Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.     

Genetic and molecular analysis of the dpy-14 region in Caenorhabditis elegans.

Summary Essential genes have been identified in the 1.5 map unit (m.u.)dpy-14-unc-29 region of chromosome I inCaenorhabditis elegans. Previous work defined nine genes with visible mutant phenotypes and nine genes with lethal mutant phenotypes. In this study, we have identified an additional 28 essential genes with 97 lethal mutations. The mutations were mapped using eleven duplication breakpoints, eight deficiencies and three-factor recombination experiments. Genes required for the early stages of development were common, with 24 of the 37 essential genes having mutant phenotypes arresting at an early larval stage. Most mutants of a gene have the same time of arrest; only four of the 20 essential genes with multiple alleles have alleles with different phenotypes. From the analysis of complementing alleles oflet-389, alleles with the same time-of-arrest phenotype were classified as either hypomorphic or amorphic. Mutants oflet-605, let-534 andunc-37 have both uncoordinated and lethal phenotypes, suggesting that these genes are required for the coordination of movement and for viability. The physical and genetic maps in thedpy-14 region were linked by positioning two N2/BO polymorphisms with respect to duplications in the region, and by localizing the right breakpoint of the deficiencyhDf8 on the physical map. Using cross-species hybridization toC. briggsae, ten regions of homology have been identified, eight of which are known to be coding regions, based on Northern analysis and/or the isolation of cDNA clones.     

The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans

Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.     

Lethal mutations defining 112 complementation groups in a 4.5 Mb sequenced region of Caenorhabditis elegans chromosome III

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2?Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans.     

Caenorhabditis elegans,a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.     

Using <Emphasis Type="Italic">C. elegans</Emphasis> to screen for targets of ethanol and behavior-altering drugs

Caenorhabditis elegans is an attractive model system for determining the targets of neuroactive compounds. Genetic screens in C. elegans provide a relatively unbiased approach to the identification of genes that are essential for behavioral effects of drugs and neuroactive compounds such as alcohol. Much work in vertebrate systems has identified multiple potential targets of ethanol but which, if any, of those candidates are responsible for the behavioral effects of alcohol is uncertain. Here we provide detailed methodology for a genetic screen for mutants of C. elegans that are resistant to the depressive effects of ethanol on locomotion and for the subsequent behavioral analysis of those mutants. The methods we describe should also be applicable for use in screening for mutants that are resistant or hypersensitive to many neuroactive compounds and for identifying the molecular targets or biochemical pathways mediating drug responses. Published: June 8, 2004.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Autophagy is Required for Dietary Restriction-Mediated Life Span Extension in C. elegans

Dietary restriction extends life span in diverse species including Canorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.     

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.     

Functional redundancy of two C. elegans homologs of the histone chaperone Asf1 in germline DNA replication

Eukaryotic genomes contain either one or two genes encoding homologs of the highly conserved histone chaperone Asf1, however, little is known of their in vivo roles in animal development. UNC-85 is one of the two Caenorhabditis elegans Asf1 homologs and functions in post-embryonic replication in neuroblasts. Although UNC-85 is broadly expressed in replicating cells, the specificity of the mutant phenotype suggested possible redundancy with the second C. elegans Asf1 homolog, ASFL-1. The asfl-1 mRNA is expressed in the meiotic region of the germline, and mutants in either Asf1 genes have reduced brood sizes and low penetrance defects in gametogenesis. The asfl-1, unc-85 double mutants are sterile, displaying defects in oogenesis and spermatogenesis, and analysis of DNA synthesis revealed that DNA replication in the germline is blocked. Analysis of somatic phenotypes previously observed in unc-85 mutants revealed that they are neither observed in asfl-1 mutants, nor enhanced in the double mutants, with the exception of enhanced male tail abnormalities in the double mutants. These results suggest that the two Asf1 homologs have partially overlapping functions in the germline, while UNC-85 is primarily responsible for several Asf1 functions in somatic cells, and is more generally involved in replication throughout development.     

Suppressors of Glp-1, a Gene Required for Cell Communication during Development in Caenorhabditis Elegans, Define a Set of Interacting Genes

The glp-1 gene is essential for two cell interactions that control cell fate in Caenorhabditis elegans: induction of anterior pharynx in the embryo and induction of mitotic proliferation in the germ line. To identify other genes involved in these cell interactions, we have isolated suppressors of two temperature sensitive alleles of glp-1. Each of 14 recessive suppressors rescues both embryonic and germline glp-1(ts) defects. These suppressors are extragenic and define a set of six genes designated sog, for suppressor of glp-1. Suppression of glp-1 is the only obvious phenotype associated with sog mutations. Mutations in different sog genes show allele-specific intergenic noncomplementation, suggesting that the sog gene products may interact. In addition, we have analyzed a semidominant mutation that suppresses only the glp-1 germline phenotype and has a conditional feminized phenotype of its own. None of the suppressors rescues a glp-1 null mutation and therefore they do not bypass a requirement for glp-1. Distal tip cell function remains necessary for germline proliferation in suppressed animals. These suppressor mutations identify genes that may encode other components of the glp-1 mediated cell-signaling pathway or regulate glp-1 expression.     

Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.     

Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.     

Autophagy genes mediate the effect of calcineurin on life span in C. elegans

Calcineurin (CaN) is a serine/threonine phosphatase, activated by Ca²⁺/calmodulin (Ca²⁺/CaM). CaN is known to regulate various cellular responses in different organisms. A recent study showed an extended life span in the calcineurin mutants of C. elegans. In this study, we report that calcineurin defective strains exhibit enhanced autophagy. In addition, we found two essential autophagy genes (bec-1 and atg-7) are required for the life-span extension in calcineurin null mutants [cnb-1(jh103)]. Thus, for the first time we suggest that autophagy genes are required for the life-span regulation in calcineurin defective C. elegans strains.     

Mitochondrial Dysfunction Confers Resistance to Multiple Drugs in Caenorhabditis elegans

In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCε.