Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans

Abstract

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes. Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes. cysteine protease cathepsin L gene green fluorescence protein expressed sequence tag RNA interference basic local alignment search tool reverse transcriptase α-amanitin-resistant gene first larval stage second larval stage third larval stage fourth larval stage polyethylene glycol β-galactosidase phosphate-buffered saline double-stranded RNA Cysteine proteases of the papain superfamily have long been recognized for their role in intracellular and extracellular protein degradation in a range of cellular processes (1Bond J.S. Butler P.E. Annu. Rev. Biochem. 1987; 56: 333-364Crossref PubMed Scopus (455) Google Scholar). Within the papain family, the cathepsins can be subdivided into more than 10 subfamilies on the basis of their primary sequence and enzymatic activity (2Santamaria I. Velasco G. Pendas A.M. Fueyo A. Lopez-Otin C. J. Biol. Chem. 1998; 273: 16816-16823Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The family includes cathepsin B, C, L, and Z, all of which contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, such as substrate specificity and pH stability. Cathepsin B-like cysteine protease genes occur as a large multigene family in a wide range of parasitic and free-living nematodes. Several cathepsin B genes were reported to be expressed in Caenorhabditis elegans, some of which were restricted to the intestines of larval and adult transgenic worms (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar, 4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar). Interestingly, the structurally similar Hemonchus contortus (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar, 4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar) and Schistosoma mansoni (6Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar) cathepsin B homologues were also expressed in the gut and were suggested to be potentially involved in feeding (5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar, 7McKerrow J.H. Drug Disc. Desi. 1994; 2: 437-444Google Scholar), such as nutrient digestion. Heterologous transformation of C. elegans with an H. contortus cathepsin B gene promoter has demonstrated also conservation of the mechanisms controlling its spatial expression in free-living and parasitic nematodes (8Britton C. Redmond D.L. Knox D.P. McKerrow J.H. Barry J.D. Mol. Biochem. Parasitol. 1999; 103: 171-181Crossref PubMed Scopus (47) Google Scholar), and therefore both enzymes were hypothesized to be not only structurally similar, but also functionally homologous and important for proper feeding (4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 7McKerrow J.H. Drug Disc. Desi. 1994; 2: 437-444Google Scholar). Cathepsin L and Z-like proteases were shown to be present in many parasitic nematodes, where they are speculated to have diverse biological functions including invasion, feeding, molting, and immune evasion (reviewed in Refs. 6Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar, 9Koiwa H. Shade R.E. Zhu-Salzman K. D'Urzo M.P. Murdock L.L. Bressan R.A. Hasegawa P.M. FEBS Lett. 2000; 471: 67-70Crossref PubMed Scopus (95) Google Scholar, and 10Shompole P.S. Jasmer D.P. J. Biol. Chem. 2000; 276: 2928-2934Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Many of these cathepsins have homologues in C. elegans suggesting that they may be involved in functions conserved across different nematode species. Yet not much is known about their precise function. In Dirofilaria immitis (11Richter J.K. Sakanari J.A. Frank G.R. Grieve R.B. Exp. Parasitol. 1992; 75: 303-307Crossref PubMed Scopus (38) Google Scholar) and Brugia pahangi 1X. Hang, C. Britton, and J. McKerrow, unpublished data.1X. Hang, C. Britton, and J. McKerrow, unpublished data. cysteine proteases were shown to be associated with molting as well as activities that might facilitate larval migration. The potential role of cysteine proteases during molting was indirectly established in Onchocerca volvulus by showing that the peptidyl monofluoromethyl ketones, low molecular weight irreversible cysteine protease inhibitors, inhibit the molting of third stage larvae (L3) in a time- and dose-dependent manner (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). These irreversible inhibitors can block cathepsin Z and L-like, but not the B-like enzyme activities, suggesting that a cathepsin L, as well as a cathepsin Z, might be involved in the molting process. The target cysteine proteases were indirectly localized in the granules of the glandular esophagus ofO. volvulus L3 using the biotin-Phe-Ala-CHN2inhibitor (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In further studies, a larval O. volvuluscysteine protease named LOVCP was cloned (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). LOVCP as well as its homologue in Toxocara canis and C. eleganswere recently classified as a novel monophylectic group in the papain family of cysteine proteases, named cathepsin Z (13Falcone F.H. Tetteh K.K. Hunt P. Blaxter M.L. Loukas A. Maizels R.M. Exp. Parasitol. 2000; 94: 201-207Crossref PubMed Scopus (20) Google Scholar). The O. volvulus cathepsin Z was shown to be required for molting and the development of fourth-stage larvae (L4) based on its localization using monospecific anti-LOVCP antibodies. The native enzyme was localized in molting L3 in the region where the separation between the cuticles of L3 and L4 takes place. The general role of the O. volvulus cathepsin Z-like enzyme as well as its C. elegans homologue (14Hashmi S. Tawe W. Lustigman S. Trends Parasitol. 2001; 17: 387-392Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) during molting was therefore hypothesized to be as a proteolytic enzyme involved in cuticle degradation, similar to that observed in the entomopathogenic fungiMetarhizium anisopliae with its endogenous cysteine protease Pr4 (15Cole Jr., S.C. Charnleg A.K. Cooper R.M. FEMS Microbiol. Lett. 1993; 113: 189-196Crossref Google Scholar). This function as well as other yet unknown functions of cathepsin Z and L cysteine proteases were previously hypothesized to be present in nematodes based on the immunolocalization of onchocystatin, an endogenous cysteine protease inhibitor, in thin sections of O. volvulus. Onchocystatin was expressed in L3, molting L3, adult worms, and eggshells around developing microfilaria (16Lustigman S. Brotman B. Huima T. Prince A.M. Mol. Biochem. Parasitol. 1991; 45: 65-75Crossref PubMed Scopus (71) Google Scholar, 17Lustigman S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar), suggesting that its target cysteine protease(s) is possibly involved in regulating activities such as molting, cuticle remodeling, and embryogenesis during the development of the parasite in the host. Importantly, C. elegans has two homologues of onchocystatin (14Hashmi S. Tawe W. Lustigman S. Trends Parasitol. 2001; 17: 387-392Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), suggesting the presence of their target enzymes in this worm as well. The indirect approaches as described above, however, do not provide conclusive information on the precise function of these proteases. Recently, a new family of cathepsin L-like sequences with similarity to previously characterized mammalian cathepsin L-like enzymes was identified in the O. volvulus as well as in other filarial L3 EST data bases, 2D. Guiliano, unpublished data.2D. Guiliano, unpublished data. and in other parasitic nematodes. 3C. Britton and L. Murray, unpublished data.3C. Britton and L. Murray, unpublished data.Their function is not as yet known. We have identified a related cathepsin L-like protease sequence (T03E6.7) within the C. elegans complete genome data base (ACeDB), and used the C.elegans powerful system for investigating its functionsin vivo during development. To determine the function of the cathepsin L protease we took advantage of methods available in C. elegans that enable analysis of gene promoter activity and gene function in individual cells. Based on the obtained information we could predict its potential physiological role in filarial parasites. This study is the first to directly demonstrate the functional importance of cathepsin L in nematode development. A BLAST search (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar) of the C. elegans genome data base (ACeDB, www.sanger.ac.uk/projects/C_elegans/wormpep/1) and the nr data base using the Ov-CPL (accession number AF331036) and other filarial nematodes amino acid sequences, Di-CPL (accession number AF001101), Bp-CPL (accession number AF031819.1) identified a predicted C. elegans cathepsin L-like gene (T03E6.7), which was namedCe-cpl-1.4 ACe-cpl-1 cDNA clone containing the full-length sequence was also identified in the EST data base (accession number C12099) and the corresponding λ ZAP II phage was obtained from Yuji Kohara (clone yk146d10, C. elegans consortium, National Institute of Genetics, Mishima, Japan). The pBluescript phagemid was excised and the DNA sequenced in both directions to confirm the predicted amino acid sequence. Signal sequences and putative cleavage sites were identified using the SignalP server (www.cbs.dtu.dk/services/SignalP). Prediction of the pro-region cleavage site as well as the active sites were based on alignment of the CPL protein sequences was made using Cluster W multiple sequence alignment. Analysis of the promoter region ofCe-cpl-1 gene was performed using Genefinder provided by BCM server (www.hgsc.bcm.tmc.edu/searchlauncher). C. elegans strains used in this study were the wild-type Bristol N2 strain and unc-76 mutant strain DR96 (unc-76(e911)V) (19Brenner M. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar), both provided by theCaenorhabditis Genetics Center. Strains were maintained on NGM agar plates as previously described (19Brenner M. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). Semi-quantitative RT-PCR (sqRT-PCR) was carried out using first strand cDNA generated from total RNA collected from synchronous L1, L2, L3, L4, and young adultC. elegans cultures at 2-h intervals, as previously described (20Johnstone I.L. Barry J.D. EMBO J. 1996; 15: 3633-3639Crossref PubMed Scopus (133) Google Scholar). The stage-specific cDNA samples were kindly provided by Iain Johnstone (Wellcome Center for Molecular Parasitology, University of Glasgow, UK) and used according to the established protocols. Gene-specific cDNA fragments were amplified using two sets of PCR primers, one set was specific forCe-cpl-1 (CPF1, sense, 5′-GTCTCCGTGCTCTGGGTCGGTTCCGTATC-3′ and CPR1, antisense, 5′-CCATGGTGTCGACACCGAGGAGTCATAC-3′) and the other set was specific for an internal control, the ama-1transcript (20Johnstone I.L. Barry J.D. EMBO J. 1996; 15: 3633-3639Crossref PubMed Scopus (133) Google Scholar). The primers were designed to span an intron to distinguish cDNA from contaminating gDNA products. The following PCR conditions, which allowed reactants to remain in excess, were used: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 3 min. Amplified products were separated on 2% agarose gels, Southern blotted, and probed with the appropriate end-labeled oligonucleotides. After autoradiography, specific bands corresponding to amplified cpl-1 and control ama-1 products were excised from the blot for each time point and counted in scintillant. The relative content of the transcript corresponding to the Ce-cpl-1 gene is expressed as the ratio of the signal for cpl-1 for each developmental stage to that ofama-1. The sqRT-PCR was carried out on two occasions, with very similar results, one of which is presented. The double-stranded RNA interference (RNAi) procedure was carried out as described by Fireet al. (21Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11651) Google Scholar) and Tabara et al. (22Tabara H. Grishok A. Mello C.C. Science. 1998; 282: 430-431Crossref PubMed Scopus (518) Google Scholar). Three different regions of the Ce-cpl-1 gene were PCR amplified from cosmid T03E6 DNA using Vent DNA polymerase (New England Biolabs, Beverly, MA) and the following PCR primers (underline indicates artificial restriction enzyme sites introduced for cloning): CPexon1F (XhoI), sense, 5′-AATCTCGAGATTCATTCTTCTGGCACTG-3′, and CPexon1R (XbaI), antisense, 5′-AAATCTAGATTCTTACCAAGTCAGC-3′ (PCR product 275 bp); CPexon3F (PstI), sense, 5′-TTTCTGCAGCCAGATGAGGTTGAC-3′ and CPexon3R (PstI), antisense 5′-ACCCTGCAGGTAAAGTTGGAAGCTGC-3′ (PCR product 453 bp); CPexons3+4F (XbaI), sense, 5′-AAATCTAGACGACTGCTCTACCAAGTAC-3′, and CPexons3+4R (XhoI), antisense, 5′-GACCTCGAGATAACTGGCCTTGGTGGC-3′ (PCR product 1.07 kb). Positions and sizes of the resulting PCR products are indicated in Fig. 1B. The three DNA fragments and the full-lengthCe-cpl-1 cDNA present in clone yk146d10 were cloned separately into pBluescript (Stratagene, La Jolla, CA) and used as templates for RNA synthesis. The double-stranded RNA (dsRNA) used for RNAi experiments was prepared following the Fire et al. (21Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11651) Google Scholar) protocol. Briefly, forin vitro transcription, each of the pBluescript plasmid DNA constructs was linearized with the appropriate restriction enzyme and single-stranded sense or antisense RNA was synthesized using the RiboMAX RNA Large-Scale Production System (Promega, Madison, WI) according to the manufacturers instructions. Equal amounts of each set of sense and antisense RNA strand were then annealed by incubation in injection buffer (20 mm KPO4, 3 mmK citrate, 2% PEG 6000, pH 7.5) for 10 min at 68 °C and 30 min at 37 °C. Double-stranded RNA (concentration 0.5 mg/ml) was then injected into the gonad of 25–30 young adult C. eleganshermaphrodites. Injected worms were left for 16–24 h at 20 °C to recover and to lay any eggs present in utero prior to microinjection, and then transferred to individual plates at 24-h intervals. The F1 progeny was quantified and examined for embryonic lethality or abnormal development. RNAi using the soaking protocol was done on L3 and L4 as described by Tabara et al. (22Tabara H. Grishok A. Mello C.C. Science. 1998; 282: 430-431Crossref PubMed Scopus (518) Google Scholar). Briefly, 15 C. elegans worms were incubated in 15 μl of 0.2 m sucrose in 0.1 × PBS containing 1 mg/ml dsRNA pre-mixed with 1 μl of Lipofectin (Invitrogen, Carlsbad, CA). After 24 h, soaked larvae were transferred to individual plates and their development examined for 4–5 days. Larvae soaked in 0.2 m sucrose in 0.1 × PBS without dsRNA served as the control. The length and width of each treated worm were measured under microscope using ocular and stage micrometer. Data were analyzed by One-way Analysis of Variance (ANOVA) and Student-Newman-Keuls Comparison Test (GraphPad InStat Software Inc.). Probability values of less than 0.05 were considered significant. Loss ofcpl-1 transcript following dsRNA injection was examined by RT-PCR using the same cpl-1 and ama-1 internal primers described above. Approximately 20 RNAi injected adult hermaphrodites or 300 RNAi mutant embryos were collected and washed twice in 1 ml of PBS. Wild-type adults and embryos were used as controls for normal gene expression levels. Adult and embryo pellets were then