Caenorhabditis Elegans Mutants Research Articles

Caenorhabditis Elegans Mutants Predict Regulation of Fatty Acids and Endocannabinoids by the CYP-35A Gene Family

Background: Cytochrome P450s (CYPs) are mono-oxygenases that metabolize endogenous compounds, such as fatty acids and lipid signaling molecules, and furthermore have a role in metabolism of xenobiotics. In order to investigate the role of CYP genes in fat metabolism at the molecular level, four Caenorhabditis elegans mutants lacking functional CYP-35A1, CYP-35A2, CYP-35A4, and CYP-35A5 were characterized. Relative amounts of fatty acids, as well as endocannabinoids, which regulate weight gain and accumulation of fats in mammals, were measured while fat contents in worms were visualized using Oil-Red-O staining. Results: The cyp-35A1 and cyp-35A5 mutants had a significantly lower intestinal fat content than wild-type animals, whereas cyp-35A2 and cyp-35A4 mutants appeared normal. The overall fatty acid compositions of CYP mutants did not alter dramatically, although modest but significant changes were observed. cyp-35A1 and cyp-35A5 mutants had significantly higher levels of C18:1n7 and lower C18:2n6c. All four mutants had higher relative amounts of C18:1n7 than the wild-type. In the cyp-35A5 mutant, the levels of the endocannabinoid anandamide were found to be 4.6-fold higher than in wild-type. Several fatty acid synthesis genes were over-expressed in cyp-35A1 including fat-2. Feeding oleic or elaidic triglycerides to wild-type animals demonstrated that cyp-35A1 transcriptional levels are insensitive to environmental exposure of these fats, while cyp-35A2, cyp-35A4, and cyp-35A5 were significantly down regulated. Conclusion: These results demonstrate a dynamic role for CYP-35A subfamily members in maintaining the diversity of fatty acid profiles in C. elegans, and more generally highlight the importance of CYPs in generating both structural and signaling fatty acid functions in other organisms.

TRIM-9 functions in the UNC-6/UNC-40 pathway to regulate ventral guidance

TRIpartite Motif (TRIM) family proteins are ring finger domain-containing, multi-domain proteins implicated in many biological processes. Members of the TRIM-9/C-I subfamily of TRIM proteins, including TRIM-9, MID1 and MID2, have neuronal functions and are associated with neurological diseases. To explore whether the functions of C-I TRIM proteins are conserved in invertebrates, we analyzed Caenorhabditis elegans and Drosophila trim-9 mutants. C. elegans trim-9 mutants exhibit defects in the ventral guidance of hermaphrodite specific neuron (HSN) and the touch neuron AVM. Further genetic analyses indicate that TRIM-9 participates in the UNC-6-UNC-40 attraction pathway. Asymmetric distribution of UNC-40 during HSN development is normal in trim-9 mutants. However, the asymmetric localization of MIG-10, a downstream effector of UNC-40, is abolished in trim-9 mutants. These results suggest that TRIM-9 functions upstream of MIG-10 in the UNC-40 pathway. Moreover, we showed that TRIM-9 exhibits E3 ubiquitin ligase activity in vitro and this activity is important for TRIM-9 function in vivo. Additionally, we found that Drosophila trim-9 is required for the midline attraction of a group of sensory neuron axons. Over-expression of the Netrin/UNC-6 receptor Frazzled suppresses the guidance defects in trim-9 mutants. Our study reveals an evolutionarily conserved function of TRIM-9 in the UNC-40/Frazzled-mediated UNC-6/Netrin attraction pathway.

3-Ketoacyl thiolase delays aging ofCaenorhabditis elegansand is required for lifespan extension mediated bysir-2.1

Studies of long-lived Caenorhabditis elegans mutants have identified several genes that function to limit lifespan, i.e., loss-of-function mutations in these genes promote longevity. By contrast, little is known about genes that normally act to delay aging and that when mutated cause premature aging (progeria). To seek such genes, we performed a genetic screen for C. elegans mutants that age prematurely. We found that loss-of-function mutations of the ketoacyl thiolase gene kat-1 result in an increased accumulation of the lipofuscin-like fluorescent aging pigment, shortened lifespan, early behavioral decline, and other abnormalities characteristic of premature aging. These findings suggest that kat-1 acts to delay C. elegans aging. kat-1 encodes a conserved metabolic enzyme that catalyzes the last step of fatty acid oxidation and was previously shown to regulate fat accumulation in worms. We observed that kat-1 is required for the extension of lifespan and enhanced thermotolerance mediated by extra copies of the deacetylase gene sir-2.1. kat-1 acts independently of other known pathways that affect longevity. Our findings suggest that defects in fatty acid oxidation can limit lifespan and accelerate aging in C. elegans and that kat-1-mediated fatty acid oxidation is crucial for overexpressed sir-2.1 to delay aging.

