wbg-volume-18-number.. - WormBook

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Triton X decreases adherence of C.elegans to pipette tips in liquidmediumGregory M. Davis, Madhu Sharma, Lloyd Low and Peter R. BoagDepartment of Biochemistry and Molecular Biology, Monash University, Melbourne, AustraliaCorrespondence to: Peter Boag (peter.boag@med.monash.edu.au)The adherence of worms to standard plastic tips makes accurate dispensing difficult. While usingglass tips can overcome this problem, their application is limited when sterility and frequent tipchanging is needed, for example, when performing liquid culture RNAi screens using 96-well plates(Lehner et al., 2006). RNAi screen protocols often require ~10 L1 worms to be dispensed into eachwell of a 96 well plate, however using standard P200 tips with worms in M9 alone resulted in an averagevariance of ± 6 L1 worms per well. We found that worms suspended in M9 with Triton X-100 decreasedthis adherence, resulting in an average variance of ± 2 L1 worms in each 10µl volume.Both Triton X-100 and Tween-20 can be used to decrease this adherence to standard P200 tips.Since autoclaving either of these reagents is not recommended, sterilization can be carried out via filtrationusing a 2µm filter. The lower viscosity of Triton X-100 over Tween-20 greatly aided filtrationand sterility tests showed no associated contamination. We found that using concentrations of Triton Xin M9 ranging from 0.1% to as low as 0.01% proved to be effective in decreasing worm adherencethroughout our assay. No negative issues regarding the growth or progeny of worms were observedwhen compared to a control plate containing no Triton X. Therefore this method could serve usefulwhen applied to protocols that require consistently small numbers of worms in large repetitions.ReferenceLehner B, Tischler J, Fraser, AG. (2006). RNAi screens in Caenorhabditis elegans in a 96-well liquid format and their applicationto the systematic identification of genetic interactions. Nat. Protoc. 1, 1617-20.- 5 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010A simple yet effective method to harvest worms from a liquid cultureTeruaki Takasaki 1 , Toshinobu Fujiwara 2 and Susan Strome 11 University of California, Santa Cruz CA, 2 Kobe University, Kobe, JapanCorrespondence to: Teruaki Takasaki (takasaki@ucsc.edu), Toshinobu Fujiwara (tosinobu@kobe-u.ac.jp)Although it is easy to grow worms in large quantities in liquid medium, harvesting worms istroublesome, especially when the culture is big. There are several options to collect worms (Hope,1999). Centrifugation is the most typical procedure, yet in many cases the residual bacteria supplied asfood in the culture is also precipitated and is hard to get rid of. A sucrose floatation step to obtain apure worm sample is effective but difficult to scale up. An alternative way to harvest worms is to chillthe culture flasks on ice, allow the worms to settle, then aspirate off the supernatant. This is easy andeffective, but not all experiments tolerate a prolonged chilling step. Another harvesting option is to collectworms with mesh, which may be hard to keep sterile.Here we show a simple and efficient method to harvestworms using a separatory funnel.1. Place a separatory funnel on a tripod.2. Close the stopcock.3. Pour the liquid culture into the funnel.4. Allow worms to settle for ~10 min at roomtemp.5. Gently open the stopcock over a collectingtube.6. Close stopcock when you have collected allworms.We use a Nalgene® separatory funnel (#4300), which is made of polypropylene with a Teflon TFEstopcock. Worms hardly stick to it. Although the funnel is autoclavable (stopcock is not autoclavable),we use ethanol to sterilize it. An 8-cm tripod (inner diameter) works well with both 500 ml and 1000ml funnels. If you fully open the stopcock, the worms will come out within 2 seconds. The worm pelletshould be pure. If you want it to be extra pure, you can repeat the steps with new buffer.ReferenceHope, IA. (1999). C.elegans. A practical approach. (New York: Oxford University Press).- 6 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Few gene expression differences between C. elegans grown in liquidversus on platesValerie Reinke 1 , Frederick G. Mann 2 , Christina M. Whittle 2 and Jason D. Lieb 21 Department of Genetics, Yale University School of Medicine, New Haven CT, 2 Department of Biology, University ofNorth Carolina at Chapel Hill, Chapel Hill NCCorrespondence to: Valerie Reinke (valerie.reinke@yale.edu), Jason Lieb (jlieb@bio.unc.edu)The two major methods for growing C. elegans are liquid and plate culture. Differences in oxygenationand other environmental variables could affect many experimentally relevant biological processes,such as metabolism, cellular structure, and gene regulation. Worms on solid support move withdeliberate sinusoidal movements, while worms in liquid thrash rapidly and almost constantly, often appearingelongated. We investigated differential gene expression under these two commonly usedgrowth conditions.Embryos were isolated by bleaching from adults grown for several generations on plates. Afterhatching in M9 media, animals were placed either on plates seeded with OP50 or in liquid supplementedwith HB101 under standard conditions. Both cultures were grown until worms reached youngadulthood, approximately 48 hours later. Total RNA was isolated independently from each growth,differentially labeled with a fluorescent dye, and mixed directly for comparative hybridization to 4-plexNimbleGen DNA microarrays, which contained 72,000 probes, which covered all predicted C. elegansprotein-coding genes (minimum 3 probes per gene). The experiment was performed in triplicate.Analysis of the expression data yielded surprisingly few genes with differential expression, about 1%.Overall, 176 genes were downregulated significantly (p

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Media osmolarity modulates dopamine-dependent, swimming-inducedparalysis (SWIP)J. Andrew Hardaway 1,4 , Sarah M. Whitaker 1 and Randy D. Blakely 1,2,3Departments of 1 Pharmacology and 2 Psychiatry, 3 Center for Molecular Neuroscience, and 4 Neuroscience Graduate Program,Vanderbilt University School of Medicine, Nashville, TNCorrespondence to: Randy Blakely (randy.blakely@vanderbilt.edu)Dopamine (DA) signaling depends upon the coordination of multiple presynaptic processes, includingthose supporting DA biosynthesis, packaging, release and reuptake. The presynaptic dopaminetransporter (DAT-1) is of particular importance in regulating both the exposure of pre- and postsynapticDA receptors as well as supporting recycling of DA for further rounds of release. Using Caenorhabditiselegans, our lab demonstrated that loss of DAT (dat-1) elicits a DA-dependent, locomotory phenotypetermed Swimming Induced Paralysis (SWIP) (Mcdonald et al., 2007). Whereas wild-type (N2) L4 stageworms swim at a continuous rate for ~20 min., dat-1 worms initiate normal swimming, but then paralyzeover the next six minutes. SWIP can be rescued by pretreatment with the vesicular DA transporter (CAT-1) inhibitor reserpine, loss of DA biosynthesis (cat-2) or loss of the DA receptor DOP-3 (dop-3). In ourinitial studies, dat-1 SWIP was thought to be evident in either water or M9 buffer. Recently, however, wetested N2 and dat-1 worms for SWIP in both media, as well as three sucrose-supplemented water solutions.As shown in Fig. 1, as the osmolarity of the aqueous media increases, dat-1 SWIP penetrance decreasessignificantly. In contrast, swimming of wild-type worms is unaffected by media osmolarity. Althoughthe effects of osmolarity on neural activity in worms have been well documented, here we showthat media composition modulates a DA-modulated C. elegans motor behavior by DA and possibly otherbiogenic amines like serotonin. Candidate, reverse or forward genetic manipulations of SWIP in water vs.M9 may provide an opportunity to further dissect DA signaling in the nematode in vivo.Figure 1 - dat-1 SWIP decreases as a function ofincreasing osmolarity of aqueous media. L4 animalswere picked using an eyelash pick into 80 µl of theindicated media, incubated for 10 min, and thenscored for paralysis. Each trial contained at least 10worms, and each point is represents 17 trials.ReferenceMcdonald PW, Hardie SL, Jessen T, Carvelli L, Matthies DS, Blakely RD. (2007). Vigorous motor activity in Caenorhabditiselegans requires efficient clearance of dopamine mediated by synaptic localization of the dopamine transporterDAT-1. J. Neurosci. 