Fiona J. Thompson

Heat shock factor functions at the convergence of the stress response and developmental pathways in Caenorhabditis elegans

Heat shock factor (HSF) is best characterized as the transcriptional regulator of heat shock protein genes, required by all cells to survive periods of stress. Recent evidence suggests that HSF also functions to regulate the expression of genes involved in growth and development under normal physiological conditions. In this study, we used RNA interference (RNAi) assays to investigate the role of HSF in Caenorhabditis elegans. Exposure of wild‐type worms to hsf dsRNAi constructs caused a temperature‐sensitive developmental arrest at the L2/L3 stage. At normal growth temperatures, hsf(RNAi) worms that developed to adults were small and scrawny, largely infertile, and showed a significant reduction in life span. These results demonstrate that HSF is required for normal postembryonic development under physiological conditions. Following heat shock, hsf(RNAi) worms were thermosensitive and displayed a significant reduction of hsp16 expression. When hsf(RNAi) was carried out in various dauer‐constitutive mutant backgrounds, a dramatic reversal of dauer formation was observed, indicating that HSF is also required in the dauer pathway. In its natural habitat of the soil, where C. elegans is exposed to a constantly fluctuating environment; the ability to integrate the stress response with development may be an essential element of its ecology.

Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti

The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown rats. Specifically, gender-biased transcription between free-living females and free-living males, and parasitic-biased transcription between parasitic females and free-living females was determined. Of the estimated 3,688 distinct transcripts represented on the microarray, 743 (20%) exhibited male-biased transcription of >1.4-fold (2(0.5)), 689 (19%) female-biased transcription, 418 (11%) parasitic-biased transcription and 305 (8%) free-living-biased transcription. Among those transcripts that exhibited the highest levels of differential transcription, an orthologue of major sperm protein was identified in males, distinct aspartic protease orthologues in either parasitic or in free-living females, and orthologues of hsp-17 chaperone in parasitic females. These 3,688 transcripts were separated into 12 clusters, such that the pattern of transcription between life-stages and biological replicates was similar among transcripts within a cluster and dissimilar between clusters. Using annotation inferred from Caenorhabditis elegans, gene ontology terms over-represented in one or more clusters were identified and showed that female-biased transcription was associated with genes involved in reproductive processes and larval development, male-biased transcription was linked to genes involved in metabolism, and free-living-biased transcription related to genes involved in the regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased transcription was less clear. The present findings for S. ratti provide a basis for a detailed exploration of differentially regulated molecules and might assist in the search for novel drug or vaccine targets in parasitic nematodes.

The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi.

A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNA during parasite development in the mosquito vector was investigated using RT-PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks.

Temperature is a cue for gene expression in the post-infective L3 of the parasitic nematode Brugia pahangi.

The temporal expression pattern of two genes, Bp-cdd and Bp-S3, was studied at defined points throughout the life cycle of Brugia pahangi. Both mRNAs were up-regulated to coincide with the transition of the L3 from the vector to the mammalian host. Bp-cdd was expressed almost exclusively in the post-infective (p.i.) L3 and L4 stages of the life cycle while Bp-S3 was also expressed in adult worms, but at a much lower level than in the larval stages. Immunogold labelling with an antiserum raised to the recombinant Bp-CDD localised the native antigen to the hypodermis in the p.i. L3 and L4. Specific labelling was not detected in the adult worm. The expression of both mRNAs could be triggered by exposure of the vector-derived L3 to a simple mammalian culture system. Analysis of the factors, which induced expression suggested that the temperature shift which accompanies the transition from mosquito to mammal was the most important cue for expression of both genes.

Stage-specific gene expression in lymphatic filarial nematodes.

Lymphatic filarial nematodes remain a significant cause of morbidity throughout much of the tropics. One approach to the development of rational control methods is an improved understanding of the basic biology of these organisms in relation to the mechanisms used to complete their life cycles. In this article, Eileen Devaney, Sam Martin and Fiona Thompson review new approaches to defining stage-specific molecules in filarial nematodes, and discuss their recent work on the isolation and characterization of stage-regulated cDNAs from Brugia pahangi.

Migration related changes in gene expression in leg muscle of the Christmas Island red crab Gecarcoidea natalis : seasonal preparation for long distance walking

SUMMARY During their annual breeding migration the Christmas Island land crab Gecarcoidea natalis sustains locomotion aerobically for up to 12 h per day compared with just 10 min during the dry season when their muscles quickly become anaerobic. A seasonal transition to an endurance-muscle phenotype would thus seem essential for migrating crabs. The current study employed a gene discovery approach comparing two expressed sequence tag (EST) libraries, one each for leg muscle from dry (non-migrating) and wet season (migrating) crabs. The 14 most abundant transcripts differed in their representation between the two libraries. The abundances of transcripts of genes predicted to code for different proteins forming contractile muscle components, including actin, troponin and tropomyosin, were significantly different between seasons and thus between physiological states. The shift in the isoform composition of the contractile elements provided evidence for a switch from slow phasic (S1) to slow tonic (S2) fatigue-resistant muscle fibres. A tropomyosin (tm) transcript aligned with a tm isoform of lobster (tmS2), and semi-quantitative RT-PCR confirmed this isoform to be more abundant in the migrating crab muscle. Two LIM protein coding genes, a paxillin-like transcript (pax) and a muscle LIM protein (mlp), were relatively up-regulated in muscle of wet season crabs. These proteins have a fundamental role in muscle development and reconstruction, and their comparative up-regulation is consistent with a remodelling of leg muscle for migration in the wet season. Such a transition would result in an increased representation of aerobic endurance-type fibres concomitant with the greater aerobic exercise capacity of the migrating red crabs.

Cloning and characterisation of mmc-1, a microfilarial-specific gene, from Brugia pahangi

Nine differentially expressed genes were cloned from Brugia pahangi in a screen which sought to identify cDNAs that were differentially expressed between the microfilariae from the mammalian host and the mosquito vector. One gene (mmc-1), that was up-regulated in mammalian-derived microfilariae, was characterised in detail. RT-PCR analysis demonstrated that mmc-1 was specific to the microfilarial stage of the life cycle and was not transcribed by developing microfilariae in utero, but only following the release of the microfilariae from the adult female. Analysis of DNA from other filarial worms suggested that mmc-1 may be a Brugia-specific gene. Using serum samples from individuals exposed to Brugia malayi infection, it was shown that MMC-1 was specifically recognised by antibodies of the IgG3 subclass. mmc-1 has no homologues in the data bases and its function in the parasite is unknown.

Biochemical and molecular characterization of two cytidine deaminases in the nematode Caenorhabditis elegans.

Two cytidine deaminases (CDDs) from the free-living nematode Caenorhabditis elegans have been cloned and characterized. Both Ce-CDD-1 and Ce-CDD-2 are authentic deaminases and both exhibit RNA-binding activity towards AU-rich templates. In order to study their temporal and spatial expression patterns in the worm, reporter gene constructs were made using approx. 2 kb of upstream sequence. Transfection of C. elegans revealed that both genes localized to the cells of the intestine, although their temporal expression patterns were different. Expression of Ce-cdd-1 peaked in the early larval stages, whereas Ce-cdd-2 was expressed in all life cycle stages examined. RNA-interference (RNAi) assays were performed for both genes, either alone or in combination, but only cdd-2 RNAi produced a consistent visible phenotype. A proportion of eggs laid from these worms were swollen and distorted in shape.