Repetitive Domain Research Articles

NMR structure note: repetitive domain of aciniform spidroin 1 from Nephila antipodiana

NMR structure note: repetitive domain of aciniform spidroin 1 from Nephila antipodiana

Characterisation of a novel high-molecular-weight glutenin subunit 1Dy12.3 from hexaploid wheat (Triticum aestivum L.)

A novel high-molecular-weight glutenin subunit encoded by the Glu-1D locus was identified in hexaploid wheat (Triticum aestivum L.) cultivar Noe and was designated as 1Dy12.3. This subunit differed in SDS-PAGE mobility from the well-known 1Dy10 and 1Dy12 subunits that are also encoded by this locus. An analysis of the gene sequences confirmed the uniqueness of 1Dy12.3 and revealed that it is most closely related to the 1Dy12 subunit. The size of the deduced protein was calculated to be 67 884 Da, which is different from the 1Dy10 and 1Dy12 subunits (67 475 Da and 68 713 Da, respectively). The 1Dy12.3 protein consists of 652 residues, with a highly conserved signal sequence and N- and C-terminal domains, although the central repetitive domain comprising motifs of hexapeptide (PGQGQQ) and nonapeptide (GYYPTSLQQ) repeats was less conserved. The 1Dy12.3 subunit demonstrates fewer QHPEQG hexapeptide motifs and exhibits an increased number of methionine residues in comparison to the other characterised high-molecular-weight glutenin subunits. The 1Dy12.3 subunit was cloned and expressed in Escherichia coli and was detected with a prolamin-specific antibody. The size of the detected immunocomplex corresponded to the native 1Dy12.3 protein isolated from grains. The existence and characterisation of this novel high-molecular-weight glutenin subunit increases the diversity of the glutenins encoded by the Glu-1D locus.

The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Molecular and Immunological Characterization of Tri a 36, a Low Molecular Weight Glutenin, as a Novel Major Wheat Food Allergen

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.

A Detailed, Hierarchical Study of Giardia lamblia's Ventral Disc Reveals Novel Microtubule-Associated Protein Complexes

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.

Characterization of HMW-GSs and their gene inaction in tetraploid wheat

In this study, we report the expression of HMW-GSs in 87 accessions of tetraploid wheat, the characterization of three inactive and one active HMW glutenin genes, and the functional verification of HMW-GSs by promoter-GUS expression. SDS-PAGE profiles revealed that tetraploid wheat has many different combinations of HMW-GSs and the number of subunits varies from 1 to 4. HMW glutenin genes at the Glu-A1x, Glu-A1y and Glu-B1y loci exhibited different frequencies of inaction while the Glu-B1x allele was expressed in all 87 accessions. Gene cloning showed that only 1Bx (Tdu-e) could express a full-length protein and its deduced protein sequence has the typical primary structure but with fewer cysteine residues. The expression of the other three HMW glutenin genes has been disrupted by stop codons in their repetitive domains. Besides short indels or mutations of one or more bases, an 85-bp deletion and a 185-bp insertion were found in the promoter regions of 1Ay (Tdu-s) and 1Bx (Tdu-e). The transient expression of promoter-GUS constructs indicated that the 1Ay promoter can drive expression of the GUS gene. We conclude that defects (stop codons or the insertion of large transposon-like elements) in the coding regions may be the most probable cause for the inaction of the HMW glutenin genes.

Molecular cloning and characterization of the partial major ampullate silk protein gene from the spider Araneus ventricosus

Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22kDa polypeptide in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100°C for at least 30min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial.

Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology.

Marker-assisted selection of high molecular weight glutenin alleles related to bread-making quality in Iranian common wheat (Triticum aestivum L.)