27, 14216-14227.- 8 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Quantifying roaming behavior in synaptic defective mutants ofC. elegansFernando Calahorro 1,2 and Manuel Ruiz-Rubio 1,21 Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Córdoba, Spain, 2 Instituto Maimónides de InvestigaciónBiomédica de Córdoba (IMIBIC) Córdoba, SpainCorrespondence to: Manuel Ruiz-Rubio (ge1rurum@uco.es)C. elegans dwells in humus where it feeds on microorganisms. Movement in the worm is theresult of environmental and internal signals creating stimulatory and inhibitory output from sensoryneurons. Mutants deficient in neurexin (nrx-1) and/or neuroligin (nlg-1) genes exhibit impairment inexcitatory and inhibitory neuronal synapses. It has been shown that nlg-1 deficient mutants are defectivein detecting osmotic strength (Calahorro et al., 2009) and other sensory behavior (Hunter et al.,2010). Here we illustrate a simple experiment that allows quantifying the roaming behaviour phenotypein neuroligin and neurexin defective mutants of C. elegans. The results of a representative experimentare shown in Figure 1.The experiment was carried out as follow: 1) Worms were grown synchronously at 20ºC onNGM agar plates to L4 stage. Animals were washed three times with CTX buffer (5 mM K 2 HPO 4 ,KH 2 PO 4 buffer pH 6.6; 1 mM CaCl 2 ; 1 mM MgSO 4 ). 2) One central circle, 1 cm of diameter, wasdrawn on the back of two types of assay plates: NGM plates without OP50 bacteria, and NGM platesseeding with 100 μl of OP50 outlining a ring (0.5 cm of thickness) in the perimeter, maintaining free ofbacteria the centre of the plate. The plates were incubated for 24 hours at 37ºC before used. 3) Around20-30 washed worms were pipetted in the middle of the agar, coinciding with the centre of the circledrawn on the back of the plate. The excess of liquid was gently removed with a small sterile filter paper.4) The animals, out of the ring, were counted every 15 minutes.Figure 1: Distinguishing roaming behavior in different strains with defects in the neuronal synapse. Differences betweenwild type N2 strain and each of the mutants were statistically significant by the t test: VC1416 (P = 0.0003),VC228 (P = 0.0130), CRR21 (P = 0.0001). Differences between the results obtained in plates without and with foodwere not statistically significant (P > 0.05). Error bars represent S.D. of at least three independent experiments.ReferencesCalahorro C, Alejandre E, Ruiz-Rubio M. (2009). Osmotic avoidance in Caenorhabditis elegans: synaptic function of twogenes, orthologues of human NRXN1 and NLGN1, as candidates for autism. J. Vis. Exp. 34, pii: 1616.Hunter JW, Mullen GP, McManus JR, Heatherly JM, Duke A, Rand JB. (2010). Neuroligin deficient mutants of C. eleganshave sensory processing deficits and are hypersensitive to oxidative stress and mercury toxicity. Dis. Model Mech.3, 366-76.- 9 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Towards an automatic method for toxicity and pharmacological testingin C. elegansSergio H. SimonettaFundación Instituto Leloir, CONICET, Buenos Aires, ArgentinaCorrespondence to: Sergio Simonett (ssimonetta@leloir.org.ar)In toxicity and pharmacological studies C. elegans are exposed to different compounds and theeffects analyzed by observing worm viability or behavior. An alternative approach is to use locomotoractivity as a readout, which has been instrumental for assessing compound toxicity (Dhawan et al.,2000). Data acquisition usually requires microscope observation and manual counting, which is timeconsuming and inconvenient for extensive compound screenings. We have previously developed anautomated method to detect C. elegans movement in which swimming worms are detected as theycross through infrared microbeams (Simonetta et al., 2007 PMID: 17207862).We are currently adapting this methodology for high throughput analyses. We have developeda 384 channel apparatus and successfully recorded the behavioral changes produced by toxic compounds(Figure 1). The effect increases with compound concentration and is dependent on exposuretime. Our "Worm Microtracker" might be useful for the community to develop easier and faster toxicityand paralysis assays, opening the possibility of performing high throughput studies in C. elegans.Figure 1. Measure of compoundtoxicity using infrared locomotortracking. A) Schematic diagram ofexperimental procedure. Young adultC. elegans (50worms/100µl aprox.)were prepared in K buffer (53mMNaCl, 32mM KCl), transferred to a96 well microplate (100µl/well) andadequate concentrations of KCladded. The microplate was registeredcontinuously for 24 hours at 20°Cusing a 384 channel infrared trackingsystem. B) Locomotor activity plotsshowing the toxic effects of KCl exposurevs. time. Each dot correspondsto the mean of activitycounts/30minutes for 8 replicates.Treatment plots are adjusted to apolynomial curve. C) Estimation ofBC50 (Concentration at which movementis reduced by 50% comparedwith controls). Locomotor activityrelative to control is plotted against KCl concentration. The BC50 was calculated graphically for 4h and 24h treatment.ReferencesDhawan R, Dusenbery DB, Williams PL. (2000). Comparison of metal-induced lethality and behavioral responses in thenematode Caenorhabditis elegans. Environ. Toxicol. Chem. 19, 3061-3067.Simonetta SH and Golombek DA. (2007). An automated tracking system for Caenorhabditis elegans locomotor behaviorand circadian studies application. J. Neurosci. Methods 161, 273-80.- 10 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Measuring reactive oxygen species in C. elegans using DCFDA– a word of cautionSheng Fong, Jan Gruber and Barry HalliwellDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, SingaporeCorrespondence to: Barry Halliwell (bchbh@nus.edu.sg)Dichlorofluorescin diacetate (DCFDA) is a popularfluorescence-based probe for reactive oxygen species (ROS)detection in vitro and in vivo, and has been used for this purposein C. elegans. DCFDA is first deacetylated by endogenousesterases to dichlorofluorescein (DCFH), which can reactwith several ROS to form the fluorophore DCF (reviewed byHalliwell and Gutteridge, 2007). The DCFDA assay in C. elegansis sometimes performed using lysed worms, followinghigh intensity sonication. This process disrupts the outer cuticleand internal membranes, causing intracellular, as well asintraorganelle, contents to be released. Worm lysis will causethe release of transition metal ions such as iron. Free iron mayparticipate in redox cycling to generate ROS, for example, byFenton chemistry (Halliwell and Gutteridge, 2007). Therefore,apparent ROS levels detected in lysed worms may beFigure 1. Linear regression curves for lysed andwhole worms.un-physiological. However, DCFDA readily diffuses into cells where it undergoes deacetylation, andthis allows ROS measurement in whole worms. If whole animals are utilized, cells are not disruptedand iron remains sequestered.Using DCFDA and a synchronized worm cohort, we measured the amount of ROS in equivalentnumbers of lysed and whole worms. The gradient of the linear regression curve (expressed asΔRFU/Δmin) (Figure 1) is the rate of fluorescent DCF production. This gradient can be, very approximately,converted to the rate of H 2 O 2 -equivalent ROS production (nmol/min) by spiking knownamounts of H 2 O 2 . It should, however, be noted that H 2 O 2 itself does not oxidize DCFH, and hence, thefree radical reactions resulting in increased DCF signal following H 2 O 2 spiking are likely complex.Therefore, the calibration ratio can only be considered to provide an approximate estimation of ROSproduced. Nevertheless, we found that the rate of H 2 O 2 -equivalent production was more than an orderof magnitude faster in lysed (4.1x10 -3 nmol/min/worm) compared to whole animals (3.6x10 -4 nmol/min/worm). Using the O 2 consumption rate measured in the same worm cohort (0.018 nmol/min/worm), the% of O 2 consumed leading to ROS generation can be estimated by dividing the rate of H 2 O 2 -equivalentproduction by the rate of O 2 consumption. Using this approach, we found apparent ROS production tobe around 23% for lysed worms, but only 2% for whole worms. Current estimates of in vivo mitochondrialROS production range from 0.2 to 2% (Balaban et al., 2005 PMID: 15734681). Assuming allROS measured using the DCFDA assay to be of mitochondrial origin, ROS production is clearly overestimatedby a factor of at least 10-fold, and may be as much as 100-fold, in lysed worms compared towhole worms. Therefore, when using the DCFDA assay, whole worms should be utilized instead.ReferencesBalaban RS, Nemoto S, Finkel T. (2005). Mitochondria, oxidants, and aging. Cell 120, 483-495.Halliwell B, Gutteridge JMC. (2007). Free radicals in biology and medicine, 4th ed. (Oxford; New York, Oxford UniversityPress).