Discriminating PCR artifacts using directed heteroduplex analysis (DHDA).
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Animals↗
Biomedical subjects
T R Unnasch
Publications and source records attributed to T R Unnasch.
Technical report. Optimizing probe selection in directed heteroduplex analysis using HDprobe 1.1.
Directed heteroduplex analysis (DHDA) has proven to be a powerful technique for rapid geotyping in human populations. This strategy should also have widespread utility in differentiating closely related organisms of medical and public health importance through identification of DNA sequence polymorphisms. Identifying an optimal probe sequence for use in DHDA has required empirical testing of both the positive and negative strands of a number of potential probes. To identify optimal probes more efficiently, a computer program has been developed that predicts the number of potential stable and unstable mismatches between a probe and its target sequences in DHDA. This information can then be used to predict--from among a group of potential probes--which one will be the most successful in differentiating closely related homologues of a targeted gene sequence. This approach was tested on a number of probe and target sequences derived from the mitochondrial NADH dehydrogenase subunit 4 gene of the West African black fly, Simulium damnosum sensu lato. The number of unstable mismatches predicted to occur in a given heteroduplex by the computer program was found to be important in differentiating closely related species. Therefore, this strategy is useful in identifying informative probes in the development of new DHDA-based assays.
Animals↗
The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.
Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.
Amino Acid Sequence↗
Truly infection-free persons are rare in areas hyperendemic for African onchocerciasis.
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Africa↗
Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family.
Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.
Americas↗
Cloning and characterization of a potentially protective chitinase-like recombinant antigen from Wuchereria bancrofti.
While there is no direct evidence demonstrating the existence of protective immunity to Wuchereria bancrofti infection in humans, the presence of individuals, in populations in areas where infection is endemic, with no clinical evidence of past or current infection despite appreciable exposure to the infective larvae, suggests that protective immunity to filarial parasites may occur naturally. Earlier work indicated that such putatively immune individuals generated antibodies to a 43-kDa antigen from larval extracts of the related filarial parasite Brugia malayi that was recognized by only 8% of the infected population. With rabbit antiserum raised against this 43-kDa antigen, this current study identified a recombinant clone, WbN43, with an insert size of 2.3 kb, from a W. bancrofti genomic expression library. The recombinant fusion protein was differentially recognized by the putatively immune individuals but not by the infected patients. The coding sequence (684 bp) from the 5' end had significant sequence similarity to chitinases from Serratia marcescens, Bacillus circulans, Streptomyces plicatus, and B. malayi. Peptide sequencing of the expressed product also defined a chitinase-like sequence. Molecular characterization indicated WbN43 to be a low-copy-number gene, with expression predominantly in infective larvae and microfilariae but not in adult parasites.
Adult↗
Epitopes of the Onchocerca volvulus RAL1 antigen, a member of the calreticulin family of proteins, recognized by sera from patients with onchocerciasis.
RAL1 is an antigen (Ag) encoded by the filarial nematode Onchocerca volvulus, the parasite causing onchocerciasis (river blindness). RAL1 shares 64.4% identity with the autoantigen calreticulin. The striking similarity of the parasite Ag and the human autoantigen has led to the hypothesis that RAL1 may induce a cross-reactive immune response to calreticulin, which in turn may be involved in the pathogenesis of onchocerciasis. To test this hypothesis, we explored the immune response to RAL1 recombinant Ag (RAL1 rAg) and human calreticulin in patients with O. volvulus infection. A total of 86% of the O. volvulus-infected individuals produced antibodies recognizing RAL1 rAg. Antibody reactivity to RAL1 rAg in patient sera was confined primarily to the central and carboxyl-terminal parts of the molecule. No significant correlations were found to associate recognition of RAL1 rAg, or any particular portion thereof, with a particular disease state. Antibodies against RAL1 thus appear to be produced as a general immune reaction to O. volvulus infection and do not necessarily lead to a cross-reacting response with the host protein. In contrast, 33% of the patient sera tested bound recombinant human calreticulin. All of these sera also recognized a polypeptide encompassing the carboxyl-terminal portion of the RAL1 rAg. These results suggest that recognition of an epitope encoded in the carboxyl-terminal portion of RAL1 is at least in part responsible for inducing a cross-reacting immune response to the host protein.
Amino Acid Sequence↗
Major retinal cell components recognized by onchocerciasis sera are associated with the cell surface and nucleoli.