Altered redox status of coenzyme Q9 reflects mitochondrial electron transport chain deficiencies in Caenorhabditis elegans

Mitochondrial disorders are often associated with primary or secondary CoQ10 decrease. In clinical practice, Coenzyme Q10 (CoQ10) levels are measured to diagnose deficiencies and to direct and monitor supplemental therapy. CoQ10 is reduced by complex I or II and oxidized by complex III in the mitochondrial respiratory chain. Therefore, the ratio between the reduced (ubiquinol) and oxidized (ubiquinone) CoQ10 may provide clinically significant information in patients with mitochondrial electron transport chain (ETC) defects. Here, we exploit mutants of Caenorhabditis elegans ( C. elegans) with defined defects of the ETC to demonstrate an altered redox ratio in Coenzyme Q9 (CoQ9), the native quinone in these organisms. The percentage of reduced CoQ9 is decreased in complex I ( gas-1) and complex II ( mev-1) deficient animals, consistent with the diminished activity of these complexes that normally reduce CoQ9. As anticipated, reduced CoQ9 is increased in the complex III deficient mutant ( isp-1), since the oxidase activity of the complex is severely defective. These data provide proof of principle of our hypothesis that an altered redox status of CoQ may be present in respiratory complex deficiencies. The assessment of CoQ10 redox status in patients with mitochondrial disorders may be a simple and useful tool to uncover and monitor specific respiratory complex defects.

Effects of TAT-conjugated platinum nanoparticles on lifespan of mitochondrial electron transport complex I-deficient Caenorhabditis elegans, nuo-1

Platinum nanoparticle (Pt-np) species are superoxide dismutase/catalase mimetics and also have an activity similar to that of mitochondrial electron transport complex I. To examine if this complex I-like activity functions in vivo, we studied the effects of Pt-nps on the lifespan of a mitochondrial complex I-deficient Caenorhabditis elegans mutant, nuo-1 (LB25) compared with wild-type N2. We synthesized a fusion protein of a cell-penetrating peptide, human immunodeficiency virus-1 TAT (48-60), C-terminally linked to a peptide with a high affinity to platinum (GRKKRRQRRRPPQ-DRTSTWR). Pt-nps were functionalized by conjugation with this fusion protein at a 1:1 ratio of TAT-PtBP to Pt atoms. Adult worms were treated with conjugated Pt-nps for 10 days. The mean lifespan of untreated N2 and LB25 was 19.6 ± 0.4 and 11.8 ± 0.3 days, respectively. Using 5 μM of conjugated Pt-nps, the lifespan of N2 and LB25 was maximally extended. This maximal lifespan extension of LB25 was 31.9 ± 2.6%, which was significantly greater than that of N2 (21.1 ± 1.7%, P < 0.05 by Student's t-test). Internalization of Pt into the whole body and mitochondria was similar between these two strains. Excessive accumulation of reactive oxygen species was not observed in the cytosol or mitochondria of untreated LB25. Treatment for five days with 5 μM conjugated Pt-nps decreased cytosolic and mitochondrial reactive oxygen species in N2 and LB25 to a similar extent. The ratio of [NAD(+)]/[NADH] was very low in the whole body and mitochondria of control LB25. After five days of treatment with 5 μM conjugated Pt-nps, the ratio of [NAD(+)]/[NADH] was increased in N2 and LB25. However, the degree of the increase was much higher in LB25 than in N2. Pt-nps function as NADH oxidase and recover the [NAD(+)]/[NADH] ratio in LB25, leading to effective extension of the lifespan of LB25.

