Differences In Number Of Repeats Research Articles

Development of an isogenic human cell trio that models polyglutamine disease.

Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain genes. To investigate the functional influence of repeat expansion on disease mechanisms, we applied a biallelic genome-engineering platform that we recently established, called Universal Knock-in System or UKiS, to develop a human cell trio, a set of three isogenic cell lines that are homozygous for two different numbers of repeats (first and second lines) or heterozygous for the two repeat numbers (third line). As an example of a polyQ disease, we chose spinocerebellar ataxia type 2 (SCA2). In a pseudodiploid human cell line, both alleles of the glutamine-encoding triplet repeat in the SCA2-causing gene, ataxin 2 or ATXN2, were first knocked in with a donor sequence encoding both thymidine kinase and either puromycin or blasticidin resistance proteins under dual drug selection. The knocked-in donor alleles were then substituted with a payload having either 22 or 76 triplet repeats in ATXN2 by ganciclovir negative selection. The two-step substitution and subsequent SNP typing and genomic sequencing confirmed that the SCA2-modeling isogenic cell trio was obtained: three clones of 22-repeat homozygotes, two clones of 22/76-repeat heterozygotes and two clones of 76-repeat homozygotes. Finally, RT-PCR and immunoblotting using the obtained clones showed that, consistent with previous observations, glutamine tract expansion reduced transcriptional and translational expression of ATXN2. The cell clones with homozygous long-repeat alleles, which are rarely obtained from patients with SCA2, showed more drastic reduction of ATXN2 expression than the heterozygous clones. This study thus demonstrates the potential of UKiS, which is a beneficial platform for the efficient development of cell models not only for polyQ diseases but also for any other genetic diseases, which may accelerate our deeper understanding of disease mechanisms and cell-based screening for therapeutic drugs.

Development of Genetic Markers for Sex and Individual Identification of the Japanese Giant Flying Squirrel (Petaurista leucogenys) by an Efficient Method Using High-Throughput DNA Sequencing.

DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.

Abstract 2806: High sensitivity qPCR microsatellite instability detection in FFPE tissue

Abstract Microsatellite instability (MSI) refers to genetic instability in short nucleotide repeats (e.g., 1-6bp tandem repeats), where a cell comprises a different number of repeats as compared to what was inherited from a progenitor cell. This genetic instability is often observed in tumor cells with impaired mismatch repair (MMR), especially in colorectal cancer (CRC) and endometrial cancer. MSI-high tumors can help identify patients that would benefit from additional genetic testing to diagnose Lynch Syndrome. Additionally, MSI can potentially be used as a biomarker to predict response to treatment with immunotherapy and CRC patients with MSI have a significantly better prognosis compared to those with intact mismatch repair. Here we present the VarTrace® MSI Research qPCR Assay, which enables high sensitivity detection of microsatellite instability. The assay uses NuProbe’s PCR-based Blocker Displacement Amplification (BDA) technology to enable the selective amplification of MSI unstable alleles with 1% analytical limit of detection. The assay is intended for the qualitative detection of mutations in 5 MSI loci (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) without the need of matched normal, and reports MSI-high (MSI-H), MSI-low (MSI-L) and Microsatellite Stable (MSS). Analytical validation of the assay has been completed on Horizon MSI/MSS formalin-fixed, paraffin-embedded (FFPE) DNA reference standard, SeraCare MSI AF5% reference panel mix, and 50 CRC FFPE samples. One FFPE section with as little as 2ng of input DNA is enough for this assay. The high sensitivity potentially allows for the detection of MSI status in liquid biopsy. Citation Format: Yan Helen Yan, Blake Young, Zhiheng Wang, Adam Yaseen, Alessandro Pinto, David Zhang. High sensitivity qPCR microsatellite instability detection in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2806.