PURPOSE: Cellular localization of the components recognized by onchocerciasis autoantibodies has not been investigated in any detail in cultured retinal cells. This study sought to examine, in cultured retinal cells, the subcellular localization of major components that cross-react with onchocerciasis sera. METHODS: Immunofluorescence confocal laser scanning microscopy and Western blot analysis were carried out on adult pig retinal cells. RESULTS: The onchocerciasis sera contain antibodies cross-reacting strongly with components of the surface and nucleoli in both the cultured retinal pigment epithelial and neural retinal cells. These epitopes are not recognized by the control sera obtained from noninfected individuals residing in an onchocerciasis hyperendemic area, and from those with or without ocular disease who have never been in any of the onchocerciasis hyperendemic countries. Double-labeling immunofluorescence microscopy does not detect any colocalization of a putative onchocerciasis autoantigen, calreticulin, and those cellular components recognized by onchocerciasis sera in either cell type. Furthermore, none of the onchocerciasis sera tested recognized recombinant calreticulin by Western blot analysis. CONCLUSIONS: Major epitopes for onchocerciasis anti-retinal autoantibodies are associated with the surface and nucleolus components of retinal cells. Interaction of the onchocerciasis antibodies with the retinal cell surface molecules may play an important role in the development of ocular diseases initiated by the damage of retinal cells. Furthermore, the finding that the cellular components recognized by onchocerciasis sera do not colocalize with calreticulin, taken together with the observation of lack of recognition of recombinant calreticulin by these sera on Western blots, suggests that calreticulin is not a major onchocerciasis autoantigen.
Animals↗
DNA sequence analysis using hierarchical ART-based Classification Networks.
Adaptive resonance theory (ART) describes a class of artificial neural network architectures that act as classification tools which self-organize, work in realtime, and require no retraining to classify novel sequences. We have adapted ART networks to provide support to scientists attempting to categorize tandem repeat DNA fragments from Onchocerca volvulus. In this approach, sequences of DNA fragments are presented to multiple ART-based networks which are linked together into two (or more) tiers; the first provides coarse sequence classification while the subsequent tiers refine the classifications as needed. The overall rating of the resulting classification of fragments is measured using statistical techniques based on those introduced by Zimmerman, et al. (1994) to validate results from traditional phylogenetic analysis. Tests of the Hierarchical ART-based Classification Network, or HABclass network, indicate its value as a fast, easy-to-use classification tool which adapts to new data without retraining on previously classified data.
Algorithms↗
DNA probe-based classification of Simulium damnosum s. l.-borne and human-derived filarial parasites in the onchocerciasis control program area.
The development of polymerase chain reaction-based methods using strain- and species-specific DNA probes for Onchocerca volvulus has permitted classification of individual parasites from every stage of the parasite's life cycle. This technology has been applied on a large scale basis by Onchocerciasis Control Program (OCP) in West Africa. The primary objective of the OCP in using the DNA probes was to obtain accurate estimates of the annual transmission potential of the blinding strain of O. volvulus. The DNA probe classification of larvae collected throughout the OCP area demonstrated that larvae of less pathogenic strains of O. volvulus and other filarial parasites carried by Simulium damnosum s.l. have resulted in a significant overestimation of the annual transmission potential for blinding onchocerciasis. This effect is particularly pronounced along the southern border of the OCP, where the blinding and less pathogenic strains of O. volvulus coexist, and in the north of the control area, where animal parasites, particularly O. ochengi, may even predominate. A second objective of the OCP in applying the DNA probe technology was to determine the distribution of blinding and less pathogenic O. volvulus in infected individuals along the southern border of the control area. Results obtained from these studies have generally confirmed the distribution pattern established by previous epidemiologic studies. In addition, DNA probe classifications have demonstrated that in areas where the blinding and less pathogenic strains of O. volvulus coexist, a single individual may simultaneously be infected with both strains of the parasite.
Africa, Western↗
Onchocerca volvulus: characterization of an immunodominant hypodermal antigen present in adult and larval parasites.