- 11 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Subcellular imaging in C. elegans and suppressor screensFrancesca Farina, Rémi Vernet, Betty Peignelin, Matthieu Y. Pasco, Cedric Bicep and Christian NériINSERM Unit 894, Neuronal Cell Biology & Pathology, Paris, FranceCorrespondence to: Christian Néri (christian.neri@inserm.fr)C. elegans is greatly suitable in suppressor screens and has strong potential to screen for the effectsof modifiers at the subcellular level as it is transparent at all stages of lifespan. Here, we describea 96-well plate assay that illustrates how high resolution imaging may be used in day-to-day researchas well as RNAi and drug screens. We used the Plate Runner HD ® (Trophos, France), a 96/384-wellplate motorized device able to collect fluorescence at resolutions ranging from 1024x1024 px (1 px is7.4 μm) to 8192x8192 px (1 px is 1 μm). This device has high depth of field (40 μm at resolution of7.4 μm; 8 μm at resolution of 1 μm), thus allowing fluorescent signals to be quantified from whole animals.This device also has a wide-field objective that allows single images of the whole well to be acquiredat once (Fig. 1A). To develop drug screening for neuromuscular disease, we used C. eleganstransgenics that co-express GFP and the oculopharyngeal muscular dystrophy (OPMD) proteinPABPN1 in body wall muscle cells. Mutant PABPN1 animals show defective motility accompanied byloss of GFP nuclei (20 signals lost in 3-day adults) and muscle cell degeneration (Catoire et al., 2008).These phenotypes are aggravated by sirtuin (sir-2.1/SIRT1) activation and ameliorated by sirtuin inhibition(Catoire et al., 2008; Pasco et al., 2010). Resveratrol, an indirect SIRT1 activator, enhances mutantPABPN1 toxicity whereas EX-527, a selective SIRT1 inhibitor, has the opposite effect (Catoire etal., 2008; Pasco et al., 2010). We used these chemicals to normalize a drug screen assay (Fig. 1B). SynchronisedL1 larvae are incubated at 20°C with bacteria and drugs (40 L1 in 50μl/well; 5 wells/point)until they reach adulthood. At 46 hours, FUDR (0.1 mg/ml) is added to prevent egg-laying and hatching.Day-3 adults are immobilized using 2,3-butanedione monoxime (0.1 M, 300 μl/well). Morphometricanalysis of images (here 2048x2048) is performed using Image J and a script developed in the laboratory.Average numbers of GFP signals/worm are integrated into a database and subjected to statisticalanalysis. Drug screening is now ongoing using this assay. Perfect immobilization of animals is requiredfor imaging at 4096x4096 and up. Similar strategies may be used in the development of a variety ofsuppressor screens.Fig. 1. A. Full-well image of PABPN1 nematodesshowing nuclear GFP (white dots) at a resolution of2048x2048 px. Scale bar is 1 mm. The insert showsone animal at high magnification. B. Effects ofchemicals known to modulate the phenotypes of mutantPABPN1 animals. Reversion is calculated as[(test-control)/control]x100 and the maximal achievableamelioration is 14%. EX-527 ameliorates mutantPABPN1 (13 Alas) animals with no effect in normalPABPN1 (10 Alas) animals. Resveratrol shows theopposite. Data are mean±SEM. **P < 0.005, *P

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010In vivo visualization of flavonoids in C. elegans using 2-aminoethyldiphenyl borateGregor Grünz, Hannelore Daniel and Britta SpanierMolecular Nutrition Unit, Technical University of Munich, Freising, GermanyCorrespondence to: Britta Spanier (spanier@wzw.tum.de)In the past decade C. elegans has become a popular model in investigating molecular effects ofphytochemicals. Among these, flavonoids represent a major group of plant secondary metabolitesshown to alter lifespan and stress resistance in C. elegans (Kampkoetter et al., 2008). However,bioavailability of these compounds is rather low. Detection in worms usually requires analytical techniquessuch as HPLC and rather large amounts of worm material but gives no information on where inworms the compound accumulates (Kampkoetter et al., 2008). Here we report a method to detect andsemi-quantify flavonoids in C. elegans by analysis of the fluorescence after derivatisation in vivo. Forthis purpose, we used 2-aminoethyl diphenyl borate (Naturstoff reagent A (NSRA)), which is employedin thin layer chromatography, plant histology and cell culture to enhance auto-fluorescence ofplant polyphenols (Neu, 1956; Ernst et al., 2010). NSRA forms a chelate with the polyphenolic compounds,leading to a shift of the spectral band to a longer wavelength (bathochromic shift) and to an intensifiedsignal.We exposed wild type N2 L4-larvae on NGM plates to the flavonoids myricetin, quercitin orkaempferol in increasing concentrations (0, 10, 50 or 100 μM) for 48 hours. Thereafter, the wormswere incubated in M9 buffer containing 10% heat-killed OP50 and 0.2% NSRA for 2 hours. Subsequently,fluorescence was monitored using a confocal laser scanning microscope with excitation at488 nm and emission at 590-620 nm (myricetin and quercitin) and at 540-560 nm (kaempferol).NSRA selectively increased the fluorescence of the flavonoids, making them detectable in C.elegans. Highest fluorescence was always obtained in gut epithelial cells (A) as the sites of flavonoiduptake and intensity reflected the flavonoid concentrations in a dose-dependent manner (B). Thisshows that NSRA is an easy-to-use and cheap tool to visualize and semi-quantify flavonoids in vivo inC. elegans.ReferencesErnst IM, Wagner AE, Lipinski S, Skrbek S, Ruefer CE, Desel C, Rimbach G. (2010). Cellular uptake, stability, visualizationby ‘Naturstoff reagent A’, and multidrug resistance protein 1 gene-regulatory activity of cyanidin in humankeratinocytes. Pharmacol. Res. 61, 253-258.Kampkötter A, Timpel C, Zurawski RF, Ruhl S, Chovolou Y, Proksch P, Wätjen W. (2008). Increase of stress resistanceand lifespan of Caenorhabditis elegans by quercetin. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149, 314-323.Neu R. (1957). Chelate von Diarylborsäuren mit aliphatischen Oxylalkaminen als Reagenz für den Nachweis on Oxyphenyl-benzo-gamma-pyronen.Naturwissenschaften 44, 181–182.- 13 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010A web-based bioinformatics solution integrated with next generationsequencing to identify molecular lesions of a C. elegans mutantDawei Lin 1 , Yu-Tai Chang 2 , Jose Boveda 1 , Zhiwei Lu 1 , Charles Nicolet 1 , Ian Korf 1,2 , Daniel Starr 21Genome Center and 2 Department of Molecular and Cellular Biology, University of California, Davis CACorrespondence to: Dawei Lin (lhslin@ucdavis.edu)The capability of generating sequences in a massive-parallel fashion by next generation sequencingtechnologies (NGS) has revolutionized C. elegans genetics, greatly accelerating the rate to gofrom mutant to molecular lesion (Hobert, 2010 PMID: 20103786; Sarin et al., 2008 PMID: 18677319).A major hurdle for many C. elegans researchers is that they lack bioinformatics skills and computinginfrastructure to analyze Terabytes of sequence data. While tools such as MAQGene (Bigelow et al.,2009 PMID: 19620971) are available, they require installation procedures and maintenance on Linuxsystems out of the reach of most genetics labs. In addition, the sheer size of the data makes it an unnecessaryburden to transfer it from sequencing facilities to local computers. We have established a onestoppipeline that offers researchers the option to send in raw DNA materials and retrieve analyzed resultsremotely after sequencing done by Illumina GAII. The researchers are able to reanalyze their datawith different parameters if necessary through a Web-base interface.The pipeline was used to attempt to identify the molecular lesion of an enhancer of the nuclearmigration defect of unc-84 (emu) allele that had been traditionally mapped to ~150 kb on X. (1) ADNA sample from the Starr lab was sent to the DNA technology Core at the UC Davis Genome Center.(2) A library was made from mutant genomic DNA and the Core sequenced 85 bp single ends. Onelane of sequencing generated ~1.3 Gb of raw sequence. (3) MAQGene aligned the data to the N2 referencegenome and characterized differences; the data and results were available through SLIMS, a webbasedLaboratory Information Management System developed by the Bioinformatics Core at the UCDavis Genome Center. (3) MAQGene output a list of mutation candidates and associated annotations inExcel format. Our sequence data covered nearly 99% of the genome at least 1X coverage and 97% at2x or greater coverage. As internal positive controls, the software correctly identified the original unc-84(n369) lesion, 25 SNPs that we had previously confirmed by traditional methods, and a SNP knownto be in the starting strain. One uncovered region of about 50 bp was identified within the mapped region.We are testing candidate mutations and small deletions in the yc21 mapped region by traditionalmethods to determine if any are the cause of the emu phenotype. The pipeline has proved that integratingDNA sequencing directly with downstream Bioinformatics analysis is an efficient way to makenew technologies more accessible to average C.elegans geneticists. The contact information and pricingis at http://bit.ly/9NgksT.ReferencesBigelow H, Doitsidou M, Sarin S, Hobert O. (2009). MAQGene: software to facilitate C. elegans mutant genome sequenceanalysis. Nat. Methods 6, 549.Hobert O. (2010). The impact of whole genome sequencing on model system genetics: get ready for the ride. Genetics 184,317-319.Sarin S, Prabhu S, O'Meara MM, Pe'er I, Hobert O. (2008). Caenorhabditis elegans mutant allele identification by wholegenomesequencing. Nat. Methods 5, 865-867.- 14 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Intelligent worm gene function prediction with GeneMANIASylva Donaldson, Quaid D. Morris and Gary D. BaderDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, CanadaCorrespondence to: Gary Bader (gary.bader@utoronto.ca), Quaid Morris (quaid.morris@utoronto.ca)The ability to collect functional data, like physical and genetic interactions and co-expression,about every gene in the genome is expanding the possibilities of biological research. However, navigatingthrough these data can be a difficult task for those without specialized computational expertise.GeneMANIA uses many large publicly available datasets to analyze gene lists and identify highthroughputdata sources in which the query genes are highly associated.GeneMANIA is useful for generating hypotheses about gene function and planning mediumscalephenotypic screens. GeneMANIA will extend your gene list with more genes that are functionallyassociated with those in the list. The genes returned by GeneMANIA often represent promising candidatesfor functional assays (Mostafavi et al., 2008). It is designed to adapt to your query, so if yourquery list consists of members of a protein complex, GeneMANIA will retrieve more potential membersof the complex. If members of your gene list function in a particular biological process (like neuronaldevelopment), GeneMANIA will return more putative contributors to that process.Using the software is easy - just choose worm, enter your set of favorite genes and press Go.Example gene lists are provided. GeneMANIA currently supports standard gene symbols (from Worm-Base) and Entrez, Ensembl, Uniprot/SwissProt and RefSeq database identifiers.The advanced options panel can be used to choose additional networks to search, change thenetwork weighting method, and increase the number of genes to retrieve. You can choose from amongmany different networks to be used in the analysis, although the default networks are reasonable formost queries. GeneMANIA currently supports 76 networks for C. elegans, including 10 co-expression,8 physical interaction, 4 genetic interaction, 1 co-localization, 2 shared protein domain and 50 predictednetworks. The predicted networks largely consist of WormNet (Lee et al., submitted) and I2D(Brown and Jurisica, 2007) data.GeneMANIA displays results as an interactive network, illustrating the functional relatednessof the query and retrieved genes. The links represent datasets in which genes are associated. The tabbedbrowser on the right side of the page can display information about the datasets or the genes in the network.Genes can be highlighted and moved around. The network visualization menu provides a rangeof options, including the ability to save the analysis. A complete report of the analysis can be generated,including a publication quality image of the network.GeneMANIA is free, web-based, open source and still a work in progress. We welcome anycomments, suggestions and collaborations.ReferencesBrown KR, Jurisica I. (2007). Unequal evolutionary conservatioin of human protein interactions in interologous networks.Genome Biol. 8, R95.Mostafavi S, Ray D, Warde-Farley D, Grouios C, Morris Q. (2008). GeneMANIA: a real-time multiple association networkintegration algorithm for predicting gene function. Genome Biol. 9, S4.- 15 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010The Caenorhabditis elegans SLC database: Solute transporter identificationusing comparative genomicsCarsten Jäckel, Hannelore Daniel and Britta SpanierMolecular Nutrition Unit, Technical University of Munich, Freising, GermanyCorrespondence to: Britta Spanier (spanier@wzw.tum.de)Comparative genomic approaches have proven to be powerfultools in identifying genes as well as categorizing them based on proteinfunctions. One product is the HGNC (Human Gene NomenclatureCommittee) Solute Carrier (SLC) System (Hediger et al., 2004; He etal., 2009). It comprises 55 families with a total of over 365 putativeproteins that act as uniporters, symporters or antiporters in the plasmacell membrane as well as in mitochondrial and vesicular membranes.The range of transported substrates covers inorganic and organic cations/anions, amino acids and peptides,sugars, bile salts, urea, biogenic amines, neurotransmitters, ammonium, choline, vitamins, nucleosides/nucleotides,fatty acids, etc. In addition, numerous orphan transporters are contained thatneed their substrates be defined. Evidently these transporters are of critical importance for cellular homeostasisand thus many of these genes are highly conserved across species. Here we present the C.elegans SLCdb (http://www.wormslc.org), a database that lists the top C. elegans homologs to themammalian SLC tables. This database allows for a quick identification of target genes by either directlysearching for worm or SLC gene names or aliases and for retrieval of available information. Thesite also features a quick overview of all SLC families and the currently published knowledge on thesetransporters in C. elegans with a reference list.To date, the database contains 1886 homology datasets. The homologous C. elegans proteinshave been selected step-wise. First the top BLASTP (Altschul et al., 1990) hits were selected, applyinga cutoff threshold of E-20, which removes most false positives. In a next step genes that have beenpublished but may have been below the threshold were manually added to the list. Since BLAST algorithmsonly do local alignments they do not account for protein lengths and overall homology. Becauseof that limitation we next used the NEEDLE algorithm (Needleman et al., 1970) for additional informationand confirmation for the results obtained. For assessing of how well conserved a protein is withinthe nematode phylum, every single protein was then compared to its closest C. briggsae homolog(using NEEDLE), which usually yielded very high sequence identities and similarities. Finally, all resultswere compiled into a uniform format in the database, presenting the results sorted by NEEDLEscore, showing the closest C. elegans homologs, a brief description of the protein functions (whenavailable), links to WormBase (http://www.wormbase.org/) and the C. briggsae homolog along with abrief description and the NEEDLE score.ReferencesAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410.He L, Vasiliou K, Nebert DW. (2009). Analysis and update of the human solute carrier (SLC) gene superfamily. Hum. Genomics3, 195-206.Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA. (2004). The ABCs of solute carriers: physiological,pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch. 447, 465-468.Needleman SB, Wunsch CD. (1970). A general method applicable to the search for similarities in the amino acid sequenceof two proteins. J. Mol. Biol. 48, 443-453.