Adhesive Protein Gene Research Articles

Quantitative PCR-Based Parasite Burden Estimation of Babesia gibsoni in the Vector Tick, Haemaphysalis longicornis (Acari: Ixodidae), Fed on an Experimentally Infected Dog

Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 10(5) to 3.5 × 10(1)/μl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 10(3) iRBCs/μl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick's genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.

Alternative PCR–RFLP methods for mussel Mytilus species identification

Mussels are the most important bivalves in both European aquaculture and market. Together with mussels produced in EU, the European market also allows the entrance of several mytilid species from up to 16 non-EU countries. Therefore, traceability methodologies are needed for the protection of consumers’ rights and for reinforce labeling regulations. The aim of this study was to establish a methodology that allows the accurate identification of 6 different mussel species, as in fresh as in processed forms. Amplification of mussel samples with primers Myti-F/R, which anneal in a region of the adhesive foot protein gene, yielded a 190-bp fragment in M. edulis, a 178-bp in M. trossulus, a 202-bp in M. californianus/M. coruscus, and a 136-bp in M. galloprovincialis/M. chilensis. Hence, differentiation among M. edulis, M. trossulus, and the rest of Mytilus species was achieved in a first step. Further digestion with Acl I in M. coruscus/M. californianus samples and with Aci I in M. galloprovincialis/M. chilensis allowed the accurate identification of these species. A second methodology based on amplification by PCR of mitochondrial cytochrome oxidase subunit I (COI) gene and digestion with Xba I was developed to further confirmation of M. chilensis samples. The current methodology was applied in cultured and natural samples and in commercial forms. The protocol described in this study provides an accurate, reliable, and a powerful tool for mussel species identification, traceability, and for reinforcement of labeling regulations.

Use of a microsatellite‐based pedigree in estimation of heritabilities for economic traits in Australian blue mussel, Mytilus galloprovincialis

This study aimed to estimate the heritabilities of three economically important traits (total weight, shell shape and meat yield) in Australian blue mussels. The estimates were derived using a pedigree reconstructed from a suite of both published and newly developed microsatellite markers. A total of 135 microsatellite loci were tested, of which 10 loci produced consistent PCR amplification and reliable results across all samples (74 full-sibling families including 74 pairs of parents and 2536 offspring). Lack of polymorphism at the non-repetitive region of the adhesive protein gene confirmed that the broodstock were derived from a single species. A total of 1538 progenies (62.5%) could be assigned to single parent pairs, and the remainder were assigned to two families or more, so were discarded from further analysis. Heritabilities for total weight, shell shape and meat yield were low (0.051 ± 0.027, 0.085 ± 0.038 and 0.049 ± 0.028, respectively) but reflected large environmental variation rather than limited genetic variation, suggesting a family-based breeding programme could improve these traits. The genetic correlation between weight and meat yield, expressed as percentage of total mussel which was not shell, was negative, while the genetic correlation between meat yield and shell shape was weakly positive.

Development of a Method for Markerless Gene Deletion in Pseudomonas putida

We developed a negative counterselection system for Pseudomonas putida based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed a P. putida strain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.

Conserved sequences of thrombospondin-related adhesive protein gene of Plasmodium vivax in clinical isolates from Korea

Thrombospondin-related adhesive protein (TRAP) from Plasmodium vivax (P. vivax) became one of the important vaccine candidates for malaria, because P. vivax TRAP (PvTRAP) is responsible for the sporozoite-host interactions. PvTRAP polymorphisms in the isolates from Republic of Korea (ROK) were analysed, setting the valuable baseline data for the future vaccine developments and clinical trials with PvTRAP, as a strong vaccine candidate. A total of 54 isolates were collected in 2010. PvTRAP genes from above isolates were amplified and sequenced, and the results were analysed and compared against Sal-1 strain. Sequencing analysis of 1424-bp-size PvTRAP PCR products revealed one major allelic type with six non-synonymous substitutions, where S81T, E95D, I121V and T127R substitutions were found in region II, and K371N and A425E substitutions from region IV. The ROK isolates revealed the limited sequence polymorphisms in PvTRAP in comparison with the reported isolates from other nations.