Bread-making quality in hexaploid wheats is a complex trait. It has been shown that the amount and composition of protein can influence dough rheological properties. The high-molecular-weight (HMW) glutenins are encoded by a complex locus, Glu-1, on the long arm of group-1 homoeologus chromosome of the A, B and D genomes. In this work we used PCR-based DNA markers as a substitution tool to distinguish wheat bread-making quality. We detected PCR-based DNA markers for coding sequence of Glu-A1x, Glu-B1x and Glu-D1x to be 2300 bp, 2400 bp and 2500 bp respectively. DNA markers related to coding sequence of Glu-A1y, Glu-B1y and Glu-D1y were; 1800 bp, 2100 bp and 1950 bp, however, the repetitive region of their coding sequence were shown to be about 1300 bp, 1500 bp and 1600 bp. The results demonstrate that the size variation was due to different lengths of the central repetitive domain. Good or poor bread-making quality in wheat is associated with two allelic pairs of Glu-D1, designated 1Dx5-1Dy10 and 1Dx2-1Dy12. The 1Bx7 allele has moderate-to-good quality score. The specific DNA markers, of 450 bp, 576 bp, 612 bp and 2400 bp respectively were characterized for 1Dx5, 1Dy10, 1Dy12 and 1Bx7 alleles. These markers are very important in screening of wheat for bread-making quality.

Cloning and Characterization of Three Genes Encoding LMW-GSs from <i>Triticum aestivum</i>, Cultival Cheyenne

Three low molecular weight subunit genes, named LMW-CND1 (GeneBank accession JQ780048), LMW-CND2 (GeneBank accession JQ779840), LMW-CND3 (GeneBank accession JQ779841), with a ORF of 1053 bp, 903 bp, 969 bp, respectively, were isolated from cv. Cheyenne and characterized detailed in molecular level. The proteins encoded by the genes, with 350, 300, 322 amino acid residues respectively, differ only in repetitive domain of sequences due to insertion or deletion of repeats in this domain. Highly similarity in amino-acid sequence between these three subunits and other published LMW-GSs was also observed, showing that all three genes published here are typical LMW-GS genes and closely related to the genes on chromosome 1D. Besides, secondary structure prediction of proteins indicated that, in the three LMW-GSs, random loop accounts for no less than 70 %, α-helix amounts to 26 %, average, and only 1.4 %~1.7 % is β-sheet.

Novel HMW glutenin genes from Aegilops tauschii and their unique structures

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx5.3t and 1Dy12.1**t were revealed and characterized from Ae. tauschii accession PI554324. SDS-PAGE band of 1Dx5.3t was between those of 1Bx6 and 1Bx7, while 1Dy12.1**t with slightly faster migration rate than that of 1Dy12. The lengths of 1Dx5.3t and 1Dy12.1**t were 2115 bp and 1986 bp, encoding 703 and 660 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. 1Dx5.3t is the shortest of the known Dx-type alleles. 1Dy12.1**t is also a special subunit since it has an additional cysteine in the front of the central repetitive domain. This cysteine that is not existed in previously reported Dy-type genes may be useful for improving bread wheat quality. Median-joining Network analysis indicated that 1Dy12.1**t may be a key site in the genealogy of the Glu-Dy.

Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding.

BackgroundHigh molecular weight glutenin subunits (HMW-GSs), encoded by the genes at Glu-1 loci in wheat and its related species, are significant in the determination of grain processing quality. However, the diversity and variations of HMW-GSs are relatively low in bread wheat. More interests are now focused on wheat wild relatives in Triticeae. The genus Aegilops represents an important germplasm for novel HWM-GSs and other useful genes for wheat genetic improvement.ResultsSix novel Glu-1 alleles and HMW-GSs were identified and characterized from three species of Aegilops section Sitopsis (S genome). Both open reading frames (ORFs) and promoter regions of these Glu-1 alleles were sequenced and characterized. The ORFs of Sitopsis Glu-1 genes are approximately 2.9 kb and 2.3 kb for x-type and y-type subunits, respectively. Although the primary structures of Sitopsis HMW-GSs are similar to those of previously reported ones, all six x-type or y-type subunits have the large fragment insertions. Our comparative analyses of the deduced amino acid sequences verified that Aegilops section Sitopsis species encode novel HMW-GSs with their molecular weights larger than almost all other known HMW-GSs. The Glu-1 promoter sequences share the high homology among S genome. Our phylogenetic analyses by both network and NJ tree indicated that there is a close phylogenetic evolutionary relationship of x-type and y-type subunit between S and D genome.ConclusionsThe large molecular weight of HMW-GSs from S genome is a unique feature identified in this study. Such large subunits are resulted from the duplications of repetitive domains in Sitopsis HMW-GSs. The unequal crossover events are the most likely mechanism of variations in glutenin subunits. The S genome-encoded subunits, 1Dx2.2 and 1Dx2.2* have independent origins, although they share similar evolutionary mechanism. As HMW-GSs play a key role in wheat baking quality, these large Sitopsis glutenin subunits can be used as special genetic resources for wheat quality improvement.