- 16 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Naming genes beyond CaenorhabditisRobin N. Beech 1 , Dante Zarlenga 2 , David Bird 3 , Matt Berriman 4 and Joe Dent 51 Parasitology and 5 Biology, McGill University, Montreal, QC, Canada, 2 Animal Parasitic Diseases, USDA, Beltsville MD,3 Plant Nematode Genomes Group, NC State University, Raleigh NC, 4 Wellcome Trust Sanger Institute, Cambridge, UK.Correspondence to: Robin Beech (robin.beech@mcgill.ca)The nomenclature of genes in Caenorhabditis elegans is based on long-standing, successfulguidelines established in the late 1970s (Horvitz et al., 1979). Over time these guidelines have maturedinto a comprehensive, systematic nomenclature that is easy to apply, descriptive and therefore highlyinformative.Recently, a flood of parasitic nematodes genome data has become available, including Brugia(Ghedin et al., 2007) and Megliodogyne (Opperman et al., 2008). Annotation of these genomes islargely based on blast search similarity with C. elegans as the closest and most thoroughly characterizednematode. Several different nomenclature systems have developed, as seemed appropriate to thoseinvolved in each genome project. A strong case can be made for the general adoption of the C. elegansnomenclature guidelines across the nematodes (Beech et al., 2010; Bird and Riddle, 1994), althoughuntil recently this has not been widely used.A two, and later, three letter species code allowed the C. elegans nomenclature to be extendedto four additional Caenorhabditis species C. japonicum (Cja), C. ramanii (Cra), C. brigssae (Cbr) andC. brenneri (Cbe). Applying the guidelines to a wide range of nematode species will require furthermodification. Using a four letter species code could identify 1364 of the 1537 nematode species listedwith NCBI, without modification, rather than 976. C. elegans genes with several paralogs in a relatedspecies can be identified with a period and number after the gene name. For example, four copies ofunc-29 in the trichostrongylid parasites are unc-29.1, unc-29.2, unc-29.3 and unc-29.4 (Neveu et al.,2010). With no defined wildtype for parasitic species, a standard for designating polymorphism needsto be developed. Finally, among the 50-60% of genes in distantly related nematodes that have noclearly identifiable C. elegans homolog, are likely to be those that are most interesting for organism biology.Naming of these species-specific genes requires coordination with the C. elegans nomenclatureto avoid conflicts. For a nomenclature to be accepted and generally applied, a wide consensus amongthose who use the guidelines is required. A workshop will be held at the ICOPA XII conference inMelbourne, August 2010, to make recommendations for new guidelines.ReferencesBeech RN, Wolstenholme AJ, Neveu C, Dent JA. (2010). Nematode parasite genes, what's in a name? Trends Parasitol.[Epub ahead of print]Bird DM, Riddle DL. (1994). A genetic nomenclature for parasitic nematodes. J. Nematol. 26, 138-143.Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, Crabtree J, Allen JE, Delcher AL, Guiliano DB, Miranda-Saavedra D, et al.(2007). Draft genome of the filarial nematode parasite Brugia malayi. Science 317, 1756-1760.Horvitz HR, Brenner S, Hodgkin J, Herman RK. (1979). A uniform genetic nomenclature for the nematode Caenorhabditiselegans. Mol. Gen. Genet. 175, 129-133.Neveu C, Charvet C, Fauvin A, Cortet J, Beech R, Cabaret, J. (2010). Genetic diversity of levamisole receptor subunits inparasitic nematodes and abbreviated transcripts associated with resistance. Pharmacogenetics and Genomics InPress.Opperman CH, Bird DM, Williamson VM, Rokhsar DS, Burke M, Cohn J, Cromer J, Diener S, Gajan J, Graham S, et al.(2008). Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proc.Natl. Acad. Sci. USA 105, 14802-14807.- 17 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010ENGM: an NGM-like solid media suitable for doing genetics on theentomopathogenic nematode Heterorhabditis bacteriophoraAndrás Fodor 1,2 , Andrea Máthé-Fodor 1,2 , Éva Lehoczky 1 , Ganpat Jagdale 2 , Parwinder S. Grewal 2 andMichael G. Klein 21 Institute of Plant Protection, Georgikon Faculty, University of Pannonia, Keszthely, Hungary, 2 Department of Entomology,Ohio State University, OARDC, Wooster OHCorrespondence to: András Fodor (fodorandras@yahoo.com)There are two “secret weapons” behind the scientific success of C. elegans: NGM media andOP50 bacteria. The Heterorhabditis bacteriophora/Photorhabdus luminescens entomopathogenicnematode/bacterium (EPN/EPB) complexes are widely used in biological control. Therefore elaborationof genetic toolkits for EPN species would have a practical impact. C. elegans is considered as amodel for genetic analysis of EPN species (Fodor et al., 1990).The genome sequence of H. bacteriophora(H. b.) is almost complete. The “secret tools” (the proper NGM-like media and OP50-like bacteria)for genetics of H. b. have, however, been missing. H. b. can only be fed on its own symbiont in labmedia, which is rich in N sources and contains oil. Neither H. b. nor its bacterial symbiont could growin NGM, even if it was supplemented with oil and an abundant peptone N-source. The results werehardly better with supplemented TSY or TSA. In a rich oily media (Woots, NA) the bacteria overgrowthe nematodes and the animals cannot be observed individually. In Woots agar media the growth wasremarkable, but the visibility of the nematodes was poor. In Woots agar C. elegans could propagate,but “feeding RNAi” (Fire et al., 1998 ) could not be induced. We elaborated a novel media, calledENGM (Entomopathogenic Nematode Growth Media) in which H. b., C. elegans, as well as their foodsourcebacteria (P. luminescens, E. coli TT01, OP50) could properly grow. The visibility of the nematodeson ENGM is almost as good as that on NGM. Feeding RNAi can be induced in C. elegans byfeeding in both NGM and ENGM with similar frequencies, suggesting that RNAi in H. b. might also bestudied in ENGM. Since then we successfully induced heterolog RNAi in H. b. (in preparation). Thefood source for H. b. is the moderately growing Tn-10-induced NS107 rifR, kmR, ap R mutant (isolatedby us) of P. luminescens TT01 (Duchaud E. et al., 2003 ) (kindly provided by Dr. T. Ciche). The recipeof ENGM is as follows: 2.5 g bacto-peptone; 1.5 g beef extract; 2.3 g brain-heart infusion; 15 g agar to1 liter of deionized water. After autoclaving: 5 g vegetable oil; 1 ml of 5 mg/ml cholesterol (dissolvedin EtOH); 2 ml of 0.5M MgSO 4 , rifampicin 100 , kanamycin 30 ) are to be added. ENGM plates should beseeded with moderately growing symbiotic bacteria, such as NS107. (For details on the isolation ofNS107, please contact the corresponding author).ReferencesDuchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S, Boursaux-Eude C, Chandler M, CharlesJF, et al. (2003). The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens Nat. Biotechnol.21, 1307 – 1313.Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998). Potent and specific genetic interference by double-strandedRNA in Caenorhabditis elegans. Nature 19, 806-811.Fodor A, Vecseri G, Farkas T. (1990). C. elegans, as a model for studying entomopathogenic nematodes. In EntomopathogenicNematodes in Biological Control, R. Gaugler and H. Kaya eds. (Boca Raton, FL, USA: CRC Press), pp. 285-300.- 18 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Potential for cytonuclear epistasis in Caenorhabditis briggsaeinter-population hybridsAnna L. Coleman-Hulbert 1 , Dana K. Howe 2 , Dee R. Denver 2 and Suzanne Estes 11 Department of Biology, Portland State University, Portland OR, 2 Department of Zoology and Center for Genome Researchand Biocomputing, Oregon State University, Corvallis ORCorrespondence to: Suzanne Estes (estess@pdx.edu)A barrier to rigorous tests for the role of mitochondrial dysfunction in aging processes has beenthe lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Towardthe goal of developing such a model, we studied life history, metabolic, and aging phenotypes in naturaland experimental populations of Caenorhabditis briggsae harboring different levels of a heteroplasmicmitochondrial ND5 deletion recently discovered to segregate among many wild C. briggsae populations(Howe and Denver, 2008). The normal product of ND5 is a central component of complex I ofthe mitochondrial electron transport chain and integral to cellular energy metabolism.Natural high-ND5 deletion C. briggsae isolates have decreased total fecundity (Howe and Denver,2008). We found that heteroplasmic C. briggsae also have reduced pharyngeal pumping ratesthroughout adulthood and grow faster and larger as larvae, but slower