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PubMed1999

Transient expression of DNA and RNA in parasitic helminths by using particle bombardment.

Parasitic helminths (worms belonging to several metazoan phyla) cause considerable morbidity and mortality in humans. They are an important veterinary problem, and they result in significant economic losses in animal grazing and agriculture. Experimental studies on parasitic helminths have been limited by a lack of parasite cell lines and methods for molecular genetic analyses. We evaluated particle bombardment (biolistics) as a strategy to introduce and express nucleic acids in these multicellular parasites. By using embryos of the parasitic nematode Ascaris as a model, we developed methods to introduce and express both DNA and RNA during several stages of Ascaris embryogenesis. Biolistic transfection will facilitate experimental strategies in Ascaris embryos complementing other biochemical tools available (e.g., in vitro whole-cell embryo extracts for transcription, RNA processing, and translation). Transfection experiments with adult schistosomes further suggest that the biolistic strategy should be applicable to a variety of other parasitic helminths. The development of these methods provides molecular genetic tools to study gene expression and the biology of a variety of types and developmental stages of important helminth parasites.

Animals↗
PubMed1998

Generation, identification, and evaluation of expressed sequence tags from different developmental stages of the Asian blood fluke Schistosoma japonicum.

Here we report 658 expressed sequence tags (ESTs) generated from the 5'-termini of clones randomly selected from directional cDNA libraries constructed from mRNAs from three developmental stages of Schistosoma japonicum. Putative identifications were assigned to 46. 2% of the ESTs; 6.4% were previously known from S. japonicum, 5.6% were previously known from S. mansoni, 34.2% were known from other organisms, and the remaining 53.8% may represent S. japonicum-specific genes. These 658 ESTs appeared to be derived from 457 unique genes, which together represent 2 to 3% of the 15,000 to 20,000 genes predicted to occur in the schistosome genome.

Animals↗
PubMed2003

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Animals↗
PubMed2003

Proteomic changes during disturbance of cholesterol metabolism by azacoprostane treatment in Caenorhabditis elegans.

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.

Animals↗
PubMed2006

A conserved sequence motif in 3' untranslated regions of ribosomal protein mRNAs in nematodes.

The 3' untranslated regions (3' UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression or localization at the post-transcriptional level. This study concerns the identification of novel, conserved elements in 3' UTRs of many ribosomal protein mRNAs in Caenorhabditis elegans and Caenorhabditis briggsae. Analysis of the region around the polyadenylation signal in many ribosomal protein mRNAs indicates the conservation of a sequence motif UUGUU occurring both before and immediately after the polyadenylation signal. Building a statistical model of this motif and searching a database of C. elegans 3' UTRs reveals that this motif is also present in the 3' UTR of some genes involved in translation and ribosome maturation, among others. We suggest that this signal may be involved in translation or other message-level regulation of ribosomal genes in C. elegans.

3' Untranslated Regions↗
PubMed1995

Gene cloning and complete nucleotide sequence of philippine Schistosoma japonicum paramyosin.

The development of an effective vaccine is recognised as a necessary adjunct to the control of schistosomiasis japonica, a disease affecting several million people in China and the Philippines. Currently, recombinant Schistosoma japonicum molecules are considered most suitable for large scale vaccine production and a number of genes encoding vaccine candidate polypeptides have been cloned and expressed (see Waine et al., 1993a). One of the molecules providing most promise as a vaccine target is paramyosin (Butterworth, 1992), a major structural protein of thick filaments in the muscle of most invertebrates; paramyosin genes have now been cloned from a range of parasitic helminths, including schistosomes (Limberger and McReynolds, 1990; Laclette et al., 1991; Dahmen et al., 1993; Landa et al., 1993; Mühlschlegel et al., 1993, Nara et al., 1994). The cloning and nucleotide sequence of S. Japonicum paramyosin is described.

Animals↗
PubMed2006

Whole genome RNAi screens for increased longevity: important new insights but not the whole story.

This article discusses the pros and cons of using RNAi screening to identify longevity genes in Caenorhabditis elegans. The discussion will focus on the results of two large-scale longevity RNAi screens that were recently published. The two screens revealed largely non-overlapping sets of candidate longevity genes. The possible reasons for such differences and their implications will be discussed.

Aging↗
PubMed1999

Complexity of developmental control: analysis of embryonic cell lineage specification in Caenorhabditis elegans using pes-1 as an early marker.

In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that approximately 100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.

Animals↗
PubMed2005

Selective sensitivity of Caenorhabditis elegans neurons to RNA interference.

RNA interference is a new approach to knockdown gene expression, but effectiveness varies depending on the organism, cell type or target sequence. Studies with Caenorhabditis elegans have shown that subsets of cells including neurons are often resistant to RNA interference. We measured RNA interference using green fluorescent protein reporter strains and feeding, soaking and injection delivery methods in a number of Caenorhabditis elegans neuron subtypes (dopaminergic, GABAergic, cholinergic, glutamatergic, touch). The sensitivity to RNA interference varied: GABAergic and dopaminergic neurons showed greater resistance while cholinergic, glutamatergic and touch neurons were more sensitive. Dysfunctional RRF-3, a putative RNA-directed RNA polymerase, had a significant effect on increasing neuron sensitivity in most subtypes. These results demonstrate that Caenorhabditis elegans neurons vary in their sensitivity to RNA interference.

Acetylcholine↗
PubMed2002

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.

3' Untranslated Regions↗