resuspended in 200 μl of lysis buffer (0.5% SDS, 5% β-mercaptoethanol, 10 mm EDTA, 10 mmTris-HCl, pH 7.5, and 0.5 mg/ml proteinase K), quick-frozen at −80 °C for 10 min, followed by incubation at 55 °C for 1 h. The RNA was extracted using Total RNA Isolation Reagent (Advanced Biotechnologies Ltd.) and the RT-PCR was carried out using SuperScript One-Step RT-PCR System (Invitrogen) according to the manufacturers instructions. Each 50-μl reaction mixture was split into two tubes into which either cpl-1 primers or ama-1 control primers were added. After 35 cycles of amplification, the RT-PCR products were separated on 2% agarose gels. Both transcriptional and translationalCe-cpl-1 reporter gene fusion constructs were generated and their expression patterns examined. For the transcriptional fusion construct, a promoter region of 1.76-kb ofcpl-1 upstream sequence, was generated by PCR on T03E6 cosmid DNA using Vent DNA Polymerase (New England Biolabs) and the following PCR primers: CPpromF1 sense, corresponding to to relative to the and (XbaI), antisense, to to relative to the restriction enzyme the PCR was cloned into reporter containing a localization signal by A. The fusion a including of the potential promoter region and all the and three This was amplified by PCR on T03E6 cosmid DNA using the following primers: F1 sense, corresponding to to relative to the and antisense, to to within the cpl-1 gene relative to the The PCR amplified gene was first cloned into the using the (Invitrogen) and then into the reporter by A. DNA of the was prepared using the System (Invitrogen) and sequenced to confirm that the of cpl-1 is with of C. elegans was performed by of plasmid DNA into the of the gonad as described previously A. EMBO J. PubMed Google Scholar, C.C. D. V. EMBO J. 1991; PubMed Scopus Google Scholar). plasmid DNA corresponding to the transcriptional or was with a plasmid DNA at containing a mutant of the C.C. D. V. EMBO J. 1991; PubMed Scopus Google Scholar). were identified by their Mol. Biol. 1990; PubMed Scopus Google Scholar). To of expression from transcriptional fusion constructs in the of any from transformation of the strain DR96 was carried out with plasmid of Center for which the wild-type expressed unc-76 in which and the were for β-galactosidase as previously described (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar) using as a to in the of was by transgenic worms on a 2% agarose in that their and under a fluorescence three were examined for each A of Ce-cpl-1 cDNA the enzyme amino was amplified and cloned into the and sites of the expression The protein was expressed in the of and was therefore from the mm pH The was in m in pH followed by separation using the according to the manufacturers instructions. containing the were identified using A was using the multiple site S.A. J.A. B. PubMed Scopus Google Scholar). Each a total of 10 of in on and For of the enzyme in C. elegans hermaphrodites were washed plates in PBS and to the The embryos were then collected and in using the protocol D. Biol. 113: PubMed Scopus Google Scholar). For of larvae and adults were collected and washed in PBS and buffer and using in buffer for 30 min and two cycles in a M. 1990; Full Text PDF PubMed Scopus Google Scholar). The and embryos or the worms were treated with a mg/ml normal in for 1 h reaction with antibodies. were used at and were used at a were under fluorescence microscope using appropriate sets in the presence of containing CA). C. elegans stage and embryos collected from 5% hermaphrodites were for min in in 0.1 m pH containing The worms were then for as previously described S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar). sections of C. elegans worms were probed with the C. fusion incubation with with Inc.). or to were used as The localization of the native CPL in O. done as previously described S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar) using thin sections of O. volvulus L3 and worms and the O. volvulus region or the or to were used as The the CPL enzymes recognized by blot their corresponding and not with the cathepsin Z-like of C. elegans or O. volvulus not The potential promoter region of was amplified by PCR using O. volvulus λ II provided by R. D. the and primers designed based on known A containing the was amplified first followed by using the and an antisense corresponding to a region within the The final region identified as the putative promoter of the gene was upstream of the of region (accession number For the transcriptional fusion construct, this was amplified from O. volvulus DNA using the following of PCR primers: (XbaI), sense, corresponding to to relative to the and antisense, to to relative to the restriction enzyme the PCR was cloned into reporter containing a localization signal by A. The fusion gene containing the putative promoter was a we C. elegans transgenic volvulus promoter as described in and expression of C. elegans reporter gene Based on search of using the region of amino acid and the other filarial nematode sequences, a related sequence was identified in cosmid (accession