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PubMed1993

In search of new mutants in cell-signaling systems of the nematode Caenorhabditis elegans. Review.

Development of multicellular organisms is controlled mainly by cell-signaling systems. In this review I first discuss methods of genetic analysis and properties of mutants of cell-signaling systems in general and in the nematode C. elegans. Then, I describe two of our approaches to isolating new mutants in cell-signaling of C. elegans. The first approach is to select for mutants that have the same visible phenotype as those in known cell-signaling genes. In a survey of larval lethal mutations we found that there are quite a few mutants in which the inner surface of the body wall is detached from the outer surface of the intestine. Some of them map in genes that are known to act in cell-signaling systems in vulval induction or sex myoblast migration, which are not essential to the growth and survival of C. elegans. Therefore, we think many of the mutations of the above phenotype disrupt cell-signaling in an unidentified essential function, and also cell-signaling in the non-essential functions. The second approach is to isolate mutants resistant to a drug expected to disturb cell-signaling. As the drug we have chosen sodium fluoride, which depletes calcium ion, activates G-proteins and inactivates some phosphatases. The mutants are grouped into two classes (three and two genes, respectively) according to degree of fluoride-resistance and growth rate of larvae. Although there is so far no direct evidence that these mutants are related to cell-signaling, they show complex epistasis that can be explained by a model consisting of a cell-signaling pathway.

Animals↗
PubMed1999

A Caenorhabditis elegans homeobox gene expressed in the male tail, a link between pattern formation and sexual dimorphism?

ceh-7 is a small Caenorhabditis elegans homeobox gene. We have shown that this gene is transcribed. Examination of the expression pattern of ceh-7 using reporter constructs revealed that it is expressed in a few cells of the male tail, which form a ring around the rectum. The most posterior member of the C. elegans Hox cluster, egl-5, an Abd-B homologue, has previously been shown to be required for the proper development of several blast cells in the male tail. We have examined the expression of ceh-7 in mutant backgrounds of egl-5 and also mab-5, an Antp/Ubx/Abd-A homologue. We find that ceh-7 is not expressed in egl-5 mutants, but is still expressed in mab-5 mutants.

Amino Acid Sequence↗
PubMed2002

Codon optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells.

Differences in codon usage can seriously hamper the expression of cloned cDNAs in heterologous systems. In this study, we show that the expression of a cloned Schistosoma mansoni cDNA in cultured HEK293 cells was dramatically increased by rewriting a portion of the cDNA according to human preferred codon usage, suggesting that codon optimization is a valuable strategy for improving the heterologous expression of helminth sequences. We further describe a simple modification of a recursive PCR-based method, which allows the rewriting of long stretches of DNA sequence in a single PCR reaction. This method can be used to optimize the codon usage of virtually any DNA from helminths and other parasites.

Animals↗
PubMed2004

Molecular cloning and characterization of cDNA encoding a ubiquitin-conjugating enzyme from Clonorchis sinensis.

The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.

Amino Acid Sequence↗
PubMed1998

Cloning and sequence analysis of a lysophospholipase homologue from Schistosoma mansoni.

A novel cDNA encoding a lysophospholipase (SmLPLH) homologue has been cloned from the human parasite, Schistosoma mansoni. The predicted primary structure of SmLPLH shares high sequence homology with mammalian lysophospholipases (62%) and also contains aconsensus signature peptide (GXSXG), which is present in all lipases. Two splice variants of SmLPLH that differ by 32 N-terminal amino acids were identified. The shorter truncated species carries a conserved S. mansoni spliced leader (SL) sequence at its 5' end and is believed to be formed by trans-splicing of the SL to an internal exon splice site. The functional significance of these two isoforms of SmLPLH is currently unknown.

Amino Acid Sequence↗
PubMed1998

Promoter trapping identifies real genes in C. elegans.

