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Find video protocols related to scientific articles indexed in Pubmed.
Identification of Genes Important for the Physical Interaction between Protein Pairs through Reverse PCA (rPCA).
Curr Protoc Cell Biol
PUBLISHED: 09-02-2014
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Cells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. Understanding physical and functional interactions between proteins in living systems is of vital importance in biology. The importance of protein-protein interactions (PPIs) has led to the development of several powerful methodologies and techniques to detect them. All of this information has enabled the creation of large protein-interaction networks. One important challenge in biology is to understand how protein complexes respond to genetic perturbations. Here we describe a systematic genetic assay termed "reverse PCA," which allows the identification of genes whose products are required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the PPI of interest can be detected by resistance to the drug methotrexate, in the context of the protein-fragment complementation assay (PCA). By combining the synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. The identification of such mutants is valuable for unraveling important regulatory mechanisms, and for defining the response of the protein interactome to specific perturbations. Curr. Protoc. Cell Biol. 64:17.15.1-17.15.11. © 2014 by John Wiley & Sons, Inc.
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Gene expression alterations in ulcerative colitis patients after restorative proctocolectomy extend to the small bowel proximal to the pouch.
Gut
PUBLISHED: 07-02-2014
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To evaluate molecular profiles in the small bowel (SB) mucosa proximal to the pouch in ulcerative colitis (UC) patients after pouch surgery.
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Is the emotional Stroop task a special case of mood induction? Evidence from sustained effects of attention under emotion.
Atten Percept Psychophys
PUBLISHED: 05-16-2014
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Sustained effects of emotion are well known in everyday experience. Surprisingly, such effects are seldom recorded in laboratory studies of the emotional Stroop task, in which participants name the color of emotion and neutral words. Color performance is more sluggish with emotion words than with neutral words, the emotional Stroop effect (ESE). The ESE is not sensitive to the order in which the two groups of words are presented, so the effect of exposure to emotion words does not extend to disrupting performance in a subsequent block with neutral words. We attribute this absence of a sustained effect to habituation engendered by excessive repetition of the experimental stimuli. In a series of four experiments, we showed that sustained effects do occur when habituation is removed, and we also showed that the massive exposure to negative stimuli within the ESE paradigm induces a commensurately negative mood. A novel perspective is offered, in which the ESE is considered a special case of mood induction.
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Ethnic effect on FMR1 carrier rate and AGG repeat interruptions among Ashkenazi women.
Genet. Med.
PUBLISHED: 05-02-2014
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Purpose:Fragile X syndrome, a common cause of intellectual disability, is usually caused by CGG trinucleotide expansion in the FMR1 gene. CGG repeat size correlates with expansion risk. Premutation alleles (55-200 repeats) may expand to full mutations in female meiosis. Interspersed AGG repeats decrease allele instability and expansion risk. The carrier rate and stability of FMR1 alleles were evaluated in large cohorts of Ashkenazi and non-Ashkenazi women.Methods:A total of 4,344 Ashkenazi and 4,985 non-Ashkenazi cases were analyzed using Southern blotting and polymerase chain reaction between 2004 and 2011. In addition, AGG interruptions were evaluated in 326 Ashkenazi and 298 non-Ashkenazi women who were recruited during 2011.Results:Both groups had major peaks of 30 and 29 repeats. Ashkenazi women had a higher frequency of 30 repeats and a lower frequency of other peaks (P < 0.0001). A higher rate of premutations in the 55-59 repeats range (1:114 vs. 1:277) was detected among the Ashkenazi women. Loss of AGG interruptions (<2) was significantly less common among Ashkenazi women (9 vs. 19.5% for non-Ashkenazi women, P = 0.0002).Conclusion:Ashkenazi women have a high fragile X syndrome carrier rate and mostly lower-range premutations, and carry a low risk for expansion to a full mutation. Normal-sized alleles in Ashkenazi women have higher average number of AGG interruptions that may increase stability. These factors may decrease the risk for fragile X syndrome offspring among Ashkenazi women.Genet Med advance online publication 29 May 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.64.
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Telomere length kinetics assay (TELKA) sorts the telomere length maintenance (tlm) mutants into functional groups.
Nucleic Acids Res.
PUBLISHED: 04-11-2014
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Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.
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Microscopic chromosome Xp distal deletions--a challenging issue in prenatal genetic counseling.
Prenat. Diagn.
PUBLISHED: 02-24-2014
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A prenatal diagnosis of chromosome X short arm deletions may present a challenge in prenatal genetic counseling. We present clinical and molecular data of carriers of Xp distal deletions.
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Variable clinical presentation of an MUC1 mutation causing medullary cystic kidney disease type 1.