Chinese women with 29–30 FMR1 CGG repeats have an earlier menopause

Purpose: A strong, well-established non-linear relationship exists between fragile X mental retardation (FMR1) premutation and menopausal age. The aim of this study is to evaluate whether this relationship continues into the normal CGG repeat range.Methods: FMR1 CGG repeats of 111 Chinese postmenopausal women from a prospective cohort and the relationship with age at menopause were analyzed. Associations of FMR1 genotypes with annually measured estradiol and follicle stimulating hormone (FSH) levels were also assessed.Results: One premutation and two intermediate carriers were identified, with a prevalence of 0.90% and 1.80%, respectively. The age at menopause differed with statistical significance (p = 0.007) between women carrying bi-allelic 29–30 repeats (49.66 ± 3.26 years) and those carrying a different number of repeats (51.26 ± 2.74 years). Age at menopause among subgroups (≤28, 29–30, and ≥31 repeats) of alleles 1 and 2 were also different (p = 0.014, p = 0.044). FSH trajectories to final menstrual period differed between women with the bi-allelic 29–30 repeats and others (p = 0.019).Conclusions: Women with 29–30 FMR1 CGG repeats may experience menopause approximately 2 years earlier than those carrying ≤28 or ≥31 CGG repeats, and have a longer FSH fluctuant period.

Comparison of Identification of Toxoplasma gondii by Commercial Realtime PCR and Inhouse Realtime PCR Methods

Objective: This study aimed to compare the diagnosis of toxoplasmosis with a commercial kit and inhouse realtime PCR methods to determine molecular methods with high diagnostic accuracy for use in addition to serologic tests for routine diagnosis.Methods: The study included a total of 116 samples of blood, CSF or amniotic fluid with 19 identified positive and 97 negatives for T. gondii sent to our laboratory. Due to the low number of positive samples, DNA samples from an external quality control program that our laboratory participates in were included in the study. First to all samples, realtime PCR method were applied with commercial kit used primers for T. gondii rep529 gene, and then inhouse realtime PCR were applied with TG-F and TG-R primers and Taqman probe, targeting the insertion sequence region of T. gondii B1 gene.Results: The results for the total of 116 samples studied with both methods was that 17 were identified as positive with commercial realtime PCR and 19 were determined as positive with inhouse realtime PCR. Accordingly, two cases with the commercial realtime PCR method were determined as false negative. The limit of detection for both methods used in our study was determined as 10-3 dilution (0.028 copy/reaction). There was a high level of compatibility determined between the inhouse and realtime PCR methods (kappa value: 0.934). Conclusion: In conclusion, though there was perfect compatibility observed between the results with the two methods, disadvantages of the commercial realtime PCR method included isolates where the target gene was not found, deletion or mutation of all or part of this gene or different numbers of repeats causing false negative results and high cost. Considering this, our laboratory decided to use the inhouse realtime PCR using primers for the B1 gene to research T. gondii with molecular methods. A significant limitation of the study is the low number of positive samples. For DNA samples belonging to the External Quality Control Program, the commercial kit was 66.66% successful, while the inhouse realtime PCR method was 100% successful.

RNA recombination at Chikungunya virus 3'UTR as an evolutionary mechanism that provides adaptability.

The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3’UTR. Sequence repeats are conserved at the CHIKV 3’UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3’UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions.

Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Mt-rps3 is an ancient gene which provides insight into the evolution of fungal mitochondrial genomes

The nuclear ribosomal protein S3 (Rps3) is implicated in the assembly of the ribosomal small subunit. Fungi and plants present a gene copy in their mitochondrial (mt) genomes. An analysis of 303 complete fungal mt genomes showed that, when rps3 is found, it is either a free-standing gene or an anchored gene within the omega intron of the rnl gene. Early divergent fungi, Basidiomycota and all yeasts but the CTG group belong to the first case, and Pezizomycotina to the second. Its position, size and genetic code employed are conserved within species of the same Order. Size variability is attributed to different number of repeats. These repeats consist of AT-rich sequences. MtRps3 proteins lack the KH domain, necessary for binding to rRNA, in their N-terminal region. Their C-terminal region is conserved in all Domains of life. Phylogenetic analysis showed that nuclear and mtRps3 proteins are descendants of archaeal and a-proteobacterial homologues, respectively. Thus, fungal mt-rps3 gene is an ancient gene which evolved within the endosymbiotic model and presents different evolutionary routes: (a) coming from a-proteobacteria, it was relocated to another region of the mt genome, (b) via its insertion to the omega intron, it was transferred to the nucleus and/or got lost, and (c) it was re-routed to the mt genome again. Today, Basidiomycota and Saccharomycetales seem to follow the first evolutionary route and almost all Pezizomycotina support the second scenario with their exceptions being the result of the third scenario, i.e., the gene’s re-entry to the mt genome.