Promoter trapping involved screening uncharacterized fragments of C. elegans genomic DNA for C. elegans promoter activity. By sequencing the ends of these DNA fragments and locating their genomic origin using the available genome sequence data, promoter trapping has now been shown to identify real promoters of real genes, exactly as anticipated. Developmental expression patterns have thereby been linked to gene sequence, allowing further inferences on gene function to be drawn. Some expression patterns generated by promoter trapping include subcellular details. Localization to the surface of particular cells or even particular aspects of the cell surface was found to be consistent with the genes, now associated with these patterns, encoding membrane-spanning proteins. Data on gene expression patterns are easier to generate and characterize than mutant phenotypes and may provide the best means of interpreting the large quantity of sequence data currently being generated in genome projects.

Animals↗
PubMed1992

Schistosoma mansoni: hycanthone/oxamniquine resistance is controlled by a single autosomal recessive gene.

Individual schistosomes of an hycanthone/oxamniquine-sensitive strain were crossed with individual schistosomes of the opposite sex and belonging either to the same sensitive population or to a different strain which exhibited high resistance to the two drugs. Schistosome crosses were performed by transfer of single worm pairs into the mesenteric veins of mice and the drug sensitivity/resistance of individual progeny worms was assessed using an in vitro test. Drug resistance behaved as an autosomal recessive trait, as shown by the results of the F1 and F2 generation and of the backcrosses. Drug-resistant worms appeared to be slightly less viable than their sensitive counterpart at all stages of the life cycle. The results are relevant for an interpretation of drug resistance and drug mechanisms and the approach used in this study may be applicable to different genetic markers in schistosomes.

Animals↗
PubMed1995

pop-1 encodes an HMG box protein required for the specification of a mesoderm precursor in early C. elegans embryos.

In C. elegans embryogenesis, the MS blastomere produces predominantly mesodermal cell types, while its sister E generates only endodermal tissue. We show that a maternal gene, pop-1, is essential for the specification of MS fate and that a mutation in pop-1 results in MS adopting an E fate. Previous studies have shown that the maternal gene skn-1 is required for both MS and E development and that skn-1 encodes a transcription factor. We show here that the pop-1 gene encodes a protein with an HMG box similar to the HMG boxes in the vertebrate lymphoid-specific transcriptional regulators TCF-1 and LEF-1. We propose that POP-1 and SKN-1 function together in the early embryo to allow MS-specific differentiation.

Amino Acid Sequence↗
PubMed1995

Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans.

Using their senses of taste and smell, animals recognize a wide variety of chemicals. The nematode C. elegans has only fourteen types of chemosensory neurons, but it responds to dozens of chemicals, because each chemosensory neuron detects several stimuli. Here we describe over 40 highly divergent members of the G protein-coupled receptor family that could contribute to this functional diversity. Most of these candidate receptor genes are in clusters of two to nine similar genes. Eleven of fourteen tested genes appear to be expressed in small subsets of chemosensory neurons. A single type of chemosensory neuron can potentially express at least four different receptor genes. Some of these genes might encode receptors for water-soluble attractants, repellents, and pheromones.

1-Octanol↗
PubMed1995

The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction.

Vulval induction in C. elegans is controlled by a highly conserved signaling pathway similar to the RTK-Ras-MAPK cascade in mammals. By screening for suppressors of the Multivulva phenotype caused by an activated let-60 ras allele, we isolated mutations in a gene, ksr-1, that acts as a positive modifier of vulval induction and is required for at least two other let-60 ras-mediated processes. Although ksr-1 mutations do not perturb vulval induction in an otherwise wild-type background, they have very strong effects on vulval induction in genetic backgrounds where Ras pathway activity is constitutively activated or compromised, suggesting that ksr-1 activity is required for maximal stimulation of vulval fates by the Ras pathway. Genetic epistasis analysis suggests that ksr-1 acts downstream of or in parallel to let-60 ras. We cloned ksr-1 and have shown that it encodes a novel putative protein kinase related to the Raf family of Ser/Thr kinases.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a highly immunogenic calponin-like protein.

