Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Conversion of DNA Sequences: From a Transposable Element to a Tandem Repeat or to a Gene.

Eukaryotic genomes are rich in repetitive DNA sequences grouped in two classes regarding their genomic organization: tandem repeats and dispersed repeats. In tandem repeats, copies of a short DNA sequence are positioned one after another within the genome, while in dispersed repeats, these copies are randomly distributed. In this review we provide evidence that both tandem and dispersed repeats can have a similar organization, which leads us to suggest an update to their classification based on the sequence features, concretely regarding the presence or absence of retrotransposons/transposon specific domains. In addition, we analyze several studies that show that a repetitive element can be remodeled into repetitive non-coding or coding sequences, suggesting (1) an evolutionary relationship among DNA sequences, and (2) that the evolution of the genomes involved frequent repetitive sequence reshuffling, a process that we have designated as a "DNA remodeling mechanism". The alternative classification of the repetitive DNA sequences here proposed will provide a novel theoretical framework that recognizes the importance of DNA remodeling for the evolution and plasticity of eukaryotic genomes.

DNA methylation in disease: Immunodeficiency, Centromeric instability, Facial anomalies syndrome.

DNA methylation is an epigenetic modification essential for normal mammalian development. Initially associated with gene silencing, more diverse roles for DNA methylation in the regulation of gene expression patterns are increasingly being recognized. Some of these insights come from studying the function of genes that are mutated in human diseases characterized by abnormal DNA methylation landscapes. The first disorder to be associated with congenital defects in DNA methylation was Immunodeficiency, Centromeric instability, Facial anomalies syndrome (ICF). The hallmark of this syndrome is hypomethylation of pericentromeric satellite repeats, with mutations in four genes: DNMT3B, ZBTB24, CDCA7 and HELLS, being linked to the disease. Here, we discuss recent progress in understanding the molecular interactions between these genes and consider current evidence for how aberrant DNA methylation may contribute to the abnormal phenotype present in ICF syndrome patients.

BEL1-like protein (StBEL5) regulates CYCLING DOF FACTOR1 (StCDF1) through tandem TGAC core motifs in potato.

Tuberization in potato is governed by many intrinsic and extrinsic factors. Various molecular signals, such as red light photoreceptor (StPHYB), BEL1-like transcription factor (StBEL5), CYCLING DOF FACTOR1 (StCDF1), StCO1/2 (CONSTANS1/2) and StSP6A (Flowering Locus T orthologue), function as crucial regulators during the photoperiod-dependent tuberization pathway. StCDF1 induces tuberization by increasing StSP6A levels via StCO1/2 suppression. Although the circadian clock proteins, GIGANTEA (StGI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (StFKF1), are reported as StCDF1 interactors, how the StCDF1 gene is regulated in potato is unknown. The BEL-KNOX heterodimer regulates key tuberization genes through tandem TGAC core motifs in their promoters. A recent study reported the presence of six tandem TGAC core motifs in the StCDF1 promoter, suggesting possible regulation of StCDF1 by StBEL5. In our study, we observed a positive correlation between StBEL5 and StCDF1 expression, whereas StCDF1 and its known repressor, StFKF1, showed a negative correlation for the tested tissue types. To investigate the StBEL5-StCDF1 interaction, we generated transgenic potato promoter lines containing a wild-type or mutated (deletion of six tandem TGAC sites) StCDF1 promoter fused to GUS. Wild-type promoter transgenic lines exhibited widespread GUS activity, whereas this activity was absent in the mutated promoter transgenic lines. Moreover, StBEL5 and StCDF1 transcript levels were significantly higher in the stolon-to-tuber stages under short-day conditions compared to long-day conditions. Using wild-type and mutated prStCDF1 as baits in Y1H assays, we further demonstrated that StBEL5 interacts with the StCDF1 promoter through tandem TGAC motifs, indicating direct regulation of StCDF1 by StBEL5 in potato.

Versatile communication strategies among tandem WW domain repeats.

Interactions mediated by short linear motifs in proteins play major roles in regulation of cellular homeostasis since their transient nature allows for easy modulation. We are still far from a full understanding and appreciation of the complex regulation patterns that can be, and are, achieved by this type of interaction. The fact that many linear-motif-binding domains occur in tandem repeats in proteins indicates that their mutual communication is used extensively to obtain complex integration of information toward regulatory decisions. This review is an attempt to overview, and classify, different ways by which two and more tandem repeats cooperate in binding to their targets, in the well-characterized family of WW domains and their corresponding polyproline ligands.

