Development of a Species-Specific PCR for Detection and Quantification of Meloidogyne hapla in Soil Using the 16D10 Root-Knot Nematode Effector Gene.

The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of numerous agricultural crops in temperate regions. Accurate detection and quantification is vital to supporting informed pest management decisions. However, traditional methods of manual nematode extraction and morphology-based identification are time-consuming and require highly specialized training. Molecular methods may expand the diagnostician's toolkit beyond those methods that rely on this disappearing specialized skillset. However, molecular assays targeting the internal transcribed spacer region may lead to inaccurate results because of intraspecific variability. The Meloidogyne spp. effector gene 16D10 was assessed as a target for a SYBR Green I quantitative PCR (qPCR) assay for detection and quantification of M. hapla. M. hapla-specific qPCR primers were developed and evaluated for specificity against five M. hapla isolates and 14 other plant-parasitic nematodes. A standard curve was generated by relating the quantification cycle (Cq) to the log of M. hapla population densities artificially introduced into soil. The influence of soil inhibitors on quantitative amplification was assessed by generating a dilution series from DNA extracted from pure nematode cultures and inoculated soil. Extracts from soil produced significantly higher Cq values than those produced from pure culture extracts. The utility of the qPCR was evaluated using soil samples collected from three naturally infested potato fields, resulting in a significant positive relationship between populations estimated using qPCR and populations derived from manual counting. The qPCR developed in this study provides a useful method for detecting and quantifying M. hapla in soil and demonstrates the utility of effector genes in plant-parasitic nematode diagnostics. The ability to use effector genes as targets for qPCR and other molecular detection and quantification methods may open additional avenues of novel research and support development of improved species-level diagnostics.

3-oxoacyl-ACP reductase from Schistosoma japonicum: integrated in silico-in vitro strategy for discovering antischistosomal lead compounds.

BACKGROUND: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR. METHODOLOGY/PRINCIPAL FINDINGS: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells. CONCLUSIONS/SIGNIFICANCE: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.

Comparison of transcript profiles in different life stages of the nematode Globodera pallida under different host potato genotypes.

The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second-stage juveniles (J2s) and sedentary parasitic-stage J2s (SJ2); (ii) a comparison of SJ2s at 8 days after inoculation (DAI) in the susceptible cultivar (Desirée) and two partially resistant lines. The results showed differential expression of G. pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines; the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30 DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30 DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.

Cu,Zn superoxide dismutase: cloning and analysis of the Taenia solium gene and Taenia crassiceps cDNA.

Cytosolic Cu,Zn superoxide dismutase (Cu,Zn-SOD) catalyzes the dismutation of superoxide (O(2)(-)) to oxygen and hydrogen peroxide (H(2)O(2)) and plays an important role in the establishment and survival of helminthes in their hosts. In this work, we describe the Taenia solium Cu,Zn-SOD gene (TsCu,Zn-SOD) and a Taenia crassiceps (TcCu,Zn-SOD) cDNA. TsCu,Zn-SOD gene that spans 2.841 kb, and has three exons and two introns; the splicing junctions follow the GT-AG rule. Analysis in silico of the gene revealed that the 5'-flanking region has three putative TATA and CCAAT boxes, and transcription factor binding sites for NF1 and AP1. The transcription start site was a C, located at 22 nucleotides upstream of the translation start codon (ATG). Southern blot analysis showed that TcCu,Zn-SOD and TsCu,Zn-SOD genes are encoded by a single copy. The deduced amino acid sequences of TsCu,Zn-SOD gene and TcCu,Zn-SOD cDNA reveal 98.47% of identity, and the characteristic motives, including the catalytic site and ß-barrel structure of the Cu,Zn-SOD. Proteomic and immunohistochemical analysis indicated that Cu,Zn-SOD does not have isoforms, is distributed throughout the bladder wall and is concentrated in the tegument of T. solium and T. crassiceps cysticerci. Expression analysis revealed that TcCu,Zn-SOD mRNA and protein expression levels do not change in cysticerci, even upon exposure to O(2)(-) (0-3.8 nmol/min) and H(2)O(2) (0-2mM), suggesting that this gene is constitutively expressed in these parasites.

[Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor].

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.

[Molecular cloning and characterization of an acetylcholinesterase gene Dd-ace-2 from sweet potato stem nematode Ditylenchus destructor].

A cDNA, named Dd-ace-2, encoding an acetylcholinesterase (AChE, EC3.1.1.7), was isolated from sweet-potato-stem nematode, Ditylenchus destructor. The nucleotide and amino acid sequences among different nematode species were compared and analyzed with DNAMAN5.0, MEGA3.0 softwares. The results showed that the complete nucleotide sequence of Dd-ace-2 gene of Ditylenchus destructor contains 2425 base pairs from which deduced 734 amino acids (GenBank accession No. EF583058). The homology rates of amino acid sequences of Dd-ace-2 gene between Ditylenchus destructor and Meloidogyne incognita, Caenorhabditis elegans, Dictyocaulus viviparous were 48.0%, 42.7%, 42.1% respectively. The mature acetylcholinesterase sequences of Ditylenchus destructor may encode by the first 701 residues of deduced 734 amino acids.The conserved motifs involved in the catalytic triad, the choline binding site and 10 aromatic residues lining the catalytic gorge were present in the Dd-ace-2 deduced protein. Phylogenetic analysis based on AChEs of other nematodes and species showed that the deduced AChE formed the same cluster with ACE-2s.

Identification of genes involved in interactions between Biomphalaria glabrata and Schistosoma mansoni by suppression subtractive hybridization.

Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, a medically important schistosome. In order to identify transcripts involved in snail-schistosome interactions, subtractive cDNA libraries were prepared, using suppression subtractive hybridization (SSH) between a parasite-exposed schistosome-resistant and a susceptible strain of B. glabrata, and also between schistosome-exposed and unexposed snails from the resistant snail line. Separate libraries were made from both haemocytes and the haemopoietic organ. Subtraction was performed in both directions enriching for cDNAs differentially expressed between parasite-exposed resistant and susceptible samples and up or down-regulated in the resistant line after challenge. The resulting eight libraries were screened and eight genes, differentially expressed between the haemocytes of resistant and susceptible snail strains, were identified and confirmed with reverse transcriptase PCR, including two transcripts expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite: the iron-storage and immunoregulatory molecule, ferritin, and HtrA2, a serine protease involved in the cellular stress response. Transcripts with elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Differential expression of two of the transcripts with no known function from the susceptible strain, was independently confirmed in a repeat exposure experiment.

MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode.

Animal microRNAs (miRNAs) are gene regulatory factors that prevent the expression of specific messenger RNA targets by binding to their 3' untranslated region. The Caenorhabditis elegans lsy-6 miRNA (for lateral symmetry defective) is required for the left/right asymmetric expression of guanyl cyclase (gcy) genes in two chemosensory neurons termed ASE left (ASEL) and ASE right (ASER). The asymmetric expression of these putative chemoreceptors in turn correlates with the functional lateralization of the ASE neurons. Here we find that a mutation in the die-1 zinc-finger transcription factor disrupts both the chemosensory laterality and left/right asymmetric expression of chemoreceptor genes in the ASE neurons. die-1 controls chemosensory laterality by activating the expression of lsy-6 specifically in ASEL, but not in ASER, where die-1 expression is downregulated through two sites in its 3' untranslated region. These two sites are complementary to mir-273, a previously uncharacterized miRNA, whose expression is strongly biased towards ASER. Forced bilateral expression of mir-273 in ASEL and ASER causes a loss of asymmetric die-1 expression and ASE laterality. Thus, an inverse distribution of two sequentially acting miRNAs in two bilaterally symmetric neurons controls laterality of the nematode chemosensory system.

Detection of apoptosis during planarian regeneration by the expression of apoptosis-related genes and TUNEL assay.