Absorption and generation of femtosecond laser-pulse excited spin currents in noncollinear magnetic bilayers

Spin currents can be generated on an ultrafast timescale by excitation of a ferromagnetic (FM) thin film with a femtosecond laser-pulse. Recently, it has been demonstrated that these ultrafast spin currents can transport angular momentum to neighbouring FM layers, being able to change both the magnitude and orientation of the magnetization in the adjacent layer. In this work, both the generation and absorption of these optically excited spin currents are investigated. This is done using non-collinear magnetic bilayers, i.e. two FM layers separated by a conductive spacer. Spin currents are generated in a Co/Ni multilayer with out-of-plane (OOP) anisotropy, and absorbed by a Co layer with an in-plane (IP) anisotropy. This behaviour is confirmed by careful analysis of the laser-pulse induced magnetization dynamics, whereafter it is demonstrated that the transverse spin current is absorbed very locally near the injection interface of the IP layer (90% within the first approx. 2 nm). Moreover, it will also be shown that this local absorption results in the excitation of THz standing spin waves within the IP layer. The dispersion measured for these high frequency spin waves shows a discrepancy with respect to the theoretical predictions, for which a first explanation involving intermixed interface regions is proposed. Lastly, the spin current generation is investigated using different number of repeats for the Co/Ni multilayer, which proves to be of great relevance for identifying the optical spin current generation mechanism.

Phylogeny of Indian Rhizophoraceae Based on the Molecular Data from Chloroplast tRNALEUUAA Intergenic Sequences

Molecular identification data of unexplored Indian Rhizophoraceae an eco-friendly mangroves are an imperative aspect in molecular phylogenetics. We describe the phylogenetic relationships among the Rhizophoraceae genus Rhizophora, Ceriops and Bruguiera using tRNA Leu (UAA) intron sequences as a molecular marker. The results of present study reveals congeneric relationship between R. apiculata, R. mucronata, B. gymnorhiza indicating a high degree of gene flow within them and the pairwise distribution of study plants among Rhizophoraceae family. The phylogram constructed using tRNA Leu sequence clearly clustered the species of the same genus in individual group. The stem-loop could be divided into two classes, both built up from two base pairing heptanucleotide repeats. Size variation was primarily caused by different numbers of repeats but some strains also contained additional sequences in this stem-loop. The statistical summaries of DNA sequence data can enable to identify the structural signature of the genome as well as to classify phylogenetic relationships among different species reflected in the difference of genetic diversity distributions within their DNA sequences.

High levels of somatic DNA diversity at the myotonic dystrophy type 1 locus are driven by ultra-frequent expansion and contraction mutations

Several human genetic diseases are associated with inheriting an abnormally large unstable DNA simple sequence repeat. These sequences mutate, by changing the number of repeats, many times during the lifetime of those affected, with a bias towards expansion. These somatic changes lead not only to the presence of cells with different numbers of repeats in the same tissue, but also produce increasingly longer repeats, contributing towards the progressive nature of the symptoms. Modelling the progression of repeat length throughout the lifetime of individuals has potential for improving prognostic information as well as providing a deeper understanding of the underlying biological process. A large data set comprising blood DNA samples from individuals with one such disease, myotonic dystrophy type 1, provides an opportunity to parameterize a mathematical model for repeat length evolution that we can use to infer biological parameters of interest. We developed new mathematical models by modifying a proposed stochastic birth process to incorporate possible contraction. A hierarchical Bayesian approach was used as the basis for inference, and we estimated the distribution of mutation rates in the population. We used model comparison analysis to reveal, for the first time, that the expansion bias observed in the distributions of repeat lengths is likely to be the cumulative effect of many expansion and contraction events. We predict that mutation events can occur as frequently as every other day, which matches the timing of regular cell activities such as DNA repair and transcription but not DNA replication.

Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG) n /(CTG) n hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG) n /(CTG) n hairpin structures during DNA metabolism.

Design, Production and Molecular Structure of a New Family of Artificial Alpha-helicoidal Repeat Proteins (αRep) Based on Thermostable HEAT-like Repeats

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable ( T m > 70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners.