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor.

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology.

Characterization of a novel spore wall protein NbSWP16 with proline-rich tandem repeats from Nosema bombycis (microsporidia).

Nosema bombycis, a pathogen of silkworm pebrine, is an obligate unicellular eukaryotic parasite. It is reported that the spore wall proteins have essential functions in the adherence and infection process of microsporidia. To date, the information related to spore wall proteins from microsporidia is still limited. Here, a 44 kDa spore wall protein NbSWP16 was characterized in N. bombycis. In NbSWP16, a 25 amino acids signal peptide and 3 heparin binding motifs were predicted. Interestingly, a region that contains 3 proline-rich tandem repeats lacking homology to any known protein was also present in this protein. The immunofluorescence analysis (IFA) demonstrated that distinct fluorescent signals were detected both on the surface of mature spores and the germinated spore coats. Immunolocation by electron microscopy revealed that NbSWP16 localized on the exospore regions. Finally, spore adherence analysis indicated that spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro. In contrast with anti-NbSWP16, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously. Collectively, these results suggest that NbSWP16 is a new exospore protein and probably be involved in spore adherence of N. bombycis.

Association between amygdala reactivity and a dopamine transporter gene polymorphism.

Essential for detection of relevant external stimuli and for fear processing, the amygdala is under modulatory influence of dopamine (DA). The DA transporter (DAT) is of fundamental importance for the regulation of DA transmission by mediating reuptake inactivation of extracellular DA. This study examined if a common functional variable number tandem repeat polymorphism in the 3' untranslated region of the DAT gene (SLC6A3) influences amygdala function during the processing of aversive emotional stimuli. Amygdala reactivity was examined by comparing regional cerebral blood flow, measured with positron emission tomography and [(15)O]water, during exposure to angry and neutral faces, respectively, in a Swedish sample comprising 32 patients with social anxiety disorder and 17 healthy volunteers. In a separate US sample, comprising 85 healthy volunteers studied with blood oxygen level-dependent functional magnetic resonance imaging, amygdala reactivity was assessed by comparing the activity during exposure to threatening faces and neutral geometric shapes, respectively. In both the Swedish and the US sample, 9-repeat carriers displayed higher amygdala reactivity than 10-repeat homozygotes. The results suggest that this polymorphism contributes to individual variability in amygdala reactivity.

Datos genéticos poblacionales de 15 marcadores tipo «short tandem repeats» empleados en las pruebas de paternidad e identificación de individuos por genética forense en el área metropolitana de la región centro de México / Population genetic data for 15 markers kind of short tandem repeats used in paternity testing and individual identification by forensic genetic in the metropolitan area from central region of Mexico

Introducción: Actualmente en México se ha incrementado el uso de marcadores del tipo «short tandem repeats» en la práctica forense para la identificación de individuos debido a su excelente poder discriminativo y a su uso extendido en los laboratorios de todo el mundo. Objetivo: El objetivo de este trabajo fue profundizar en el conocimiento de la frecuencia de los alelos pertenecientes a los marcadores D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S17, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 y FGA en la población del área metropolitana de la región centro de México. Material y métodos: Las frecuencias alélicas fueron obtenidas a partir de una muestra de 300 individuos sanos y no relacionados entre sí. Los datos fueron analizados estadísticamente. Resultados y conclusiones: Todos los loci se encuentran en equilibrio de Hardy-Weinberg y muestran un poder de discriminación de 0,999999999. Los resultados contribuyen a establecer una base de datos representativa de las frecuencias alélicas característica de la región y muestran la heterogeneidad genética que existe entre las distintas poblaciones latinoamericanas (AU)

Introduction: Nowadays in Mexico the use of short tandem repeats has increased in Forensic practice for individual identification, due to its excellent discriminative power and its widespread use in laboratories around the world. Objective: The aim of this research work was to delve into the knowledge of allelic frequencies from markers such as D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA in the metropolitan area in the central region of Mexico. Material and methods: A study about the allelic frequencies obtained from 300 healthy and unrelated individuals was developed. Data were analyzed statistically. Results and conclusions: The combined power of discrimination was 0.999999999. All loci are in Hardy-Weinberg equilibrium, and show high discrimination for paternity analysis and forensic genetic applications. Results contribute to establishing a representative database of genome admixture in the region, and show the marked genetic heterogeneity that characterizes Latin American populations (AU)

Conocimiento del estado vascular para la toma de decisiones terapéuticas en el ictus isquémico agudo: ¿cuál es el papel de la neurosonología? / Knowledge of vascular status for therapeutic decision-making in acute ischemic stroke: which is the role of neurosonology?