as adults, compared to zerodeletionisolates. Hybrid strains were constructed by reciprocally crossing pairs of high- and lowheteroplasmypopulations in order to isolate the mitochondrial genome of one population onto the nucleargenetic background of another population. F10 individuals were assayed for the above traits withthe expectation that hybrid means would match those of the mitochondrial source population if ND5deletion heteroplasmy level is responsible for the phenotypic variation among C. briggsae isolates.However, C. briggsae hybrid means often differed from those of maternal lineage and from each otherfor all traits (e.g., Fig. 1). Because coordination between nuclear and mitochondrial gene products isrequired for optimal energy metabolism, our results may indicate cytonuclear conflict and heterosis inhybrid C. briggsae, but paternal transmission of mitochondria has yet to be eliminated as a potentialcause of these results.Figure 1. Lifetime fecundity inhigh- (HK105) and low-ND5 deletion(PB800) C. briggase isolatesand reciprocal hybrids. mHnP_1-3are hybrid replicates in whichHK105 was the mitochondrial donorand PB800 was the nuclear donor.The reverse is true for mPnH_1-3.Lines = medians, boxes encompass50% of data, vertical lines encompass10th-90th percentiles, circles =individual outliers.ReferencesHowe DK, and Denver DR. (2008). Muller's ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution.BMC Evol. Biol. 8, 62.- 19 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Comparison of Caenorhabditis elegans and Caenorhabditis briggsae forthermotolerance featuresMarat Kh.Gainutdinov, Alya Kh.Timoshenko, Rufina R.Kolsanova, Timur M.Gainutdinov andTatyana B.KalinnikovaResearch Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences,Kazan, Russian FederationCorrespondence to: Marat Gainutdinov (mgainutdinov@nm.ru)Thermotolerance evolution of poikilothermic organisms consists in adaptation to temperatureconditions of habitats of such physiological features as the lower and upper limits of temperature forreproduction, development and behavior and organism's resistance to short-term temperature extremes.Previous researches of Caenorhabditis revealed both interspecific and intraspecific significantdifferences for the upper limits of temperature for reproduction and development. These differences arein accordance with temperature conditions of habitats (Fatt and Dougherty, 1963; Fodor et al., 1983).Nevertheless interspecific differences in tolerance to temperature extremes are still uninvestigated.Therefore we compared resistance to high temperature extremes of C.elegans (strain N2) and C.briggsae (strain AF16). Experiments were carried out with young adults grown in Petri dishes withNGM and E.coli OP50 at 23°C. Resistance to constant temperature 36°C or 37°C was measured forworms incubated individually in 1 ml of liquid medium (NGM without agar, peptone, cholesterol).Results of our experiments show that organism's resistance to extreme high temperature is significantlyhigher in C.briggsae than in C.elegans:1. Mean time of reversible disturbances of swimming induced by intensive mechanic stimulus (shakingof test tube) was similar at 36°C in C.elegans and at 37°C in C.briggsae.2. Mean time of heat death was rather more in C.briggsae at 37°C than in C.elegans at 36°C.Rapid adaptations of worms to temperature elevation by 2 hour incubation at 30°C or by heathardening (1 hour at 33°C followed by 1 hour at 23°C) increased behavior resistance to short-term heatstress (36°C for C.elegans and 37°C for C.briggsae). Efficiency of both adaptations was similar in C.elegans and C.briggsae.Therefore we conclude that adaptation of C.elegans and C.briggsae to different temperatureconditions of habitats changed only base resistance to extreme high temperature.It is known that the upper temperature limit for reproduction of C.elegans N2 is 26.5°C (Fatt andDougherty, 1963), while C.briggsae AF16 can grow at 27.5°C (Gupta et al., 2007). Therefore our dataindicate that evolution of Caenorhabditis thermotolerance involves unidirectional adaptive changes ofthe upper limits of temperature for reproduction and resistance to short-term heat stress. This patternreveals in the evolution of most poikilotherms.Thus soil nematodes Caenorhabditis may be used as convenient model organisms for studythermotolerance evolution.ReferencesFatt HV, Dougherty EC. (1963). Genetic control of differential heat tolerance in two strains of the nematode, Caenorhabditiselegans. Science. 141, 266-267.Fodor A, Riddle DL, Nelson FK, Golden JW. (1983). Comparison of a new wild-type Caenorhabditis briggsae with laboratorystrains of C. briggsae and C. elegans. Nematologica 29, 203–217.Gupta BP, Johnsen R, Chen N. (2007). Genomics and biology of the nematode Caenorhabditis briggsae, Wormbook, ed.The C.elegans Research Community, Wormbook, doi/10.1895/wormbook.1.136.1, http//www.wormbook.org.- 20 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Examining interactions of unc-8 with the fainter mutant unc-80Michael Ailion 1 , Kim Schuske 1 , Phil Morgan 2 and Erik Jorgensen 11 Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City UT,2 Department of Anesthesiology, University of Washington, Seattle WACorrespondence to: Michael Ailion (ailion@biology.utah.edu)unc-79 and unc-80 encode large proteins necessary for proper localization of the NCA ionchannel (Humphrey et al., 2007; Jospin et al., 2007; Yeh et al., 2008). unc-79 and unc-80 also show geneticinteractions with mutants in the ENaC ion channel unc-8 (Rajaram et al., 1999). Loss-of-functionmutants in unc-8 were reported to suppress the fainter and anesthetic sensitivity phenotypes of unc-79and unc-80. Additionally, the exceptional allele unc-8(n491 n1193) was reported to have a fainter phenotypeon its own. Thus, it seemed possible that unc-79 and unc-80 regulate both the UNC-8 and NCAchannels. To investigate this further, we reexamined interactions of unc-8 and unc-80 in more detail.We conclude that unc-8 does not suppress the fainter phenotype of unc-80.The fainter phenotype of unc-8(n491 n1193) is in fact due to a background mutation in unc-80.First, we genetically separated the fainter phenotype of MT2612 unc-8(n491 n1193) from the unc-8chromosome, using bli-6 dpy-20 to balance unc-8. We isolated a fainter mutant that did not carry unc-8(n491 n1193) and an unc-8(n491 n1193) mutant that did not carry the fainter phenotype. The fainterphenotype was unlinked to unc-8(n491 n1193). Second, we found that the fainter mutation in this strainfailed to complement mutations in unc-80. Third, we sequenced unc-80 in this mutant (named ox374)and found a C to T change that leads to a premature stop at Q2811. The unc-80(ox374) single mutant isa fainter indistinguishable from other unc-80 mutants. The unc-8(n491 n1193) single mutant appears tobe grossly wild-type, similar to other unc-8(lf) mutants. The unc-8(n491 n1193); unc-80(ox374) doublemutant is a fainter, like the unc-80(ox374) single mutant. The genotypes of the unc-8 and unc-80 lociwere confirmed in all single and double mutants by sequencing.We were surprised that the unc-8(n491 n1193); unc-80(ox374) double mutant is a fainter sincethe unc-8(e15 lb145) deletion mutant was reported to suppress the fainter and anesthetic phenotypes ofunc-79 and unc-80. One possibility to explain this discrepancy is that unc-8(n491 n1193) carries anE552K missense mutation and may not be as strong as a null. To test this, we built a double mutant betweenunc-8(e15 lb145) and unc-80(ox330), an early stop mutant. Similar to the unc-8(n491 n1193);unc-80(ox374) double, we observed no suppression of the fainter phenotype in an unc-8(e15 lb145);unc-80(ox330) double mutant. We are in the process of reexamining the anesthetic phenotypes of thesedouble mutants.ReferencesHumphrey JA, Hamming KS, Thacker CM, Scott RL, Sedensky MM, Snutch TP, Morgan PG, Nash HA. (2007). A putativecation channel and its novel regulator: cross-species conservation of effects on general anesthesia. Curr. Biol. 17,624-629.Jospin M, Watanabe S, Joshi D, Young S, Hamming K, Thacker C, Snutch TP, Jorgensen EM, Schuske K. (2007). UNC-80and the NCA ion channels contribute to endocytosis defects in synaptojanin mutants. Curr. Biol. 17, 1595-1600.Rajaram S, Spangler TL, Sedensky MM, Morgan PG. (1999). A stomatin and a degenerin interact to control anesthetic sensitivityin Caenorhabditis elegans. Genetics 153, 1673-1682.Yeh E, Ng S, Zhang M, Bouhours M, Wang Y, Wang M, Hung W, Aoyagi K, Melnik-Martinez K, Li M, et al. (2008). Aputative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans. PLoS Biol.6:e55.