Oligonucleotide Microarray and QRT-PCR Study of Adhesion Protein Gene Expression in Acute Coronary Syndrome Patients

Cardiovascular diseases, including acute coronary syndrome (ACS), are the leading cause of death among humans. Adhesion proteins, owing to their involvement in the initiation and progression of atherosclerotic lesions, contribute to the progression of coronary disease and ACS occurrence. Considering ambiguosity of results reported to date, we decided to conduct a preliminary investigation of adhesion protein gene expression in ACS patients as well as in healthy subjects by making use of oligonucleotide microarray technology. Analysis of eight microarrays revealed ten upregulated genes differentiating between the two groups: intercellular adhesion molecule-2, platelet/endothelial cell adhesion molecule-1, zyxin, integrin-linked kinase, calcium and integrin binding protein-1 (calmyrin), integrin beta 2, integrin beta 3 (ITGB3), integrin beta 7, integrin alpha 2b, and selectin P ligand. The expression of ITGB3 was found to have been downregulated.

Sequence and Phylogenetic Analysis of the Thrombospondin-Related Adhesive Protein (TRAP) Gene of Babesia gibsoni Isolates from Dogs in Taiwan

The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.

Cloning and sequencing of adhesion protein gene of Trichomonas gallinae from pigeon

The adhesion protein (AP) gene of Trichomonas gallinae from pigeon was cloned and sequenced. The first-strand cDNA of the AP gene of T. gallinae from pigeon was amplified by reverse transcription polymerase chain reaction (RT-PCR) with total RNA extracting kit and cloned in the vector pMD18-T. The recombinant plasmid was identified by PCR and restriction endonuclease, and the positive clone was sequenced and analysed by comparing the sequence similarity with other sequences in the GenBank. The AP gene of T. gallinae had a length of 1032 bp, which contained a complete open reading frame (ORF) of 930 bp long, coding for 309 amino acids. The sequence analysis revealed that the homology with three AP genes of Trichomonas vaginalis (i.e., TVU87096, TVU87097 and TVU87098) was 94.2%, 92.6% and 92.0%, respectively. It is concluded that the successfully cloned AP gene from T. gallinae will provide the basis for the expression of the AP gene in prokaryotic and eukaryotic cells and the preparation of its recombinant protein.

Development of a real-time PCR assay for detection of Mytilus species specific alleles: Application to a sampling survey in Scotland

Shellfish aquaculture is a growing industry in Scotland, dominated by the production of the mussel Mytilus edulis, the native species. Recently the discovery of Mytilus galloprovincialis and Mytilus trossulus together with M. edulis and all 3 hybrids in cultivation in some Scottish sea lochs led to questions regarding the distribution of mussel species in Scotland. The establishment of an extensive sampling survey, involving the collection of mussels at 34 intertidal sites and 10 marinas around Scotland, motivated the development of a high-throughput method for identification of Mytilus alleles from samples. Three Taqman®–MGB probes and one set of primers were designed, based on the previously described Me 15/16 primers targeting the adhesive protein gene sequence, and samples were screened for the presence of M. edulis, M. galloprovincialis and M. trossulus alleles using real-time PCR. Mytilus edulis alleles were identified in samples from all 44 sites. Mytilus galloprovincialis alleles were found together with M. edulis alleles extensively in northern parts of the west and east coasts. Mytilus trossulus alleles were identified in samples from 6 sites in the west and south-west of Scotland. Because M. trossulus is generally undesirable in cultivation and therefore preventing the geographical spread of this species across Scotland is considered beneficial by the shellfish aquaculture industry, these 6 samples were further analysed for genotype frequencies using conventional PCR. Although distribution of the non-native species M. galloprovincialis and M. trossulus have proven to be more widespread than previously thought, there is no evidence from our study of either M. trossulus or M. galloprovincialis acting as an invasive species in Scotland. The real-time PCR method developed in this study has proven to be a rapid and effective tool for the identification of M. edulis, M. galloprovincialis and M. trossulus alleles from samples and should prove useful in future surveys, ecological or aquaculture management related studies in both unispecific and mixed species areas of these species.

Distribution of Adhesive Protein Gene and the Protein Antigen among Avian Pasteurella multocida Isolates in Japan

The distribution of the adhesive capsular protein (Cp39) gene, the protein antigen, and its genetic variation among avian clinical isolates of Pasteurella multocida in Japan were determined by the gene sequencing, the PCR-restriction fragment length polymorphism (PCR-RFLP) and immunoblot analysis. The results demonstrated that the gene distributed among all the somatic serotype 3 isolates. Adhesive protein gene-based PCR-RFLP categorized the clinical isolates into three patterns by digestion with HindIII or three patterns with EcoRI, respectively. Genetic diversity of the adhesive protein gene was observed among those isolates but it did not influence their virulence to hosts. Whole-cell lysate of all clinical isolates incubated with sample buffer at 37°C were immunostained with chicken antiserum against recombinant Cp39 (rCp39) by immunoblot analysis. Therefore, the rCp39 may be an emerging candidate of effective component for fowl cholera vaccine in Japan.

PCR-Múltiple para el diagnóstico de Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum y Ureaplasma urealyticum

Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum are species related with some pathologies of the urogenital tract, and particularly with non gonococal urethritis (NGU) in men. The culture of these microorganisms is very dificult, that’s why the molecular techniques, mainly the polymerase chain reaction, have become in the main detection method of this organisms. Objectives: To improve a molecular method based in technology of genes for the diagnosis of this four genital micoplasma species, applying the same in clinical samples from patients with NGU. Materials and methods: The conditions were created for a Multiplex-PCR for identify this species, using as sample reference DNA, the pairs of primer complementary to the adhesion protein gene of M. genitalium (MgPa), the rRNA 16S of M. hominis, an specific region between rRNA 16S and rRNA 23S genes of U. parvum, and the adjacent region to the urease gene specific for U. urealyticum, being a specific and sensitive method. Results: When analyzing 34 urethral sawbs samples, 27 corresponded to the Mollicutes Class, being 14,8% positive to M. genitalium, 18,5% to M. hominis, 11,1% to U. urealyticum and 3,7% to U. parvum. It was realized for fist time the diagnosis of M. genitalium, M. hominis, U. parvum and U. urealyticum in urethral samples from Cuban patients. Conclusion: It is recommended to include the diagnosis of these species in other Cuban patients with urethral symptoms, to validate the proposed method and to know the relation of these microorganisms with the NGU.

Development of a Method for the Genetic Identification of Mussel Species Belonging to Mytilus, Perna, Aulacomya, and Other Genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.

Dysfusion Transcriptional Control of Drosophila Tracheal Migration, Adhesion, and Fusion

The Drosophila dysfusion basic-helix-loop-helix-PAS transcription factor gene is expressed in specialized fusion cells that reside at the tips of migrating tracheal branches. dysfusion mutants were isolated, and genetic analysis of live embryos revealed that mutant tracheal branches migrate to close proximity but fail to recognize and adhere to each other. Misexpression of dysfusion throughout the trachea further indicated that dysfusion has the ability to both inhibit cell migration and promote ectopic tracheal fusion. Nineteen genes whose expression either increases or decreases in fusion cells during development were analyzed in dysfusion mutant embryos. dysfusion upregulates the levels of four genes, including the shotgun cell adhesion protein gene and the zona pellucida family transmembrane protein gene, CG13196. Misexpression experiments with CG13196 result in ectopic tracheal fusion events, suggesting that it also encodes a cell adhesion protein. Another target gene of dysfusion is members only, which inhibits protein nuclear export and influences tracheal fusion. dysfusion also indirectly downregulates protein levels of Trachealess, an important regulator of tracheal development. These results indicate that fusion cells undergo dynamic changes in gene expression as they switch from migratory to fusion modes and that dysfusion regulates a discrete, but important, set of these genes.

Multifarious Transcriptional Regulation of Adhesion Protein Gene ap65 - 1 by a Novel Myb1 Protein in the Protozoan Parasite Trichomonas vaginalis

The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.

Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, and Mycoplasma genitalium in First-void Urine Specimens by Multiplex Polymerase Chain Reaction.

Background: Sexually transmitted diseases are often caused by one or more microorganisms, and asymptomatic carriage and transmission may be of significance. Testing for more than one organism in a single assay could be a useful approach to laboratory diagnosis. Methods and Results: A multiplex polymerase chain reaction (PCR) assay was developed that employed specific primers targeted to the 7.5-kb cryptic plasmid of Chlamydia trachomatis, the cppB gene of the 4.2-kb cryptic plasmid of Neisseria gonorrhoeae, the 140-kd major adhesion protein gene of Mycoplasma genitlium, and the urease gene of Ureaplasma urealyticum. All four polymerase chain reaction products were detectable by agarose gel electorphoresis and were confirmed by Southern hybridization using fluorescein isothiocyanate-labeled oligonucleotide probes and enhanced chemiluminescent detection. Using purified DNA preparations, multiplex PCR had a reproducible detection limit of 1 fg of C. trachomatis DNA, 100 fg of N. gonorrhoeae DNA, and 10 fg U. urealyticum DNA and M. genitalium DNA, which converts to 1-2 genomic equivalents (ge) of C. trachomatis and N. gonorrhoeae, 4 ge of M. genitalium, and 10 ge U. urealyticum. Multiplex PCR was compared with individual uniplex polymerase chian reaction PCR assays by testing 117 first-void urine samples (91 men, 26 women) from Canadian or Kenyan patients. Multiplex PCR detected 45 of 46 (97.8%) urines with C. trachomatis DNA, 42 of 42 (100%) urines with N. gonorrhoeae DNA, 17 of 17 (100%) urines with U. urealyticum DNA, 4 of 4 (100%) urines with M. genitalium DNA, 12 of 12 urines that had DNA from two bacteria, and 2 of 2 urines with DNA from three bacteria. Multiplex PCR correctly identified bacteria in 92 of 93 urines for an overall sensitivity of 98.9%. Specificity calculations were 100% for C. trachomatis (71/71), N. gonorhoeae (75/75), U. urealyticum (100/100), and M. genitalium (113/113). Conclusions: Multiplex PCR provided a single sensitive and specific test for the detection of four bacteria in first-void urine samples. Testing of first-void urine samples by multiplex PCR could facilitate studies aimed at improving our understanding of the epidemiology of these important sexually transmitted diseases.

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis.

To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA). Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A+) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing approximately 5,000 known human genes and approximately 3,000 expressed sequence tags (ESTs). A > or =2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1alpha, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.

PCR-IMS detection and molecular typing of Cryptosporidium parvum recovered from a recreational river source and an associated mussel (Mytilus edulis) bed in Northern Ireland.

PCR-IMS was used to detect Cryptosporidium spp. in environmental water samples in Northern Ireland which had previously tested negative by a conventional IFA staining method. Oocysts of C. parvum detected in river water and final treated sewage effluent collected from various sites along the river Lagan were identified as genotype 2 (animal origin) based on polymorphisms observed at the thrombospondin related adhesion protein gene locus. Similarly, genotype 1 (human origin) oocysts of C. parvum were detected in the marine filter feeder mussel, Mytilus edulis, collected from the shores of Belfast Lough. Detection of the human genotype of Cryptosporidium in mussels destined for human consumption identifies the organism's serious potential as a foodborne pathogen. This work highlights the possible value of monitoring filter feeder systems, in conjunction with specific molecular epidemiological tools, as an alternative monitoring system for the parasite within the aquatic environment.

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550 bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7–21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70 kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.

Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodiumvivax

We analyzed 22 clinical isolates of Plasmodiumvivax from Thailand and 17 from Brazil to investigate the extent of sequence variation in the thrombospondin-related adhesive protein of Plasmodiumvivax (PvTRAP), a homologue of P.falciparum TRAP (PfTRAP) which has been considered to be a promising vaccine candidate. In total 54 haplotypes were identified from 73 distinct gene clones. Coexistence of different PvTRAP in circulation occurred in 10 and 13 isolates from Thailand and Brazil, respectively. Forty out of 48 substituted nucleotides are non-synonymous changes. Most of the substituted residues reside in the von Willebrand factor type A-domain (region II), a sulfated glycosaminoglycan-binding domain (region III) and a proline-rich region (region IV). All nucleotide substitutions are dimorphic. Two haplotypes from Thailand contain an inserted sequence encoding aspartic acid-serine-proline in the proline-rich region. Sequence analysis has revealed that nucleotide diversity in PvTRAP is low although Brazilian isolates display a higher degree of variation than those from Thailand. Phylogenetic construction using the neighbor joining method has shown that most of the Thai and the Brazilian isolates appear to be mainly clustered into distinct groups. Significantly greater than expected values of the mean number of non-synonymous (dn) than synonymous (ds) nucleotide substitutions per site were observed in regions II and III of PvTRAP. Analysis of the published PfTRAP sequences has shown a similar finding in regions II and IV suggesting that positive selection operates on the regions. Hence, different regions in PvTRAP and PfTRAP could be under different pressures in terms of immune selection, structural and/or functional constraints.

P157 Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodium vivax

P157 Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodium vivax