Apoptosis is a tightly organized cell death process that plays a crucial role in metazoan development, but it has not yet been revealed whether apoptotic events are involved in the process of regeneration. Here, we tried to detect apoptotic cells during planarian regeneration using the TdT-mediated dUTP nick-end labeling (TUNEL) assay as well as the expression of apoptosis-related genes. Three novel cDNAs were isolated from a planarian cDNA library and shown to be closely related to other metazoan caspases at the amino acid sequence level. One of these cDNAs, Caspase-like gene 3 (DjClg3), was expressed primarily in apoptotic cells by double detections with the TUNEL assay. Whole mount in situ studies indicated that DjClg3 was expressed in the cells of the mesenchymal space and also around the pharynx of the intact body. Its expression in the regenerating head piece was seen in the blastema and less significantly in the brain, while in the regenerating tail piece, DjClg3 expression was detected uniformly throughout the entire region. In parallel experiments, we performed in situ TUNEL assays to localize the regions where cell death occurred during regeneration and comparable results to the DjClg3 expression patterns were obtained. This is the first report to show that planarians have apoptosis-related genes and the results suggest that the apoptotic mechanism probably takes place to a large extent in normal intact worms as well as during their regeneration. We hypothesize that the presence of apoptosis in planarians may have a role in controlling cell numbers, eliminating unnecessary tissues or cells and remodeling the old tissues of regenerating body parts.

Mapping and sequencing of acetylcholinesterase genes from the platyhelminth blood fluke Schistosoma.

Acetylcholinesterase (AChE) on the surface of the parasitic blood fluke Schistosoma is the likely target for schistosomicidal anticholinesterases. Determination of the molecular structure of this drug target is key for the development of improved anticholinesterase drugs and potentially a novel vaccine. We have recently cloned the cDNA encoding the AChE from the human parasite Schistosoma haematobium and succeeded in expressing functional recombinant protein. We now describe the cloning and molecular characterisation of homologues from two other schistosome species-Schistosoma mansoni and Schistosoma bovis, which are important parasites of man and cattle, respectively, but which differ in their sensitivity to the therapeutic anticholinesterase metrifonate. Comparison of the deduced amino acid sequences revealed that the AChE from all three species posses a high degree of identity, with conservation of all of the residues known to be important for substrate binding and catalytic activity. Also conserved is a unique C-terminal domain which is unusual in that it lacks the consensus for GPI modification, even though the native protein is considered to be GPI-anchored. We have also established the AChE gene structures for all three species and cloned the complete gene for S. haematobium AChE. The gene structure is relatively complex, comprising nine coding exons; the location of the splice sites is identical in all three species, but the size of the introns varies considerably. The two C-terminal splicing sites that are conserved in all species are also present in Schistosoma, but a third C-terminal conserved splicing site which is located 11-13 amino acids upstream of the histidine of the catalytic triad in all invertebrate AChE genes characterised to date is absent. We discuss our findings in the context of the molecular phylogeny of the AChE genes and the potential application to the control of schistosomiasis.

The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans.

Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans. Two cDNAs encoding highly similar proteins to human PARP-1 (huPARP-1) and huPARP-2 are described, and we propose to name the corresponding enzymes poly(ADP-ribose) metabolism enzyme 1 (PME-1) and PME-2 respectively. PME-1 (108 kDa) shares 31% identity with huPARP-1 and has an overall structure similar to other PARP-1 subfamily members. It contains sequences having considerable similarity to zinc-finger motifs I and II, as well as with the catalytic domain of huPARP-1. PME-2 (61 kDa) has structural similarities with the catalytic domain of PARPs in general and shares 24% identity with huPARP-2. Recombinant PME-1 and PME-2 display PARP activity, which may partially account for the similar activity found in the worm. A partial duplication of the pme-1 gene with pseudogene-like features was found in the nematode genome. Messenger RNA for pme-1 are 5'-tagged with splice leader 1, whereas those for pme - 2 are tagged with splice leader 2, suggesting an operon-like expression for pme - 2. The expression pattern of pme-1 and pme-2 is also developmentally regulated. Together, these results show that PARP-1 and -2 are conserved in evolution and must have important functions in multicellular organisms. We propose using C. elegans as a model to understand better the functions of these enzymes.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein.