- 21 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Molecular biology of the N2 allele of glb-5Sasha De Henau 1 , David Hoogewijs 2 and Jacques R. Vanfleteren 11 Department of Biology, Ghent University, B-9000 Ghent, Belgium, 2 Institute of Physiology, University of Zürich, Zürich,SwitzerlandCorrespondence to: Sasha De Henau (Sasha.DeHenau@UGent.be)A few years ago (Hoogewijs et al., 2007) we reported the amino acid sequence of GLB-5,which had been determined based on mRNA isolated from the Bristol (N2) strain of C. elegans. Thetranslated protein sequence, 258 aa long, was deposited at EMBL (Accession no. EF471982, http://www.ebi.ac.uk/cgi-bin/emblfetch?style=html&id=EF471982&Submit=Go). Unfortunately, the incorrectannotation 358 was entered in Table 1 of our 2007 paper. As a result, incorrect predictions ofmRNA and amino acid sequences were provided in WormBase (http://www.wormbase.org/db/gene/gene?name=WBGene00015964;class=Gene, release WS210). We have repeated this experiment andconfirm our earlier cDNA sequence, with the exception of one base and one corresponding amino acidsubstitution (red). The correct amino acid sequence for the N2 allele of glb-5 is:MNETTVGVGKMYEEFRITQLVNETLTTVIEKVQTNHEKISTKAKPRPLSAINEEIREYDQLSRELEKDYRRSMRIVDDDFELARTHWIQLQKSNKQGLAIRGCFLTMLEKYPQVRPIWGFGKRIEGRGDETWKPEIVEDFYFRHHCASLQAALNMIIQNKDDKSGMRRMLNEMGAHHFFYDACEPHFEVFQDSLLESMKLVLNGGDSLDDDIEQSWICTSLRIPPGSFEYDNSKQRRQKWNAADAQRNGSSSLFLRCMThe encoding cDNA sequence is identical to the one published by McGrath et al., (2009):atgaacgagacgacagtcggagttggaaaaatgtatgaagagtttcgaatcacacagcttgtgaatgaaactctaactactgttatcgaaaaagtgcaaaccaatcatgaaaaaatttctactaaagcaaagccgagacctttgagtgcaattaacgaggaaattcgtgaatacgatcaattgagcagagaacttgaaaaagattaccgaaggagtatgcgaattgttgatgatgatttcgaattggccaggacccattggatccaattacaaaaatcgaataaacaggggttggcaattcggggatgcttcctaacaatgttagaaaagtatccacaagttcgtccaatttgggggtttggaaaaagaattgagggaaggggtgatgagacatggaagcctgagatcgtggaggatttttattttagacatcattgcgcatccctccaggcagctttgaatatgataattcaaaacaaagacgacaaaagtggaatgcggcggatgctcaacgaaatgggagctcatcactttttctacgatgcatgtgaaccacattttgaagtttttcaagacagtctcctagaatcaatgaagcttgtattaaatggtggtgactcgttggatgatgatattgagcaatcttggatttgtacatcattgcgcatccctccaggcagctttgaatatgataattcaaaacaaagacgacaaaagtggaatgcggcggatgctcaacgaaatgggagctcatcactttttctacgatgcatgtgaaccacattttgaagtttttcaagacagtctcctagaatcaatgaagcttgtattaaatggtggtgactcgttggatgatgatattgagcaatcttggatttgtctgctccaaacaatccgactacatatgggagaaggaatcgaaattcaaagagccaactatctaacgcaatgcttgattccaaaagaaatggaagaagtacgtgcgaattggatgcaagtcgaaaattacgggtttcgaaaagccgggcttttattgtgtcaatcagcttttgacaactactcggaacttttgaaaactcataatttatcaatgactcttccaattgaagcaaacaagactagtgatagttttgttgcattatcagatcaaattatgcaggcactggacaaaacaatacaatcctacactcccgaagaaggattcttgaatttaattcaagaaattaaagactttgtgataaagttcttagtcgttgaagtatgccctccgcttattcggaaatcttttatcgatggattaatccacatgctttgtaaaatattaagcatcaagcatgttaaggaagattttttgcacatttggaaaaaagtgtatcgtgtcatggaacaggcaatggttgcaaatatcgtagagtattagThe genomic sequence of the N2 glb-5 gene contains a duplication leading to a duplicated exon (bold)and a premature stop codon (red), resulting in a 258 aa protein. The gene of the Hawaiian strain of C.elegans lacks this duplication, resulting in a protein that is 398 aa long and identical with the N2 proteinin its first 218 amino acids. Persson et al., (2009) also reported on GLB-5 from the Bristol and Hawaiianstrains, but provided no full cDNA or protein sequence data.ReferencesHoogewijs D, Geuens E, Dewilde S, Vierstraete A, Moens L, Vinogradov S, Vanfleteren JR. (2007). Wide diversity instructure and expression profiles among members of the Caenorhabditis elegans globin protein family. BMC Genomics8, 356.McGrath PT, Rockman MV, Zimmer M, Jang H, Macosko EZ, Kruglyak L, Bargmann CI. (2009). Quantitative mapping ofa digenic behavioral trait implicates globin variation in C. elegans sensory behaviors. Neuron 61, 692-699.Persson A, Gross E, Laurent P, Busch KE, Bretes H, and de Bono M. (2009). Natural variation in a neural globin tunes oxygensensing in wild Caenorhabditis elegans. Nature 458, 1030-1033.- 22 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Apparently normal DNA repair and transcript expression in the RB885strain carrying an intronic deletion in the xpc-1 geneJoel N. Meyer 1 and Bennett Van Houten 21 Nicholas School of the Environment, Duke University, Durham NC,2 Hillman Cancer Center, University of Pittsburgh, Pittsburgh PACorrespondence to: Joel Meyer (joel.meyer@duke.edu)xpc-1 is the C. elegans homolog of the human xeroderma pigmentosum complementation groupC gene, which plays a key role in the identification of DNA damage in the nucleotide excision repair(NER) pathway. Stergiou et al. (2005) found thatthe RB885 strain of C. elegans, which carries anintronic deletion in the xpc-1 gene, showed analtered apoptotic response to ultraviolet C (UVC)radiation. We tested whether the RB885 strainwould exhibit decreased repair of UVC-inducedDNA damage, as we observed for another NERmutant, xpa-1 (Meyer et al., 2007). We useddauer larvae in order to eliminate cell division asa potential confounder for our DNA repair assay.DNA repair was not detectably altered in RB885as compared to either N2 or glp-1 (JK1007) dauers(Fig. 1), using the F33H2.6 target to measureDNA damage. Similarly normal repair was observedin 9 additional nuclear targets. Dauer larvae,nuclear targets used to assess DNA damage/repair, UVC exposures and DNA damage measurements,were as described in Meyer et al.(2007).We confirmed the presence of the deletion in the RB855 genome using previously-developedprimers (http://aceserver.biotech.ubc.ca/cgi-bin/generic/allele?class=Allele;name=ok734), and detectedonly one unique full-length xpc-1 mRNA in dauer and mixed-stage xpc-1 nematode populations, alsousing previously-developed primers (http://worfdb.dfci.harvard.edu/searchallwormorfs.pl?by=name&sid=Y76B12C.2). Furthermore, we detected no differences in levels of xpc-1 mRNA expressioneither in unexposed or UV-exposed dauer or mixed-stage xpc-1 and N2 nematodes. xpc-1mRNA expression was induced modestly (1.5- to 2-fold) in both strains 24 h after UVC exposure(dauers exposed to 50 J/m 2 UVC; mixed-stage exposed to 200 or 400 J/m 2 UVC). For mRNA levelanalysis, we designed primers (forward, 5’- CGGAAGATGAATGGGAAGAA-3’ and reverse, 5’-GTCCATTGCGTATCGTTGTG -3’; product size 962 nt) for use in RT (reverse transcription) PCR.Those primers were designed to include exon-exon junctions to preclude the amplification of any contaminatingDNA.ReferencesMeyer JN, Boyd WA, Azzam GA, Haugen AC, Freedman JF, Van Houten B. (2007). Decline of nucleotide excision repaircapacity in aging Caenorhabditis elegans. Genome Biol. 8, R70.Stergiou L, Doukoumetzidis K, Sendoel A, Hengartner MO. (2007). The nucleotide excision repair pathway is required forUV-C-induced apoptosis in Caenorhabditis elegans. Cell Death Differ. 14, 1129-1138.- 23 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Differential expression of daf-16 splice variantsFrancis R.G. Amrit and Robin C. MaySchool of Biosciences, University of Birmingham, Birmingham, UKCorrespondence to: Robin May (r.c.may@bham.ac.uk)The transcription factor DAF-16 regulates a plethora of phenotypes in C. elegans, includinglongevity, stress tolerance and pathogen resistance. Wormbase (WS212) predicts seven alternativeisoforms of DAF-16, but little is known about the differential expression or regulation of theseisoforms.We set out to analyse the expression of the DAF-16 isoforms using quantitative real-time PCR(qRT-PCR). We were able to design isoform-specific primer sets for six of the seven isoforms; theseventh (R13H8.1e.1) is identical to R13H8.1e.2 with the exception of one missing exon and thereforecannot unambiguously be identified. We isolated RNA from mixed stage populations grown at 20°Con OP50, checked for genomic DNA contamination and linearly converted RNA to cDNA usingreverse transcriptase. Isoform specific PCR on this cDNA pool detected isoforms R13H8.1a,R13H8.1b, R13H8.1c and R13H8.1d. Additional PCR investigations and cDNA sequencingdemonstrated that isoforms R13H8.1e.2 and R13H8.1f are expressed but, under the conditions wetested, both isoforms retain introns currently annotated as being spliced out. R13H8.1e.2 retains theintron in the 5’UTR, whilst R13H8.1f retains the intron between exons one and two. We havesubmitted a revision of this