The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5' end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O.volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40-60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.

Amino Acid Sequence↗
PubMed1993

Thinking about genetic redundancy.

Partial functional redundancy among genes is frequently observed in a wide range of organisms and processes, but the selective value of such redundancy is not immediately apparent. Any fully redundant function should be evolutionarily unstable: unless selection acts to maintain the redundancy it will tend to be lost by mutational drift. I discuss four possible mechanisms by which selection might act to maintain genetic redundancy.

Animals↗
PubMed1994

Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations.

Despite its simple body form, the nematode C. elegans expresses homeotic cluster genes similar to those of insects and vertebrates in the patterning of many cell types and tissues along the anteroposterior axis. In the ventral nerve cord, these genes program spatial patterns of cell death, fusion, division and neurotransmitter production; in migrating cells they regulate the direction and extent of movement. Nematode development permits an analysis at the cellular level of how homeotic cluster genes interact to specify cell fates, and how cell behavior can be regulated to assemble an organism.

Animals↗
PubMed1992

Homeoboxes in flatworms.

A search for homeobox-containing genes was done in the genome of a primitive metazoan, the parasitic tapeworm Echinococcus granulosus. Five different homeoboxes were isolated, none of them belonging to the classical Antennapedia-type. Three of the homeodomains are similar to those from the Drosophila melanogaster NK-type genes. The fourth homeodomain shares extensive identity with that of the recently reported homeobox-containing gene goosecoid from Xenopus laevis. The third helix (the recognition helix) of the fifth isolated homeodomain is identical to that of the Xlim-1 gene of X. laevis and the lin11 gene of Caenorharbditis elegans. Using PCR, some Antennapedia-type homeoboxes were cloned from the genome of two other Platyhelminthes, Dugesia tigrina (planaria) and Fasciola hepatica. These data suggest that, contrary to what is found for the majority of the more complex metazoans, Platyhelminthes contain few homeobox genes belonging to the Antennapedia-type.

Amino Acid Sequence↗
PubMed1995

Detection of planarian Antennapedia-like homeobox genes expressed during regeneration.

Using the polymerase chain reaction with degenerate oligo primers we identified six new Antennapedia-like homeobox sequences expressed in a regenerating planarian. cDNA fragments containing the entire homeobox sequences for four of these genes were obtained. Two of them, Dutarh-3 and Dutarh-4, belong to the classical Antennapedia type and display extensive identity with Antennapedia- and Deformed-type homeodomains respectively. The third homeodomain, Dutarh-1, exhibits some similarity to the Hox1.5-, AbdB- and Dfd-type homeodomains and a fourth gene, Dutarh-6, is very similar to Mox2 in the mouse. Our results suggest that, in spite of their simple body plan, planarians contain a number of Antennapedia-like homeobox genes, probably enough to fill a cluster such as found in higher animals.

Amino Acid Sequence↗
PubMed1995

A sea urchin homologue of ceh-19, an unusual homeobox-containing gene from a nematode.

When screening for homeobox-containing genes from the sea urchin Heliocidaris erythrogramma (He), we isolated an exon of a gene which appears to be a homologue of the homeobox-containing gene, ceh-19, of Caenorhabditis elegans (Ce). The predicted translation of the sea urchin sequence shows 77% identity and 92% similarity to the first 53 amino acids of the homeodomain of ceh-19. The ceh-19 gene exhibits an intron in an unusual location in the 3' end of the homeobox; the He gene shares this feature.

Amino Acid Sequence↗
PubMed1993

Control of morphogenesis and differentiation by HOM/Hox genes.

HOM/Hox genes are master regulatory switches that specify axial identity and control the growth and differentiation of groups of cells related by position. HOM/Hox genes function combinatorially and hierarchically to specify cell fate. Some of the genes they regulate and that mediate specific identify functions have been identified. Research in Drosophila has shown that HOM genes are continuously required during development for correct axial identity.

Animals↗