Isolation and characterization of Glu-1 genes from the St genome of Pseudoroegneria libanotica

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.

Translation and validation of the developmental, dimensional and diagnostic interview (3Di) for diagnosis of autism spectrum disorder in Thai children

This study aimed to examine the effectiveness of a translated version of the short version of the Developmental, Dimensional and Diagnostic Interview (3Di) in discriminating children with autism spectrum disorders (ASDs) from typically developing children. Two groups, comprising 63 children with clinically ascertained ASDs and 67 typically developing children, were interviewed with the short 3Di translated version. Mean 3Di scale scores in each domain of autistic symptoms (social reciprocity, communication, and repetitive/stereotyped behaviors) were significantly higher in the ASD group than in the typically developing group. The optimal receiver operating characteristics curve cut-off scores were found to be 10, 8, and 3 for social reciprocity domain, communication domain, and repetitive/stereotyped behaviors domain, respectively, which are identical to the original English standardization. Corresponding sensitivities and specificities were 76.2% and 80.9% for the social reciprocity domain; 85.7% and 73.5% for the communication domain; and 66.7% and 80.9% for the repetitive behaviors domain. The areas under the curve were 0.89 (95% CI = 0.84-0.94), 0.88 (95% CI = 0.82-0.94), and 0.79 (95% CI = 0.71-0.87), respectively. The short 3Di-Thai version is found to be a useful diagnostic instrument for differentiating between clinically diagnosed children with ASDs and typically developing children, although further replication is needed.

Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties

Author SummaryThe contraction and relaxation cycles of active muscles generate substantial mechanical forces, both axially and radially, that place extraordinary stress on the molecular structures within the muscle fibers. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein that is thought to form bridges between the main contractile filaments of the muscle, providing the muscle structure with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, we determined the overall architecture of its complete repetitive domain array using a combination of four complementary structural biology methods. Our study reveals a long, dimeric tail-to-tail filament structure folded into an irregular superhelical coil arrangement of almost identical domain modules separated by short linkers. When we applied tension to these myomesin filaments, we found they could stretch to about 2.5 times their original length by unfolding these linkers, and then return to their original state when the tension was removed. Our findings explain how myomesin might adapt its overall length in response to the changing dimensions of the contracting and relaxing muscle, so acting as a highly elastic ribbon that maintains the overall structural organization of the muscle fibers. More generally, these findings demonstrate how repetitive domain modules, such as those in myomesin, can provide elasticity to highly organized biological structures.

Sex differences in children with autism spectrum disorders compared with their unaffected siblings and typically developing children

This study examined the nature of cognitive and behavioral sex differences in children with autism spectrum disorders (ASDs) and two comparison groups: a group of typically developing (TD) children and a group of unaffected siblings of ASD children. Sex differences in core autistic symptoms, co-occurring behavioral symptoms, and cognitive styles were assessed in each group. Females with ASD were less severely affected than IQ- and age-matched males with ASD in the communication and repetitive stereotyped behavior domains, as measured by the ADI-R, but such sex differences were less significant than they were in the unaffected sibling group. Several behavioral/emotional symptom scores were significantly higher in male siblings than in female siblings. However, the ASD and TD groups did not show sex differences in any behavioral/emotional symptom scores. Males were superior in systemizing relative to empathizing, while the opposite was true for females in the unaffected sibling group and the TD children group; however, both males and females were superior in systemizing relative to empathizing in the ASD group. Our findings support the extreme male brain theory of autism, and further studies are needed to understand the mechanisms behind and developmental perspectives on the nature of sex differences in ASD.