Existe en los últimos años un interés creciente en el conocimiento del estado vascular de los pacientes con ictus agudo. La detección y localización de una oclusión arterial resultan de gran interés para un enfoque pronóstico adecuado y la selección del tratamiento recanalizador agudo más apropiado. La neurosonología constituye una herramienta diagnóstica útil en el estudio del estado vascular del paciente con ictus agudo. En el presente trabajo se revisan diversas situaciones en las que los ultrasonidos aportan información diagnóstica valiosa, como en el caso de la oclusión de la arteria cerebral media (ACM), oclusión ‘en T’ de la arteria carótida interna (ACI), oclusión ‘en tándem’ ACI-ACM, monitorización de oclusiones de arterias intracraneales, oclusión aguda de la ACI extracraneal y presencias de trombos flotantes intracarotídeos. La neurosonología aporta ventajas evidentes frente a otras técnicas diagnósticas: es rápida, dinámica, económica, inocua, accesible, permite la monitorización del estado vascular del paciente en tiempo real, evita retrasos en la administración de tratamientos agudos y posee un efecto terapéutico (sonotrombólisis). Por todo ello, la neurosonología ocupa un papel fundamental en el diagnóstico del estado vascular y en la toma de decisiones terapéuticas en el paciente con ictus isquémico agudo (AU)

In the last years there is an increasing interest in the knowledge of vascular status in patients with acute stroke. Detection and localization of an artery occlusion is of great interest for an accurate prognosis and the selection of the most appropriate recanalizing therapy. Neurosonology is a useful diagnostic tool for vascular status study in patients with acute stroke. Different situations where ultrasounds offer a valuable diagnostic information are reviewed, such as middle cerebral artery (MCA) occlusion, ‘T’ internal carotid artery (ICA) occlusion, ‘tandem’ ICA-MCA occlusion, monitoring of intracranial artery occlusions, acute occlusion of extracranial ICA, and free-floating thrombus in the ICA. Neurosonology offers evident advantages compared with other diagnostic techniques: it is faster, dynamic, cheaper, harmless, and accessible, allows real-time monitoring of patients vascular status, avoids delays in acute treatments and has a therapeutic effect (sonothrombolysis). Neurosonology has an essential role in the diagnosis of vascular status and in therapeutic decisionmaking of acute ischemic stroke patients (AU)

Links between DRD4, executive attention, and alphabetic skills in a nonclinical sample.

BACKGROUND: The dopamine D4 receptor gene (DRD4) has been linked to attention deficit hyperactivity disorder (ADHD) and reading disorders. In this study, we examined whether diminished anticipatory dopamine cell firing - typical of the long variant of the DRD4 allele - is related to emergent and advanced alphabetic skills, and whether executive attention is a mediator between this allele and alphabetic skills. METHOD: We tested alphabetic skills in a normative sample of 159 children in both kindergarten and Grade 1, and executive attention 1 year earlier. Cheek cells were collected and genomic DNA was isolated from the samples using the Chemagic buccal swab kit on a chemagen Module I workstation. RESULTS: Thirty-seven percent of the children were carriers of at least one DRD4 7-repeat allele. Carriers of the long variant scored lower on alphabetic skills, and executive attention appeared to be a mediator of the relation between characteristics of DRD4 and alphabetic skills in kindergarten and first grade. CONCLUSION: This study shows how a genetic factor which has been shown to relate to variation in attention and regulatory behavior can explain delays in alphabetic skills. A practical implication is that in many cases early interventions should not only target reading skills, but also support children's engagement in tasks.

Loss of the wild-type allele contributes to myeloid expansion and disease aggressiveness in FLT3/ITD knockin mice.

Clinical evidence has shown that FLT3 internal tandem duplication (ITD) mutation confers poor prognosis in acute myeloid leukemia. Loss of the FLT3 wild-type (WT) allele is associated with even worse prognosis. We have previously reported that heterozygous FLT3(wt/ITD) "knockin" mice develop a slowly fatal myeloproliferative neoplasm (MPN). To study the roles of the WT FLT3 and ITD alleles in the development of MPNs, we generated FLT3/ITD homozygous (FLT3(ITD/ITD)) and hemizygous (FLT3(-/ITD)) mice. FLT3(-/ITD) mice, with the loss of WT allele, display a more severe MPN, as evidenced by even larger spleen, higher white blood cell counts, and shorter survival, compared with FLT3(wt/ITD) mice. Reintroduction of the WT FLT3 allele into FLT3(-/ITD) BM slowed the progression of MPN in recipient mice. FLT3(ITD/ITD) mice had an even severe MPN compared with the FLT3(-/ITD) and FLT3(wt/ITD) mice. Spontaneous leukemia developed in a small fraction of the FLT3(ITD/ITD) mice but was never observed in the FLT3(-/ITD) and FLT3(wt/ITD) mice. Our results suggest that loss of the WT allele contributes to the development of a more severe phenotype. Thus, the WT FLT3 allele seemingly functions as a tumor suppressor, attenuating the function of the FLT3/ITD allele in leukemia harboring FLT3/ITD mutations.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation.

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.

[Chicken lampbrush chromosomes: transcription of tandemly repetitive DNA sequences].

The transcribed part of the genome includes both protein-coding sequences and a variety of sequences with unknown functions. Amphibian and avian lampbrush chromosomes represent a convenient experimental system for studying cell functions and the regulation of transcription of protein-noncoding DNA. Taking lumpy loops formed on chicken (Gallusgallus domesticus) chromosome 2 at the lampbrush stage as an example, we applied an approach allowing RNA sources to be identified in the lateral loops of lampbrush chromosomes. This approach involves a bioinformatic analysis of data from the chicken genome sequencing project and a high-resolution mapping of transcripts on microsurgically isolated bivalents. As a result, a novel tandemly repetitive DNA sequence, LL2R (lumpy loop 2 repeat), of approximately 440 bp in size was identified in the chicken genome, its transcripts taking part in the formation of lumpy loops with a massive RNP matrix on chromosome 2 in growing oocytes.

Effects of epitope sequence tandem repeat and proline incorporation on polyclonal antibody production against cytochrome 1A2 and 3A4.

We describe a method for producing polyclonal antibodies against peptide antigen cytochrome P450 1A2 and 3A4 using a tandem repeat of the epitope region and incorporation of proline residue between the repeated sequences. An ELISA assay revealed more efficient generation of polyclonal antibodies to tandem repeat peptide antigens than mono-epitope peptides. The incorporation of proline residues further stimulated antibody production.

Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects. RESULTS: In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation. CONCLUSION: We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats.

Extrachromosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms, and has been noted for more than 3 decades. eccDNA occurs in normal tissues and in cultured cells, is heterogeneous in size, consists of chromosomal sequences and reflects plasticity of the genome. Two-dimensional (2D) gel electrophoresis has been adapted for the detection and characterization of eccDNA. It shows that most eccDNA consists of chromosomal tandem repeats, both coding genes and satellite DNA and is organized as circular multimers of the repeating sequence. 2D gels were unable to detect dispersed repeats within the population of eccDNA. eccDNA, organized as circular multimers, can be formed de novo in Xenopus egg extracts, in the absence of DNA replication. These findings support a mechanism for the formation of eccDNA that involves intra-chromosomal homologous recombination between tandem repeats and looping-out. Furthermore, eccDNA appears to undergo extrachromosomal replication via a rolling circle mechanism. Hence, the formation of eccDNA from arrays of tandem repeats may cause deletions, and the possible re-integration of rolling-circle replication products could expand these arrays. This review summarizes recent experimental data which characterizes eccDNA in several organisms using 2D gel electrophoresis, and discusses its possible implications on the dynamics of chromosomal tandem repeats.

Tandem stop codons in ciliates that reassign stop codons.

Tandem stop codons are extra stop codons hypothesized to be present downstream of genes to act as a backup in case of read-through of the real stop codon. Although seemingly absent from Escherichia coli, recent studies have confirmed the presence of such codons in yeast. In this paper we will analyze the genomes of two ciliate species--Paramecium tetraurelia and Tetrahymena thermophila--that reassign the stop codons TAA and TAG to glutamine, for the presence of tandem stop codons. We show that there are more tandem stop codons downstream of both Paramecium and Tetrahymena genes than expected by chance given the base composition of the downstream regions. This excess of tandem stop codons is larger in Tetrahymena and Paramecium than in yeast. We propose that this might be caused by a higher frequency of stop codon read-through in these species than in yeast, possibly because of a leaky termination machinery resulting from stop codon reassignment.