Biochemical and immunological data suggest that a relatively limited number of polypeptide antigens of viable Onchocerca volvulus-infective larvae are available to be recognized by the host's immune system. A partial cDNA clone encoding one such antigen, designated lambda RAL-2, was isolated by screening an expression cDNA library with antisera raised against viable O. volvulus L3. The antigen encoded by this clone was subsequently found to be immunogenic in the majority of individuals exposed to O. volvulus. In the present study, the native antigen corresponding to lambda RAL-2 (Ov17) has been characterized. Immunolocalization and in situ hybridization techniques have been used to localize Ov17 in adult and larval stages of the parasite. In adult females, Ov17 was localized primarily in the hypodermis. Ov17 was accessible to surface labeling reagents in viable adult parasites. Full-length cDNA clones encoding Ov17 suggested that the nascent protein contains a putative leader sequence, which is almost immediately followed by a polyglutamine tract. Analysis of antibody reactivity to recombinant proteins containing and lacking the polyglutamine tract demonstrated that this structure was not a significant B cell epitope in individuals exposed to O. volvulus.
Amino Acid Sequence↗
Design of Onchocerca DNA probes based upon analysis of a repeated sequence family.
Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.
Animals↗
Onchocerca volvulus: frequency of codon usage.
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Animals↗
Onchocerca volvulus DNA probe classification correlates with epidemiologic patterns of blindness.
Onchocerciasis, or river blindness, results from infection with Onchocerca volvulus. The parasite is endemic to West Africa, in both rain forest and savanna bioclimes. Several lines of evidence suggest that different strains of the parasite exist in the rain forest and savanna. Furthermore, epidemiologic evidence indicates that ocular onchocerciasis is most severe in savanna regions. This has led to the hypothesis that there is a strain association with ocular pathology. To test this hypothesis, parasites from villages in which severe and mild onchocerciasis were endemic were classified with two strain-specific DNA probes. A strong correlation (P less than .001) was found between disease severity and probe recognition, supporting the hypothesis that pathogenicity is strain related. The results suggest that pFS-1 and pSS-1BT may be used to predict the pathogenic potential of parasite populations throughout much of West Africa.
Africa, Western↗
Characterization of the autoantigen calreticulin.
Anti-Ro/SS-A antibodies are commonly found in the sera of patients with Sjögren's syndrome and SLE. These antibodies also occur in the mothers of children with neonatal lupus and congenital heart block. Ro/SS-A is a ribonucleoprotein complex whose cellular function remains unknown. To study its cellular function and to characterize its immunoreactivity, we have used an oligonucleotide designed after the published amino terminal sequence of a putative 60-kDa Ro/SS-A autoantigen to isolate its cDNA. This cDNA encodes a polypeptide that is the human homologue of calreticulin, a calcium binding protein of the endoplasmatic reticulum. The encoded polypeptide also shows a 64.4% identity with RAL-1, an Ag of the river blindness pathogen Onchocerca volvulus. Contrary to the data published by other authors, our results indicate that calreticulin is not a Ro/SS-A autoantigen. Moreover, we show that anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis.
Amino Acid Sequence↗
Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite.
Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.
Animals↗
Transformation of DNA sequence data into geometric symbols.
Comparative analysis of related DNA sequences has been simplified by the transformation of data in the standard A, G, C, T format into a set of geometric symbols that promote pattern recognition. Previously, comparing more than 2 or 3 sequences simultaneously has been difficult because of the monotonous patterns established by letters. Here 33 sequences are simultaneously compared to demonstrate the ease with which nucleotide substitutions are accurately identified. This has been accomplished by writing a Word-Perfect macro program to facilitate this transformation. Since this word processing program is widely used, performing this kind of analysis is readily achievable in most laboratories involved in DNA sequence analysis.
Animals↗
Isolation and characterization of form specific DNA sequences of O. volvulus.
Maintenance of the control of O. volvulus in Africa would be assisted by the development of assays to identify the origin of outbreaks of transmission within the controlled regions. The isolation of pFS-1, a DNA sequence specific for forest form O. volvulus, has suggested that it might be possible to develop such an assay based on form specific DNA probes. The isolation and characterization of pSS-1, a clone which hybridized preferentially to savannah form O. volvulus is described here. DNA sequence analysis demonstrated that pSS-1 was a representative of a highly repeated sequence family of O. volvulus. Comparison of the sequence of pSS-1 to other known examples of this family has identified a region of pSS-1, designated pSS-1BT, which appeared to be specific for savannah form O. volvulus. Tests of isolates from different areas demonstrated that the specificity of pFS-1 and pSS-BT were not confined to a small geographic region. However, the close relationship of pFS-1 and pSS-BT to less specific members of the repeat family means that their specificity and sensitivity are affected by small changes in the conditions under which they are used.
Animals↗