Evidence for T Cell Help in the IgG Response against Tandemly RepetitiveTrypanosoma cruziB13 Protein in Chronic Chagas Disease Patients

The tandemly repetitive Trypanosoma cruzi B13 protein is an immunodominant antigen among Chagas disease patients. Such repetitive domains may behave as T-independent antigens. However, T cells can recognize B13 epitopes in an HLA class II-restricted fashion and could potentially provide cognate T cell help and boost antibody titers. We assessed whether the presence of HLA class II molecules able to present B13 epitopes to T cells could affect anti-B13 IgG levels in a cognate fashion, in both major clinical forms of chronic Chagas disease. We found no difference between anti-B13 IgG antibody levels between patients carrying HLA class II molecules associated to T cell responses or other alleles. The predominant anti-B13 IgG subclass was IgG1, with negligible IgG2, suggesting a T-dependent, noncognate help for antibody production. In addition, the finding of increased anti-B13 IgG levels in sera from CCC patients indicates that clinical presentation is associated with increased anti-B13 antibody levels.

Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs.

Phylogenetic analysis of the human thyroglobulin regions

Thyroglobulin is a large protein present in all vertebrates. It is synthesized in the thyrocytes and exported to lumen of the thyroid follicle, where its tyrosine residues are iodinated . The iodinated thyroglobulin is reintegrated into the cell and processed (cleaved to free its two extremities) for thyroid hormone synthesis. Thyroglobulin sequence analysis has identified four regions of the molecule: Tg1, Tg2, Tg3 and ChEL. Structural abnormalities and mutations result in different pathological consequences, depending on the thyroglobulin region affected. We carried out a bioinformatic analysis of thyroglobulin, determining the origin and the function of each region. Our results suggest that the Tg1 region acts as a binding protein on the apical membrane, the Tg2 region is involved in protein adhesion and the Tg3 region is involved in determining the three-dimensional structure of the protein. The ChEL domain is involved in thyroglobulin transport, dimerization and adhesion. The presence of repetitive domains in the Tg1, Tg2 and Tg3 regions suggests that these domains may have arisen through duplication.

GhAGP31, a cotton non‐classical arabinogalactan protein, is involved in response to cold stress during early seedling development

Arabinogalactan proteins (AGPs), a superfamily of highly glycosylated hydroxyproline-rich glycoproteins, are widely implicated in plant growth and development. A gene (including its cDNA), designated GhAGP31, encoding a non-classical AGP protein was isolated from cotton (Gossypium hirsutum). The deduced GhAGP31 protein contains the conserved features of non-classical AGPs: a putative signal peptide, N-terminal histidine-rich stretch, middle repetitive proline-rich domain and a cysteine-containing 'PAC' domain. GFP fluorescence assay demonstrated that GhAGP31 protein was localised on cell walls. GhAGP31 transcripts were mainly detected in roots, hypocotyls and ovules, but little or almost none were detected in other tissues. In particular, expression of GhAGP31 was developmentally regulated in roots. Further study demonstrated that GhAGP31 expression in cotton roots was remarkably up-regulated by cold stress. Expression of the GUS gene driven by the GhAGP31 promoter was also dramatically enhanced in roots of transgenic Arabidopsis seedlings under cold treatment. Additionally, overexpression of GhAGP31 in yeast and Arabidopsis significantly improved the freezing tolerance of yeast cells and cold tolerance of Arabidopsis seedlings. These data imply that GhAGP31 protein may be involved in the response to cold stress during early root development of cotton.