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1

Characterisation of a collagen gene subfamily from the potato cyst nematode Globodera pallida.

We have isolated two full-length genomic DNA sequences, which encode the cuticle collagen proteins GP-COL-1 and GP-COL-2, from the potato cyst nematode Globodera pallida. A third, partial collagen gene ORF termed gp-col-t(t=truncated) has also been isolated and appears to represent an unexpressed pseudogene. The gp-col-1 and gp-col-2 genes both contain three short (<97 bp) introns which disrupt coding regions predicted to specify proteins with molecular weights of 33 and 32.7 kDa respectively. All three sequences show high similarity to each other and to the previously isolated G. pallida cDNA clone gp-col-8. The conserved pattern of cysteine residues and non-(Gly-X-Y)(n) region sequence similarity observed in all four G. pallida genes suggests that these molecules form part of the same subfamily of collagens. Southern analysis indicates that this subfamily is likely to contain further members. The G. pallida collagen sequences show striking similarity to twelve genes from Caenorhabditis elegans which collectively represent the recently classified Group 1a collagen subfamily. No data exists on the function of this subfamily in C. elegans. gp-col-1 and gp-col-2 are developmentally regulated with transcripts of both genes detected in adult virgin and gravid females but not in pre-parasitic second stage juveniles. A similar expression pattern is observed for the Group 1a collagen lemmi 5 from Meloidogyne incognita perhaps indicating a generic link between subfamily and function during the various changes in cuticular structure which accompany nematode growth and reproduction. Immunochemical studies indicate that the GP-COL-1 protein is specifically located in the hypodermis of G. pallida adult females.

CLC chloride channels in Caenorhabditis elegans.

The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism.

Identification and characterization of a putative C. elegans potassium channel gene (Ce-slo-2) distantly related to Ca(2+)-activated K(+) channels.

Two putative homologues of large conductance Ca(2+)-activated K(+) channel alpha-subunit gene (slowpoke or slo) were revealed by C. elegans genome sequencing. One of the two genes, F08B12.3 (Ce-slo-2), shows a relatively low amino acid sequence similarity to other Slo sequences and lacks key functional motifs, which are important for calcium and voltage sensing. However, its overall structure and regions of homology, which are conserved in all Slo proteins, suggest that Ce-SLO-2 should belong to the Slo channel family. We have cloned a full-length cDNA of the Ce-slo-2, which encodes a protein containing six putative transmembrane segments with a K(+)-selective pore and a large C-terminal cytosolic domain. Green fluorescent protein (GFP) and whole-mount immunostaining analyses revealed that Ce-slo-2 is specifically expressed in neuronal cells at the nerve ring, at the ventral nerve cord of the mid-body, and at the tail region. We have also identified a putative human counterpart of Ce-slo-2 from a human brain EST database, which shows a stretch of highly conserved amino acid residues. Northern blot and mRNA dot blot analyses revealed a strong and specific expression in brain and skeletal muscle. Taken together, our data suggest that Ce-slo-2 may constitute an evolutionarily conserved gene encoding a potassium channel that has specific functions in neuronal cells.

A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species.

