) Genetics 139, 1567-1583, (3) Dorman et al. (1995) Genetics 141, 1399-1406
-
[
International C. elegans Meeting,
1997]
Both
age-1 and
daf-23 mutants show a life-extension (Age) phenotype under some conditions.
age-1 mutants form dauer only at semi-lethal 27 oC, whereas
daf-23 mutants are non-conditional dauer formers with a strong maternal effect. Recently, Malone et al., 1996 and Morris et al, 1996 observed that
age-1(
hx546) fails to complement
daf-23 alleles for Daf-c and Age phenotype. They concluded that
age-1 and
daf-23 are allelic. The latter also found mutation sites in a PI3 kinase gene in
daf-23 alleles but not in
age-1(
hx546). We have sought the location of
age-1 mutation sites, because such information could provide an insight into the !healthy! life extension of
age-1 mutants and an absebce of the maternal effect. Direct sequencing has been used to determine the mutation sites in four
age-1 alleles,
hx546,
z10,
z12, and
z25. We have completed sequencing the PI3 kinase gene including the introns in
hx546 and
z25 and almost completed
z10 and
z12; no mutation site has been found. This argues against the conclusion that
age-1 and
daf-23 are the same gene. Thus, we have re-tested the genetic complementation. Though we have obtained similar results to the previous work, we found two separable Daf-c Age mutants in the
age-1(
hx546) strain (we provisionally call them
age-1(L) and
age-1(R); see also 1996 West Coast Meeting Abstract 137). Both fail to complement
daf-23 mutations. This implicates non-allelic non-complementation between
age-1(L),
age-1(R) and
daf-23. Since only the complementation data suggest
age-1/daf-23 allelism, it is not clear that
age-1 and
daf-23 are the same gene.