Joyce Moore

The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties.

The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.

Messenger RNA degradation in Saccharomyces cerevisiae.

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.

Identification of abundant mRNAs from the third stage larvae of the parasitic nematode, ostertagia ostertagi.

Reverse transcriptase-PCR (RT-PCR) was carried out on total RNA prepared from the third-stage larvae (L3) of Ostertagia ostertagi in order to clone and characterize the major transcripts expressed in this larval stage, as an initial investigation of arrested larval development in the parasite. Distinct bands were visible on an agarose gel and four of these were cloned and sequenced. Three of the bands represented multiple transcripts, while the fourth band encoded the enzyme GTP cyclohydrolase I (GTP-CH), which catalyses the first and rate-limiting step in pteridine biosynthesis. Northern blot analysis and RT-PCR demonstrated that GTP-CH is highly up-regulated in the L3 stage and undetectable in either the L2 or adult stages. Using immunogold electron microscopy, GTP-CH was shown to be predominantly localized to the cell body of the body wall muscles and the cells of the intestine in the L3.

Cloning and characterization of two nuclear receptors from the filarial nematode Brugia pahangi.

Nuclear receptors (NRs) encompass a superfamily of cytoplasmic/nuclear localized receptors that on ligand binding (or by phosphorylation) directly regulate the transcription of target genes. NRs are involved in many developmental processes, including moulting in insects and dauer larva formation in Caenorhabditis elegans. Here we report the isolation of two genes related to NRs from the filarial nematode Brugia pahangi. Bp-nhr-1 is a member of the NGF1-B subfamily of NRs and is expressed at very low levels in post-infective larval stage 3 (L3) after their transmission to the mammalian host. The second gene, Bp-nhr-2, is related to XR78E/F of Drosophila, a gene involved in the ecdysone response, over the region of its DNA-binding domain. cDNA and genomic clones have been isolated that correspond to Bp-nhr-2. The most striking feature of the encoded protein is that, although there is a DNA-binding domain similar to that of other NRs, the ligand-binding domain is absent. To investigate the pattern of transcription of Bp-nhr-2 in the filarial life cycle, semi-quantitative reverse-transcriptase-mediated PCR was performed; this analysis demonstrated that the gene is expressed in early stages after infection and in the adult and microfilariae, and is up-regulated just before the moult between L3 and L4 but is not expressed during the moult between L4 and adult. Antibodies raised against a peptide corresponding to the transactivation domain of Bp-nhr-2 demonstrate that the protein is expressed in microfilariae and adult samples and that another cross-reactive protein is expressed in these life-cycle stages.

Characterisation ofSaccharomyces cerevisiae genes encoding ribosomal protein YL6

We have characterised aSaccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 ofSchizosaccharomyces pombe. ‘mRNA13’ also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-up shift), and declines when cells are subjected to a mild heat-shock. Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature. The gene(s) corresponding to cDNA13, like other ribosomal protein genes ofS. cerevisiae, thus contain an intron. Southern blot analysis indicates the presence of two separate loci related to cDNA13 in theS. cerevisiae genome. From the sequence of one of these, a complete polypeptide sequence was deduced. The first 40 amino acids are identical to those of YL6, aS. cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis. There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of otherS. cerevisiae ribosomal protein genes.

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.

A quantitative study of the susceptibility of cockroach species to Moniliformis moniliformis (Acanthocephala)

We exposed 19 species of cockroaches to Moniliformis moniliformis by administering shelled acanthors orally and by injecting hatched acanthors directly into the body cavity. The exposure method had little influence on susceptibility: with one exception, species that were susceptible to oral exposure were also susceptible to injected acanthors and, in no case, did acanthors develop in injected animals when they did not develop in orally exposed conspecifics. We found no support for the hypothesis that the intestine may be a barrier to M. moniliformis establishment in cockroaches. There is, however, clear interspecific variation in susceptibility to M. moniliformis infection.

The relationship between mRNA half-life and gene function in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae (Sc) mRNAs have been described as falling into two major classes with respect to mRNA half-life [Santiago et al., Nucleic Acids Res. 14 (1986) 8347-8360]. We have used DNA sequence analysis to address the functional roles of eleven of the thirteen cDNAs upon which Santiago et al. based their conclusions. Eight had been described as copies of short half-life and five as copies of long-half-life mRNAs. We show here that five members of the short-half-life class encode known Sc cytosolic ribosomal proteins (rp). One further short-half-life cDNA appears to encode a new Sc rp related to higher eukaryotic rp S12. Among the long-half-life cDNAs, one encodes the glucose-inducible glycolytic enzyme enolase, while another is related to the mouse housekeeping gene MER5.