Long-lived mitochondrial (Mit) mutants of Caenorhabditis elegans utilize a novel metabolism

The Caenorhabditis elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain (ETC) functionality, yet, surprisingly, they are long lived. We have previously proposed that Mit mutants supplement their energy needs by exploiting alternate energy production pathways normally used by wild-type animals only when exposed to hypoxic conditions. We have also proposed that longevity in the Mit mutants arises as a property of their new metabolic state. If longevity does arise as a function of metabolic state, we would expect to find a common metabolic signature among these animals. To test these predictions, we established a novel approach monitoring the C. elegans exometabolism as a surrogate marker for internal metabolic events. Using HPLC-ultraviolet-based metabolomics and multivariate analyses, we show that long-lived clk-1(qm30) and isp-1(qm150) Mit mutants have a common metabolic profile that is distinct from that of aerobically cultured wild-type animals and, unexpectedly, wild-type animals cultured under severe oxygen deprivation. Moreover, we show that 2 short-lived mitochondrial ETC mutants, mev-1(kn1) and ucr-2.3(pk732), also share a common metabolic signature that is unique. We show that removal of soluble fumarate reductase unexpectedly increases health span in several genetically defined Mit mutants, identifying at least 1 alternate energy production pathway, malate dismutation, that is operative in these animals. Our study suggests long-lived, genetically specified Mit mutants employ a novel metabolism and that life span may well arise as a function of metabolic state.

Intracellular Phospholipase A1and Acyltransferase, Which Are Involved inCaenorhabditis elegansStem Cell Divisions, Determine thesn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A(1) and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Genetic and cellular characterization of Caenorhabditis elegans mutants abnormal in the regulation of many phase II enzymes.

BackgroundThe phase II detoxification enzymes execute a major protective role against xenobiotics as well as endogenous toxicants. To understand how xenobiotics regulate phase II enzyme expression, acrylamide was selected as a model xenobiotic chemical, as it induces a large number and a variety of phase II enzymes, including numerous glutathione S-transferases (GSTs) in Caenorhabditis elegans.Methodology/Principal FindingsTo begin dissecting genetically xenobiotics response pathways (xrep), 24 independent mutants of C. elegans that exhibited abnormal GST expression or regulation against acrylamide were isolated by screening about 3.5×105 genomes of gst::gfp transgenic strains mutagenized with ethyl methanesulfonate (EMS). Complementation testing assigned the mutants to four different genes, named xrep-1, -2, -3, and -4. One of the genes, xrep-1, encodes WDR-23, a nematode homologue of WD repeat-containing protein WDR23. Loss-of-function mutations in xrep-1 mutants resulted in constitutive expression of many GSTs and other phase II enzymes in the absence of acrylamide, and the wild-type xrep-1 allele carried on a DNA construct successfully cured the mutant phenotype of the constitutive enzyme expression.Conclusions/SignificanceGenetic and cellular characterization of xrep-1 mutants suggest that a large number of GSTs and other phase II enzymes induced by acrylamide are under negative regulation by XREP-1 (WDR-23), which is likely to be a functional equivalent of mammalian Keap1 and a regulator of SKN-1, a C. elegans analogue of cap-n-collar Nrf2 (nuclear factor erythroid 2-related factor 2).

Caenorhabditis elegansbehavioral genetics: where are the knobs?

Thousands of behavioral mutants of Caenorhabditis elegans have been studied. I suggest a set of criteria by which some genes important in the evolution of behavior might be recognized, and identify neuropeptide signaling pathways as candidates.

Functional analysis of nematode nicotinic receptors

Parasitic nematodes pose a significant threat to the health and economic welfare of communities worldwide, both directly through human disease and indirectly through infection of livestock and crop. At present, anthelmintic drugs such as the nicotinic agonists, which target nematode nicotinic acetylcholine receptors (nAChRs), offer a facile and cost-effective method of controlling both human and animal infection. Unfortunately, the continued heavy reliance on such compounds has led to the inevitable emergence of resistance in many different Nematoda species, thus making the subject of nematode nicotinic receptors of great importance. Using various levamisole-resistant, nAChR subunit mutants of the soil-dwelling nematode Caenorhabditis elegans (viz. unc-29, unc-38 and unc-63) and well-established resistance-monitoring assays, the responses of the different strains to the nicotinic agonists levamisole, morantel, oxantel and pyrantel have begun to be characterized. Wild-type adult C. elegans are relatively unaffected by 1 mM oxantel, contrasting with a developmental retardation of larvae. Also, unc-38 mutants appear to be less refractory to the anthelmintics than the unc-29 and unc-63 animals. In preparation for the use of C. elegans as an ectopic transformation system for parasitic receptor DNA, preliminary biolistic transformation experiments were performed using microparticle bombardment. It was found that transformation of unc-38 animals with myo-3::gfp construct has no significant effect on their ability to thrash in suspension. The mutant characterization data may possibly reflect differential expression of nAChRs at various stages of development. The transformation data suggest that microparticle bombardment has little or no effect on C. elegans, which is essential for future transgenic experiments. In summary, important baseline data on C. elegans nicotinic receptors have been generated and a fundamental transgenic control line in unc-38;myo3::gfp has been established. This will allow for exciting rescue experiments using parasitic nematode nAChR DNA.