General methods to conduct tissue specific analysis are largely lacking for nematodes. An approach is described that focused on isolation of membrane and secreted protein genes from the gut of the parasitic nematode Haemonchus contortus. The approach capitalized on a monoclonal antibody that recognizes multiple membrane and secreted worm proteins. Polyclonal antisera made against these proteins were used to screen expression cDNA libraries made either from adult worm gut or whole worm. The genes identified encode predicted or known membrane and secreted proteins from gut, including a cysteine protease, a zinc metallopeptidase and a previously described GA1 protein. Another gene, Hc40, was isolated from the whole worm cDNA library and is nearly identical to a vaccine patent sequence pBTA879. Tissue analysis demonstrated the intended focus on membrane and secreted proteins from parasite gut was achieved. Proteins related to each of those described were identified from other nematode species through data base analysis. Additionally, this analysis led to (1) identification of homologues of each gene in C. elegans; (2) deduction of a dimorphic structure in the Hc40 protein; (3) recognition of both monomorphic and dimorphic families of Hc40-related proteins; and (4) detection of two apparent classes of transcripts (mep1a and mep1b) that would each encode a divergent version of the putative zinc metallopeptidase MEP1. The tissue specific approach and information base described should generally contribute to investigations on nutrient digestion and related secretory processes in nematode gut.

The tpa-1 gene of Caenorhabditis elegans encodes two proteins similar to Ca(2+)-independent protein kinase Cs: evidence by complete genomic and complementary DNA sequences of the tpa-1 gene.

The gene tpa-1 on chromosome IV of the nematode Caenorhabditis elegans plays a major and definitive role in the adversary action of tumour-promoting phorbol esters, which induce growth arrest and locomotory distress in the animal. The gene was deduced to code for a protein kinase C (PKC) homologue by molecular cloning. We have now sequenced the complete genomic and complementary DNAs for tpa-1 and have analysed their structural features in detail: (1) tpa-1 spans over 20 kb consisting of eleven exons and ten introns; (2) two different-sized mRNAs are generated from the tpa-1 locus; (3) both mRNAs are trans-spliced to the trans-spliced leader SL1; (4) both mRNAs encode PKC isoforms, which are most similar to Ca(2+)-independent novel PKC0; (5) the two PKC isoforms differ from each other in that the smaller lacks the amino-terminal region of the larger corresponding to the first four exons and a portion of the fifth exon; and (6) three introns are located at; identical positions in the polypeptide sequences aligned between the C. elegans tpa-1 product and a PKC of the fruit fly Drosophila melanogaster.

MEK-2, a Caenorhabditis elegans MAP kinase kinase, functions in Ras-mediated vulval induction and other developmental events.

Activated Ras initiates a cascade of sequential phosphorylation events, including the protein kinases Raf, MEK, and MAP kinase. The Let-60 Ras-mediated signal transduction pathway controls vulval induction in Caenorhabditis elegans. Both Lin-45 Raf and Sur-1 MAP kinase have been determined to be essential factors during vulval induction; however, the C. elegans mek gene has not been identified. In this paper, we have cloned a C. elegans mek gene, mek-2, and demonstrated that the MEK-2 protein possesses the biochemical properties of MAP kinase kinases: The C. elegans MEK-2 protein can phosphorylate and activate a human MAP kinase (ERK1), and MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. The mek-2 gene plays a key role in the let-60 ras-mediated vulval induction pathway, as loss-of-function mutations in the gene (ku114 and h294) significantly reduce the signal transmitted through Ras. mek-2(ku114) completely suppressed the Multivulva (Muv) phenotype of a hyperactive let-60 ras mutation, and animals homozygous for mek-2(ku114) also displayed a partial larval lethal phenotype. Animals homozygous for mek-2(h294) exhibited a highly penetrant sterile and Vulvaless phenotype. Microinjection of a gain-of-function mek-2 mutation resulted in Muv and other mutant phenotypes, whereas microinjection of a dominant-negative mutation not only suppressed the Muv phenotype of an activated let-60 ras mutation but also caused an egg-laying defective phenotype in otherwise wild type animals. Our results demonstrate that mek-2 acts between lin-45 raf and sur-1/mpk-1 in a signal transduction pathway used in the control of vulval differentiation and other developmental events.

Cloning and functional expression of a Shaker-related voltage-gated potassium channel gene from Schistosoma mansoni (Trematoda: Digenea).

We have isolated a cDNA (SKv1.1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56.5 kDa) contains 6 putative membrane-spanning domains (S1-S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64-74% identity in the core region (S1-S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2.8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1.1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdotoxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths.