Number Of Tandem Repeat Units Research Articles

Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion

BackgroundThrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis.MethodsThe 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays.ResultsThree members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro.ConclusionsWe have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.Graphical

Complete mitochondrial genome of a blue-tailed skink Plestiodon capito (Reptilia, Squamata, Scincidae) and comparison with other Scincidae lizards.

Vertebrate mitochondrial genomes (mitogenomes) are valuable for studying phylogeny, evolutionary genetics and genomics. To date, however, compared to other vertebrate groups, our knowledge about the mitogenomes of skinks (the family Scincidae), even of reptile, has been relatively limited. In the present study, we determined the complete mitogenome of a blue-tailed skink Plestiodon capito for the first time, and compared it with other skinks available in GenBank. The circular genome is 17,344bp long, showing a typical vertebrate pattern with 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region (CR). The gene organization, nucleotide composition, and codon usage are similar to those from skinks previously published. Twelve out of 13 PCGs initiates with canonical start codon (ATG), while COX1 starts with GTG. The codon usage analysis revealed a preferential use of the LeuCUN (Leu1), Pro, and Thr codons with the A/U ending. All tRNAs in P. capito were predicted to fold into typical clover-leaf secondary structure, except tRNA-Ser AGY. The secondary structures of 12S rRNA and 16S rRNA comprises 34 helices and 56 helices, respectively. The alignment of the Plesitodon species CRs exhibited high genetic variability and rich A + T content. Besides, variable types and numbers of tandem repeat units were also identified in the CR of Plestiodon. Phylogenetic analyses recovered P. capito as the sister species to P. tunganus; monophyly of the Scincidae is well supported. Our results will help to better understand structure and evolution of the mitochondrial DNA control region in reptiles as well as the evolutionary status of P. capito, and to lay foundation for further phylogenetic study of skinks in a mitogenomic framework.

Self-Assembly of Topologically Networked Protein-Ti3C2Tx MXene Composites.

Hierarchical organization plays an important role in the stunning physical properties of natural and synthetic composites. Limits on the physical properties of such composites are generally defined by percolation theory and can be systematically altered using the volumetric filler fraction of the inorganic/organic phase. In natural composites, organic materials such as proteins that interact with inorganic filler materials can further alter the hierarchical order and organization of the composite via topological interactions, expanding the limits of the physical properties defined by percolation theory. However, existing polymer systems do not offer a topological parameter that can systematically modulate the assembly characteristics of composites. Here, we present a composite based on proteins and titanium carbide (Ti3C2Tx) MXene that manifests a topological network that regulates the organization, and hence physical properties, of these biomimetic composites. We designed, recombinantly expressed, and purified synthetic proteins consisting of polypeptides with repeating amino acid sequences (tandem repeats) that have the ability to self-assemble into topologically networked biomaterials. We demonstrated that the interlayer distance between MXene sheets can be controlled systematically by the number of tandem repeat units. We varied the filler fraction and number of tandem repeat units to regulate the in-plane and out-of-plane electrical conductivities of these composites. Once Ti3C2Tx MXene sheets are separated enough to facilitate formation of cross-links in our proteins with the number of tandem repeat units reaching 11, the linear I-V characteristics of the composites switched into nonlinear I-V curves with a distinct hysteresis for out-of-plane electron transport, while the in-plane I-V characteristics remained linear. This highlights the impact of synthetic protein templates, which can be designed to modulate electronic transport in composites both isotropically and anisotropically.

Polymerase Chain Reaction-Free Variable-Number Tandem Repeat Typing Using Gold Nanoparticle–DNA Monoconjugates

In this work, we report a novel polymerase chain reaction (PCR)-free variable-number tandem repeat (VNTR) typing method using a T-shaped gold nanoparticle-DNA monoconjugate, called the "watching-gene assay". The T-shaped DNA probe was synthesized by "click" chemistry and linked with the gold nanoparticle to form the gold nanoparticle-DNA monoconjugate (a VNTR probe). Through a simple annealing and ligation reaction of the VNTR probe on a synthetic DNA template mimicking the human D1S80 VNTR locus, the number of tandem repeat units could be deciphered by counting the self-assembled gold nanoparticles. The number of tandem repeat units could be identified with more than 50% yield if the repeat number was less than four. In the case of the real human genomic DNA, the 18 repeat unit number could be successfully revealed by observing the 18-gold-nanoparticle cluster, which exactly corresponded to the number of tandem repeats of the real sample. Our "watching-gene assay" is rapid, simple, and direct for data interpretation, thereby providing an advanced PCR-free genetic polymorphism analysis platform.

The Complete Mitochondrial Genome of the Wild Type of Antheraea pernyi (Lepidoptera: Saturniidae)

Abstract Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae), a well-known economically important insect, was domesticated from its wild type. In this study, the complete mitochondrial genome (mitogenome) of the wild type of A. pernyi was determined and compared for nucleotide variation with its domesticated type. The mitogenome of the wild type of A. pernyi is 15,537 bp in size, thus 29 bp smaller than that of the domesticated type. The gene content, order, and orientation of the complete mitogenome of the wild type are identical to those of the domesticated type, as are those of the other completely sequenced lepidopteran mitogenomes. A striking difference between the two mitogenomes was found in the A+T-rich region because of the numbers of tandem repeat units. The wild type has five tandem repeat units, whereas the domesticated type has six. Comparative analysis of the two mitogenomes revealed a relatively lower level of sequence divergence (1.70%). Within the two mitogenomes, there are no significant differences in nucleotide substitution rate for the 13 protein-coding genes except for the nad4L gene, which is different from those differences observed between the domesticated silkmoth Bombyx mori (L.) (Lepidoptera: Bombycidae) and its wild-type ancestor Chinese B. mandarina Moore. The divergence time between the two Antheraea mitochondrias was estimated to be between 0.74 ± 0.13 and 0.97 ± 0.17 million years ago, based on the genes cox1+cox2 sequences. To our knowledge, this is the first report on sequence variation of the complete mitogenomes between the domesticated insect and its wild-type ancestor, within a single species.

Molecular Cloning and Characterization of the Circumsporozoite Protein Gene of Plasmodium inui Isolated from Formosan Macaques (Macaca cyclopis) in Taiwan

We characterized the complete nucleic and amino acid sequences of the Plasmodium inui circumsporozoite protein (Pincsp) gene and analyzed nucleotide diversity across the entire Pincsp gene by using 7 field isolates and strains Taiwan I and II obtained from Formosan macaques (Macaca cyclopis) in Taiwan. The length of the circumsporozoite protein ( CSP ) gene ranged from 1077 to 1125 bp. Size polymorphisms were due to variations in the number of tandem repeat units. The non-repetitive (NR) region exhibited high homology (99.1 ∼ 100 and 98.7 ∼ 100% at the nucleotide and amino acid levels, respectively) and was conserved among the variants (nucleotide diversities, π, of the 5'NR and 3'NR regions were 0.00364 and 0.00392, respectively). In the central repetitive (CR) region, we decomposed the sequences into 2 kinds of repeating amino acid motifs, i.e., a repeat unit R1, PA(P/A)(P/A)A(E)GG (n = 11-13), and a following repeat unit R2: P(A/G)(A/P/G)(P/Q)AQ(N/K) (n = 9-10). Analyzing these repeat sequences showed evidence of 3 genetic mechanisms for generating variations in the repeats of the Pincsp gene, i.e., point mutation, insertion, and recombination. These findings suggest that polymorphisms in the Pincsp gene are essentially limited to the CR region, which showed much greater variability in terms of length, number of repeats, and sequence.

Transcription of clpP Is Enhanced by a Unique Tandem Repeat Sequence in Streptococcus mutans

Streptococcus mutans, the primary causative agent of human dental caries, contains a single copy of the gene encoding ClpP, the chief intracellular protease responsible for tolerance to various environmental stresses. To better understand the role of ClpP in stress response, we investigated the regulation of clpP expression in S. mutans. Using semiquantitative reverse transcription-PCR analysis, we observed that, under nonstressed conditions, clpP expression is somewhat constant throughout the growth phases, although it gradually decreases as cells enter the late stationary phase. The half-life of the clpP transcript was found to be less than 1 minute. Sequence analysis of the clpP locus reveals the presence of a 50-bp tandem repeat sequence located immediately upstream of the clpP promoter (PclpP). PCR and DNA sequence analyses suggest that the number of tandem repeat units can vary from as few as two to as many as nine, depending on the particular S. mutans isolate. Further analysis, using a transcriptional reporter fusion consisting of PclpP fused to a promoterless gusA gene, indicates that the presence of the repeat sequence region within PclpP results in an approximately fivefold increase in expression from PclpP compared to the repeat-free transcriptional reporter fusion. CtsR, a transcriptional repressor that negatively regulates clpP expression, has no effect on this repeat-mediated induction of clpP transcription. Furthermore, the repeat sequence is not necessary for the induction of clpP under stress conditions. Database searches indicate that the region containing the tandem repeats is absent in the clpP loci in other bacteria, including other closely related Streptococcus spp., suggesting that the repeat sequences are specific for the induction of clpP expression in S. mutans. We speculate that a host-specific transcriptional activator might be involved in the upregulation of clpP expression in S. mutans.

Microsatellite Polymorphisms in DNA Repair GenesXRCC1, XRCC3andXRCC5in Patients with Gynecological Tumors: Association with Late Clinical Radiosensitivity and Cancer Incidence

This study investigates the association of microsatellite polymorphisms in XRCC1, XRCC3 and XRCC5 with the development of late radiation-induced radiotherapy reactions and examines the correlation between these microsatellites and cancer incidence. Sixty-two women with cervical or endometrial cancer treated with radiotherapy were included in the study. According to the CTCAEv3.0 scale, 22 patients showed late adverse radiotherapy reactions (grade 2 or more). PCR on lymphocyte DNA followed by automated fragment analysis was performed to examine the number of tandem repeat units at each locus. No significant association was found between the repeat length at any of the microsatellites in XRCC1, XRCC3 or XRCC5 and the incidence of late radiotherapy complications. Since higher odds ratios (ORs) were found for the rare XRCC1 [AC]11 and [AC]21 repeats (OR = 2.65, P = 0.325 and OR = 8.67, P = 0.093, respectively), the possible involvement of these small and large repeats in clinical radiosensitivity cannot be completely ruled out. When specific numbers of repeats were examined, no significant correlation was found between the microsatellite repeat length in XRCC1 and XRCC5 and cancer incidence. A weak correlation between XRCC3 [AC]16 homozygotes and cancer incidence was found (OR = 2.56, P = 0.055). A large-scale multicenter study of cancer patients with a high number of radiosensitive individuals is needed to clarify the value of rare polymorphic microsatellite repeats in XRCC1 and XRCC3 as a biomarker of clinical radiosensitivity or increased cancer risk.

Complete sequence of mitochondrial DNA control region of the Japanese serow Capricornis crispus (Bovidae: Caprinae)

The Japanese serow, Capricornis crispus, is an indigenous species to Japan. It has been strictly protected for nearly half a century, whereas culling for control has been permitted in some areas since the late 1970s. In order to investigate the intraspecific genetic diversity of the Japanese serow, a study on the mtDNA control region was conducted. In the present study, the complete nucleotide sequences of the control region (1031 bp) and its flanking proline tRNA gene (65 bp) from six serows were determined. In total, ten variable sites were observed in control region sequences, resulting in four different haplotypes. All of the substitutions among these haplotypes were transitions. Central, left and right domains of the control region were identified by comparison with cow sequences. Conserved sequence block-1 (CSB-1) and a polypyrimidine tract were also identified in the right domain. Two tandemly repeated sequences of 76 bp were found in the left domain. These features were compared with four Caprinae species. Sequences of the central domain, CSB-1 and polypyrimidine tract were homologous among them. The copy number of tandem repeat units varied among species. The estimated genetic distances suggest that the Formosan serow is probably a distinct species from the Japanese serow.

Heterogeneity in the circumsporozoite protein gene of Plasmodium malariae isolates from sub-Saharan Africa

Polymorphism of the circumsporozoite protein (CSP) of Plasmodium malariae was characterized by comparing gene sequences of twelve field isolates obtained in Yaoundé, Cameroon, Central Africa, and one clinical isolate originating from Côte d'Ivoire, West Africa. The length of the CSP gene ranged from 1266 to 1302 bp. The size polymorphism was due to variation in the number of tandem repeat units. All P. malariae isolates displayed a highly conserved 5′ non-repeat region. Seven non-synonymous and two synonymous nucleotide variations were observed in the 3′ non-repeat region. In the deduced amino acid sequence the repetitive sequences consisted of a varying number of major (Asn Ala Ala Gly (NAAG); range between 42 and 46 units) and minor (Asn Asp Ala Gly (NDAG) or Asn Asp Gln Gly (NDEG); n=six or seven units) tetrapeptide units. None of the isolates had an identical sequence at nucleotide level. These findings suggest that polymorphism in CSP is essentially limited to the tandem repeat domain.

Differential effects of [ 3H]nemonapride and [ 3H]spiperone binding on human dopamine D4 receptors

We compared some binding parameters of [ 3H]nemopride and [ 3H]spiperone in human dopamine D4 (hD4) receptors with three different numbers of tandem repeat units. Although both of the radioligands showed similar affinity constants for each hD4 receptor variant, the maximal number of binding sites labeled by [ 3H]nemonapride was approximately 1.35-fold higher than that by [ 3H]-spiperone for all variants. Estimated K i values for the inhibition of [ 3H]nemonapride binding by a series of dopaminergic ligands were highly correlatedto respective values obtained for the inhibition of [ 3H]spiperone binding to each hD4 receptor. These results suggest that the hD4 receptor, as shown for the D2 receptor, may exist in multiple molecular forms as a monomer-dimer equilibria, and that [ 3H]spiperone may discriminate in the multiple molecular forms.

Molecular basis for surface antigen size polymorphisms and conservation of a neutralization-sensitive epitope in Anaplasma marginale.

Anaplasmosis is one of several tick-borne diseases severely constraining cattle production and usage in many parts of the world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginale carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates of A. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the mspl alpha gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.

Frequency of telomere repeat units in the Drosophila miranda genome

Cloned DNA fragments of Drosophila miranda which label all chromosome ends show a basic tandem repeat unit of 4.4 kb. The D. miranda telomere specific tandem repeats do not cross-hybridize with genomic D. melanogaster DNA which itself contains telomere repeat units of 3 kb. For a more detailed analysis of the functional criteria of telomere specific sequences we determined the repetition frequency of the tandem repeat units. As a low estimate we found a repetition frequency of 20 for female D. miranda DNA. This is on average equivalent to 2 telomere repeat units per chromosome end in the female D. miranda karyotype. However, a variable number of tandem repeat units per chromosome end would describe more closely the obtained differences in the labeling intensity between the individual chromosomes (X1L-5). For the D. miranda male DNA we determined a repetition frequency of 90. The frequency difference of 70 copies between male and female DNA must be due to the Y-chromosome.