Mitochondrial Dysfunction Confers Resistance to Multiple Drugs inCaenorhabditis 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 PKCepsilon.

Mutations inCaenorhabditis elegans him-19Show Meiotic Defects That Worsen with Age

From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.

Fatty acid composition and gene expression profiles are altered in aryl hydrocarbon receptor-1 mutant Caenorhabditis elegans

The aryl hydrocarbon receptor (AHR) is a eukaryotic transcription factor that plays an essential role in neuronal, immune, vascular, hepatic and hematopoietic development. In mammals, AHR induces metabolism-associated genes in response to xenobiotics. AHR is evolutionarily conserved, and the C. elegans AHR ortholog likely shares many physiologic functions with the mammalian version. While the role of AHR in development is known, the molecular basis of AHR action is less well understood. To understand the physiologic role of AHR in C. elegans, a combination of fatty acid profiling, transcriptomics, and phenotyping approaches was used. Fatty acid profiles from L4 larval stage whole animals indicated that C17isoA, C18:1n9t, C20:3n6 and C20:4n6 were significantly increased in an ahr-1 mutant compared to wild-type. Consistent with these changes, we observed a significant 5.8 fold increase in fat-7, and 1.7–1.9 fold increases in elo-5, nhr-49, and mdt-15 gene expression during the L4 stage. The ahr-1(ju145) mutant displayed deficits in growth and development including a reduced number of eggs laid, a higher proportion of dead embryos, delay in time to reach L4 stage, and movement deficits including a fewer number of body bends and a longer defecation cycle. To understand global effects of AHR-1 on transcription, microarray analysis was performed on L1 stage animals. Expression changes (324 under- and 238 over-expressed) were found in genes associated with metabolism, growth, and development. These results indicate a role for C. elegans AHR in regulating fatty acid composition and in contributing to some aspects of development. Since the transcriptional control of AHR targets may be evolutionarily conserved, these results provide a deeper understanding of the molecular actions of AHR in a model invertebrate system that may be informative for higher organisms.

Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca2+ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.

Decline in oxygen consumption correlates with lifespan in long-lived and short-lived mutants of Caenorhabditis elegans

In humans, the basal energy metabolism is thought to decline linearly with age. On the other hand, in the nematode Caenorhabditis elegans, two research groups reported independently that it declined exponentially. In this study, furthermore, we used various lifespan-mutant strains to determine whether the previous conclusion is more likely to be true. We can indirectly estimate the metabolic energy by conveniently measuring the oxygen consumption rates of C. elegans using an optical apparatus. From the profile of respiratory rates as a function of age, we can quantitatively isolate the physiological decline rate, λ, that exponentially represents the decay rate of respiratory activity with age. In addition, quantitative analysis indicates that the respiratory activity of worms has a finite value in advanced age. We also show that the maximum and mean lifespans strongly correlate with the reciprocal of the λ. These findings offer crucial biochemical evidence for a molecular mechanism at work in biological aging. Consequently, we here propose a mechanism based on a chemical reaction and offer a definition of the physiological decline rate and the finiteness of respiratory activity in advanced age.

Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2

Despite a key role for dense core vesicles (DCVs) in neuronal function, there are major gaps in our understanding of DCV biogenesis. A genetic screen for Caenorhabditis elegans mutants with behavioral defects consistent with impaired DCV function yielded five mutations in UNC-108 (Rab2). A genetic analysis showed that unc-108 mutations impair a DCV function unrelated to neuropeptide release that, together with neuropeptide release, fully accounts for the role of DCVs in locomotion. An electron microscopy analysis of DCVs in unc-108 mutants, coupled with quantitative imaging of DCV cargo proteins, revealed that Rab2 acts in cell somas during DCV maturation to prevent the loss of soluble and membrane cargo. In Rab2 null mutants, two thirds of these cargoes move to early endosomes via a PI(3)P-dependent trafficking pathway, whereas aggregated neuropeptides are unaffected. These results reveal how neurons solve a challenging trafficking problem using the most highly conserved animal Rab.