annotation to Wormbase for inclusion in WS215.Using these splice form-specific primer sets, we determined the expression level of each of thesix isoforms relative to the housekeeping gene gpd-3 under resting (20°C) and heat-shock (37°C forthree hours immediately prior to RNA extraction) conditions (Table 1). Under both conditions,isoform R13H8.1b is by far the most strongly expressed and is the only isoform to significantlyincrease its expression upon heat-shock. This isoform was also the only variant to show increasedexpression in response to heavy metal stress (7mM CuCl 2 ) or pathogen challenge (Staphylococcusaureus exposure). Thus, the dominant DAF-16 form likely to be present in wildtype C. elegans isR13H8.1b. Determining the role(s) of the alternative isoforms of this key transcription factor will be ofgreat relevance for our understanding of longevity and stress response in C. elegans.IsoformLength(bp)Fold expression relative to gpd-320 o C 37 o CR13H8.1a 2575 1 0.44R13H8.1b 3021 18621.20 274998.94R13H8.1c 3035 0.39 0.31R13H8.1d 1616 1.68 0.96R13H8.1e.1 1128Cannot be discriminated from R13H8.1e.2R13H8.1e.2* 1027 0.18 0.10R13H8.1f* 2852 0.03 0.11Table 1. Expression of daf-16 isoforms at 20°C and following 3hr heat-shock at 37°C. Expression levels were normalisedto gpd-3 and then presented as fold-expression relative to R13H8.1a under resting (20°C) conditions. *Note that isoformsR13H8.1e.2 and R13H8.1f retain introns currently annotated as spliced out - see text for details.- 24 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010An alternatively spliced version of dop-2, a D2-like receptor genePratima Pandey, Tim Pierpont and Harbinder S. DhillonDepartment of Biological Sciences, Delaware State University, Dover DECorrespondence to: Harbinder Dhillon (hsdhillon@desu.edu)Specific neurotransmitters, their receptors and ion channels localized to synaptic termini, playimportant roles in behavior and synaptic plasticity. Dopamine receptors, characterized by seven transmembranedomains, are known to act through G-protein coupled pathways. In C. elegans, dopaminehas been implicated in a variety of behavioral processes, including both non-associative and associativelearning. The worm dop-2 gene represents a dopamine auto-receptor expressed in dopaminergic neurons(Suo et al., 2003). D2-like auto-receptors have been proposed to regulate the release of dopaminefrom the pre-synaptic neurons as well as reuptake by DAT (Williams and Galli, 2006; Voglis and Tavernarakis,2008 ). We are interested in interacting partners of dop-2 and are using its gene product asbait in a ubiquitin-based yeast two hybrid screen.In the process of reverse transcriptase PCR amplification of worm total RNA we serendipitouslyamplified an additional splice variant of dop-2, besides the 2 variants K09G1.4a and K09G1.4b(Suo et al., 2003). This third full-lengthmRNA (K09G1.4c, Figure-1) has 27 additionalnucleotides at the 3’ end of exon-8neighboring the exon:intron junction. Theadditional sequence codes for 9 aminoacidsin the large intracellular loop betweentrans-membrane domains 5 and 6.The resulting motif (GDLPLPMLL) doesnot display similarity to any known motifthrough Pfam match. Interestingly, the regionalsplicing of all three variants includingintron-7 and intron-8 is the conservedGC-AT junction sequence, and not GC-AG commonly found in splice variants inworms and humans (Farrer et al., 2002).Figure-1: Three alternately spliced forms of K09G1.4 (dop-2),showing the additional sequence in K09G1.4c, the longest variant(Figure modified from Worm-Base Genome Browser).ReferencesFarrer T, Roller AB, Kent WJ, Zahler AM. (2002). Analysis of the role of Caenorhabditis elegans GC-AG introns in regulatedsplicing. Nucl. Acids Res. 30, 3360-3367.Suo S, Sasagawa N, Ishiura S. (2003). Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor.J. Neurochem. 86, 869-878.Voglis G, Tavernarakis N. (2008). A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopaminesignaling. EMBO J. 27, 3288-3299.Williams JM, Galli A. (2006). The dopamine transporter: a vigilant border control for psychostimulant action. Handb. Exp.Pharmacol. 175, 215-232.- 25 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010A sensitized screen for identifying inhibitors of EGF vulval inductionactivityClay Nelson 1 , Kimberly B. Monahan 3 , Channing J. Der 2,3 ,David J. Reiner 2,31 Departments of Biology, 2 Pharmacology, and 3 the Lineberger Comprehensive Cancer Center, University of North CarolinaSchool of Medicine, Chapel Hill NCCorrespondence to: David Reiner (dreiner@med.unc.edu)C. elegans EMS mutagenesis screens are an important tool for identifying novel components ofbiological processes. Here, we report a screen to identify novel gain-of-function alleles promoting EGFpathway activity in vulval development. Simultaneous loss of the lin-15A and lin-15B loci confers astrong herniated multivulva (Muv) defect consistent with over-induction of 1° vulval fates, and lin-15(n765ts) animals are null for one locus and temperature sensitive mutant for the other (Huang et al.,1994). This hyper-induced vulval phenotype is due to de-repressed LIN-3/EGF expression in the hyp7hypodermis surrounding the vulval precursor cells (VPCs), such that all VPCs receive an inappropriatelyhigh EGF signal (Cui et al., 2006). We reasoned that using temperature-sensitive lin-15(n765)mutant animals, we could titrate EGF activity to the level just barely insufficient for ectopic vulval induction.In this background an F 1 screen should allow screening through tens of thousands of haploidgenomes to uncover heterozygous activating mutations (gain-of-function alleles) sufficient to induceectopic vulval formation.To identify a non-inducing condition, we first assayed lin-15(ts) animals at a number of temperatures.We found that at 15°C, lin-15(ts) animals were not Muv, while Muv animals were observedat higher temperatures. We applied the standard 50 mM EMS mutagenesis protocol to late L4 lin-15(n765ts) animals, plated mutagenized P 0 animals at 15°C, and screened F 1 adult progeny for the Muvphenotype. We found an extremely high rate of positives, with 44/455 (9.7%) Muv F 1 animals. Manyputative mutants (19/44, 43.2%) segregated >5 Muv progeny, indicating that picked animals did notrepresent background noise, but rather were bona fide enhancer mutations that conferred a dominantMuv phenotype in this background at 15°C. Given this exceptionally high positive rate, we are probablyidentifying genes that are haploinsufficient for lin-3/EGF repression or EGFR pathway inhibition,which is similar to the sensitized Drosophila enhancer screen for haploinsufficiency in sevenless function,the E(sev) screen (Simon et al., 1991). Based on the estimated identifiable loss-of-function mutationrate of 5X10 -4 for this EMS mutagenesis protocol, we estimate the gene target size of our screen tobe roughly 84 genes.Clearly this screen is unsuitable for our purposes. We speculate that we have identified a verylarge group of genes required for the transcriptional repression of lin-3/EGF, the synMuv genes, or anotherrepressive function in vulval induction. Increased stringency should be achieved by using a lowertemperature, thus making the screen less sensitive.ReferencesCui M, Chen J, Myers TR, Hwang BJ, Sternberg PW, Greenwald I, Han M. (2006). SynMuv genes redundantly inhibit lin-3/EGF expression to prevent inappropriate vulval induction in C. elegans. Dev. Cell. 10, 667-72.Huang LS, Tzou P, Sternberg PW. (1994). The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulvaldevelopment. Mol. Biol. Cell, 5, 395-412.Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM. (1991) Ras1 and a putative guanine nucleotide exchangefactor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67, 701-716.- 26 -

THE WORM BREEDER’S GAZETTE VOLUME 18 NUMBER 2 | JUNE 2010Announcements:From András Fodor, Institute of Plant Protection, Georgikon Faculty, Pannonia University, Keszthely,Hungary (fodorandras@yahoo.com)I would like to let our community to know that I am back in Hungary in a new position at PannoniaUniversity in Keszthely, Hungary, where I am resuming my research on C. elegans, C. briggsae andother nematodes. I would like to thank Ann Rougvie, Aric Daul and Theresa Stiernagle from the CGCand Prof. Ralf-Udo Ehlers from the University of Kiel, Germany, for providing strains and protocols. Itis great to be back, and I am proud to be teaching real Mendelian-Brennerian Genetics to my studentsin my Homeland.- 27 -