Larry A. McReynolds

Draft Genome of the Filarial Nematode Parasite Brugia malayi

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ∼90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ∼11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ∼350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.

Species-specific oligonucleotide probes for the identification of human filarial parasites

Species-specific oligonucleotide probes have been constructed for the filarial parasites Brugia malayi and Brugia pahangi. Both parasites contain a 322 base pair repeated DNA sequence that is cleaved once by the restriction endonuclease HhaI. A consensus repeat sequence was determined from the DNA sequence of 15 cloned isolates of each species. Although the two repeats have an average homology of 89%, half the differences are clustered in a region of 66 nucleotides that has a homology of only 72%. Within this region, two probes, a 29-mer that is B. malayi specific and a 21-mer that is B. pahangi specific, were constructed. The sequence of both probes was chosen to obtain the maximum difference between the consensus sequences of the two species. The probes were also selected to be GC rich to increase their stability as a DNA hybrid. In a filter hybridization assay, the B. malayi probe has a 500-fold preference for B. malayi DNA versus B. pahangi DNA and a sensitivity of 200 pg. The B. pahangi probe has similar specificity and sensitivity for B. pahangi DNA. A rapid lysis procedure allows the probes to detect 1-2 third stage larvae of either B. malayi or B. pahangi in a filter hybridization assay.

Filarial paramyosin: cDNA sequences from Dirofilaria immitis and Onchocerca volvulus

The nucleotide sequence of a cDNA copy of the Dirofilaria immitis paramyosin gene was determined. The sequence was 2545 nucleotides in length, consisting of a single open reading frame of 848 amino acids capable of encoding a protein with a calculated molecular weight of 98,000. The cDNA clone was not complete, but probably includes over 97% of the coding region of the gene. We have previously observed that the cloned D. immitis paramyosin is recognized by sera from humans infected with Onchocerca volvulus. To determine the extent of homology at the protein level, we screened a cDNA library of O. volvulus with an antiserum made against D. immitis paramyosin. Ten recombinant clones were partially sequenced, comprising a total of 1186 nucleotides or 389 amino acids. The amino acid sequence of D. immitis paramyosin was 99% identical to the O. volvulus paramyosin. We also compared the amino acid sequence to other cloned paramyosins, and noted that 92% of the amino acids were identical to those of Caenorhabditis elegans, and 34% identical to those of Schistosoma mansoni. Comparison of the paramyosin sequence between different species revealed a hierarchy of similarities: (1) a 7-amino-acid repeat with apolar residues in the a and d position as the most conserved, followed by (2) the amino acid sequence and (3) the DNA sequence.

Cloning and bioinformatic identification of small RNAs in the filarial nematode, Brugia malayi.

Characterization of small RNAs from the filarial nematode Brugia malayi is the initial step in understanding their role in gene silencing. Both RNA cloning and bioinformatics were used to identify 32 microRNAs (miRNAs) belonging to 24 families. One family, miR-36 only occurs in helminths including B. malayi. Several of the miRNAs are arranged in clusters and are coordinately expressed as determined by northern blot analysis. In addition, small RNAs were identified from Pao/Bleo retrotransposons and their associated repeat sequences indicating that B. malayi uses an RNAi mechanism to maintain genome integrity. Analysis of these data provides a first glimpse into how small RNA-mediated silencing pathways regulate the parasitic life cycle of B. malayi.

Protein-mediated miRNA detection and siRNA enrichment using p19

p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs.

Detection of miRNAs with a nanopore single-molecule counter

miRNAs are short noncoding RNA molecules that are important in regulating gene expression. Due to the correlation of their expression levels and various diseases, miRNAs are being investigated as potential biomarkers for molecular diagnostics. The fast-growing miRNA exploration demands rapid, accurate, low-cost miRNA detection technologies. This article will focus on two platforms of nanopore single-molecule approach that can quantitatively measure miRNA levels in samples from tissue and cancer patient plasma. Both nanopore methods are sensitive and specific, and do not need labeling, enzymatic reaction or amplification. In the next 5 years, the nanopore-based miRNA techniques will be improved and validated for noninvasive and early diagnosis of diseases.

The polyprotein allergens of nematodes.

Symptoms of infection with a number of parasitic nematodes may be associated with IgE antibody and associated immunopathologies. This has drawn attention to parasite allergens and provoked discussion on the wisdom of their inclusion in recombinant vaccines. Here, Larry McReynolds, Malcolm Kennedy and Murray Selkirk review work on a prominent set of allergens recently characterized in several nematode species.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ∼21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by ∼90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.