한국 재래닭의 MHC 영역 유전자형 분석

닭의 MHC 유전자 내에 있는 microsatellite marker LEI0258은 혈청의 구분 및 질병 저항성 유전자로 많은 연구가 되어 있다. 본 연구는 유전변이가 있는 microsatellite marker LEI 0258을 한국 재래계 흑색종, 한국 재래계 갈색종, 코니쉬종, 로드아일랜드 레드종에 적용하여 품종 특이 유전자형 및 allele을 탐색하고, 품종 구분 활용에 가능하지 여부를 실험하기 위하여 실시하였다. 한국 재래계만이 가지고 있는 특이 대립 유전자는 찾을 수는 없었지만 품종간 유전자형의 빈도 차이를 보이는 대립 유전자들을 확인할 수 있어 마커의 조합을 통하여 품종을 구분할 수 있는 가능성을 제시하였으며, 질병 저항성 연구의 기초 자료로 그 이용성이 높을 것으로 사료된다. The chicken major histocompatibility complex (MHC) is known to be associated with disease resistance and susceptibility to several pathogens. The microsatellite marker LEI0258 is physically located between the BG and BF of MHC region and variations near this marker have been well documented. In this report, the LEI0258 marker was used to find specific alleles for the Korean native chicken. The MHC haplotype was analyzed by PCR screening and sequencing of LEI0258 region in four different breeds including black Korean native chicken, brown Korean native chicken, Cornish and Rhode island red. The serologically same MHC haplotypes showed the differences in repeat numbers, a few indels or single nucleotide polymorphisms by sequencing analysis. Even though we could not identify specific alleles for Korean native chickens, the genotypes analyzed in these breeds can give valuable information for the relationships with disease resistance and establishment of breeding strategies for the Korean native chicken.

A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR

Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely α-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.

Fusion of Ross Chicken Complement Component C3d-P29 Repeats with a Chicken Viral Newcastle Disease Virus F Gene Enhances Immune Responses Following DNA Immunization

C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response. The gene fragment coding for Ross chicken C3d gene (chC3d) was cloned and expressed as a component of fusion proteins destined for its application in the vaccine study of chicken, and for in vitro experiments. Three potential vaccine construct units were engineered to contain two, four, and six copies of chC3d-P29 coding gene, which was linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties (OIE). The cloned chC3d protein and different repeats of C3d-P29 proteins that contained the F gene of NDV (C3d-P29.n-F) was generated separately in Escherichia coli and CEF cells with the help of expression vectors. All recombinant proteins were analyzed by SDS-PAGE and Western blot. The result of immunogenicity detection of different numbers of repeats of C3d-P29.n-F revealed that C3d-P29 could enhance immunogenicity. Six or more repeats of C3d-P29 may be necessary for efficient enhancement of antigen-specific immune responses.

Accurate determination of copy number variations (CNVs): Application to the α- and β-defensin CNVs

The human genome is rich in genomic regions that are repeated several times. These regions are known as CNVs (copy number variations) and can be polymorphic. Moreover, the number of copies may play a role in the predisposition to particular diseases. It is therefore important to accurately determine the copy number of those CNVs in individuals.We have developed a strategy, using concatemeric constructs containing different numbers of repeats as internal standards, to accurately determine the number of repeats in the α-defensin and β-defensin region on chromosome 8p23 by real-time PCR. The test was validated by FISH in DNA of 13 individuals.Comparison with previously published methods shows that this approach provides more accurate results for the determination of the exact number of repeats when they exceed 3 copies. This strategy can be easily transferred to any CNV. With this method we structurally analyzed the α- and β-defensin region in 334 Belgian individuals.

Tracing misalignment and unequal crossing over during evolution of the repetitive region of the SM50 gene in sea urchins

The location of misalignment and unequal crossover involved in concerted evolution of tandemly repetitive sequences is difficult to document owing to the homogeneity of sequences that are subject to this process. However, the repetitive domain of the SM50 gene in sea urchins contains variation, within the gene itself, between alleles, and between species that has allowed us to determine where misalignment and unequal crossing over occurred during evolution of this gene. We have therefore analyzed the SM50 repeat regions in a variety of species to determine where recent changes in repeat numbers have occurred, and from this have deduced the mechanisms that lead to these changes. We next tried to determine whether recent misalignment and unequal crossover has produced allelic variation in current populations of sea urchins. We found SM50 alleles within three species that have different numbers of repeats. This marks the first reported documentation of allelic variation in the number of repeats in the SM50 gene. We also show how a single unequal crossover event could have produced the allelic variation. We have found that substitutions and small deletions in the sequences within the repeats can substantially affect how misalignment occurs, resulting in different patterns of repeats after concerted evolution.