A cadmium toxicity assay using stress responsive Caenorhabditis elegans mutant strains

To test the applicability of Caenorhabditis elegans mutant for toxicity screening, the sensitivity of cadmium (Cd) in C. elegans was investigated on 14 mutant strains using median lethal concentration (LC50) tests, with further analysis on growth and reproduction conducted on five selected strains. The 24 h LC50 of Cd observed on the wildtype and mutant strains of C. elegans was in the order of age-1( hx546) > mtl-2( gk125) > sod-3( gk235) > daf-21( p673) > cyp35a2( gk317) > skn-1( or13) > daf-12( rh62rh157) > hsp-16.2( gk249) > daf-18( e1375) > ctl-2( ok1137) > wildtype( N2) > sod-1( or13) > daf-16( mu86) > cep-1( gk138) > cdr-2( ok1996). Compared to the wildtype response, a decreased reproduction potential was observed in mtl-2( gk125), sod-3( gk235), cdr-2( ok1996) and cep-1( gk138) strains. To gain a mechanistic understanding of different sensitivities of the mutant strains, a time-course gene expression analysis was also performed on the five genes. A dramatic increase in the expression of the mtl-2 gene due to Cd exposure confirmed the importance of this gene in C. elegans Cd toxicity. An increased expression of the sod-3 gene at the longer exposure time period (48 h) suggests that oxidative stress may not be a direct toxic mechanism, but may rather be a consequence of Cd toxicity. Even though, LC50 values for the age-1( hx546) mutant strain were the highest among the tested strains, the response on the reproduction potential in age-1( hx546) mutant was unchanged compared to the wildtype, and the age-1 gene expression remained unaltered on exposure to Cd, which may be interpreted as the maintenance of age-1 expression level is needed for the exertion of Cd toxicity; however, the role of the age-1 gene in Cd toxicity may not be via a reproduction-related pathway. The overall results suggest that the C. elegans mutant assay seems to be a promising tool for the study of toxic mechanisms, as well as for toxicity screening in ecotoxicological research.

Neurotransmitter release: a docking role for UNC‐13 proteins (Commentary on Siksou et al.)

Neurotransmitter release: a docking role for UNC‐13 proteins (Commentary on Siksou <i>et al.</i>)

A soma-to-germline transformation in long-lived Caenorhabditis elegans mutants.

Unlike the soma which ages during the lifespan of multicellular organisms, the germline traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation toward reproduction at the expense of cellular senescence. We report here that C. elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germline. Decreased insulin-like signaling causes the somatic misexpression of germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. DAF-16/FoxO, the major transcriptional effector of insulin-like signaling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also cause somatic misexpression of PGL-1. These results suggest that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival.

Cis-regulatory Mutations in the Caenorhabditis elegans Homeobox Gene Locus cog-1 Affect Neuronal Development

We apply here comparative genome hybridization as a novel tool to identify the molecular lesion in two Caenorhabditis elegans mutant strains that affect a neuronal cell fate decision. The phenotype of the mutant strains resembles those of the loss-of-function alleles of the cog-1 homeobox gene, an inducer of the fate of the gustatory neuron ASER. We find that both lesions map to the cis-regulatory control region of cog-1 and affect a phylogenetically conserved binding site for the C2H2 zinc-finger transcription factor CHE-1, a previously known regulator of cog-1 expression in ASER. Identification of this CHE-1-binding site as a critical regulator of cog-1 expression in the ASER in vivo represents one of the rare demonstrations of the in vivo relevance of an experimentally determined or predicted transcription-factor-binding site. Aside from the mutationally defined CHE-1-binding site, cog-1 contains a second, functional CHE-1-binding site, which in isolation is sufficient to drive reporter gene expression in the ASER but in an in vivo context is apparently insufficient for promoting appropriate ASER expression. The cis-regulatory control regions of other ASE-expressed genes also contain ASE motifs that can promote ASE neuron expression when isolated from their genomic context, but appear to depend on multiple ASE motifs in their normal genomic context. The multiplicity of cis-regulatory elements may ensure the robustness of gene expression.