Clin J Am Soc Nephrol
PUBLISHED: 02-07-2014
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The genetic cause of medullary cystic kidney disease type 1 was recently identified as a cytosine insertion in the variable number of tandem repeat region of MUC1 encoding mucoprotein-1 (MUC1), a protein that is present in skin, breast, and lung tissue, the gastrointestinal tract, and the distal tubules of the kidney. The purpose of this investigation was to analyze the clinical characteristics of families and individuals with this mutation.
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Mechanism, prevalence, and more severe neuropathy phenotype of the Charcot-Marie-Tooth type 1A triplication.
Am. J. Hum. Genet.
PUBLISHED: 01-24-2014
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Copy-number variations cause genomic disorders. Triplications, unlike deletions and duplications, are poorly understood because of challenges in molecular identification, the choice of a proper model system for study, and awareness of their phenotypic consequences. We investigated the genomic disorder Charcot-Marie-Tooth disease type 1A (CMT1A), a dominant peripheral neuropathy caused by a 1.4 Mb recurrent duplication occurring by nonallelic homologous recombination. We identified CMT1A triplications in families in which the duplication segregates. The triplications arose de novo from maternally transmitted duplications and caused a more severe distal symmetric polyneuropathy phenotype. The recombination that generated the triplication occurred between sister chromatids on the duplication-bearing chromosome and could accompany gene conversions with the homologous chromosome. Diagnostic testing for CMT1A (n = 20,661 individuals) identified 13% (n = 2,752 individuals) with duplication and 0.024% (n = 5 individuals) with segmental tetrasomy, suggesting that triplications emerge from duplications at a rate as high as ~1:550, which is more frequent than the rate of de novo duplication. We propose that individuals with duplications are predisposed to acquiring triplications and that the population prevalence of triplication is underascertained.
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Gene expression profiles of ileal inflammatory bowel disease correlate with disease phenotype and advance understanding of its immunopathogenesis.
Inflamm. Bowel Dis.
PUBLISHED: 10-11-2013
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Pouchitis may develop in patients with ulcerative colitis undergoing pouch surgery. We aimed to evaluate the de novo inflammation developing in the ileal pouch, hypothesizing that it may be similar to ileitis in Crohns disease (CD).
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Reverse PCA, a systematic approach for identifying genes important for the physical interaction between protein pairs.
PLoS Genet.
PUBLISHED: 10-01-2013
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Protein-protein interactions (PPIs) are of central importance for many areas of biological research. Several complementary high-throughput technologies have been developed to study PPIs. The wealth of information that emerged from these technologies led to the first maps of the protein interactomes of several model organisms. Many changes can occur in protein complexes as a result of genetic and biochemical perturbations. In the absence of a suitable assay, such changes are difficult to identify, and thus have been poorly characterized. In this study, we present a novel genetic approach (termed "reverse PCA") that allows the identification of genes whose products are required for the physical interaction between two given proteins. Our assay starts with a yeast strain in which the interaction between two proteins of interest can be detected by resistance to the drug, methotrexate, in the context of the protein-fragment complementation assay (PCA). Using synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify those mutations that disrupt the physical interaction of interest. We were able to successfully validate this novel approach by identifying mutants that dissociate the conserved interaction between Cia2 and Mms19, two proteins involved in Iron-Sulfur protein biogenesis and genome stability. This method will facilitate the study of protein structure-function relationships, and may help in elucidating the mechanisms that regulate PPIs.
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Pitch Memory and Exposure Effects.
J Exp Psychol Hum Percept Perform
PUBLISHED: 07-22-2013
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Recent studies indicate that the ability to represent absolute pitch values in long-term memory, long believed to be the possession of a small minority of trained musicians endowed with "absolute pitch," is in fact shared to some extent by a considerable proportion of the population. The current study examined whether this newly discovered ability affects aspects of music and auditory cognition, particularly pitch learning and evaluation. Our starting points are two well-established premises: (1) frequency of occurrence has an influence on the way we process stimuli; (2) in Western music, some pitches and musical keys are much more frequent than others. Based on these premises, we hypothesize that if absolute pitch values are indeed represented in long-term memory, pitch frequency of occurrence in music would significantly affect cognitive processes, in particular pitch learning and evaluation. Two experiments were designed to test this hypothesis in participants with no absolute pitch, most with little or no musical training. Experiment 1 demonstrated a faster response and a learning advantage for frequent pitches over infrequent pitches in an identification task. In Experiment 2, participants evaluated infrequent pitches as more pleasing than frequent pitches when presented in isolation. These results suggest that absolute pitch representation in memory may play a substantial, hitherto unacknowledged role in auditory (and specifically musical) cognition. (PsycINFO Database Record (c) 2013 APA, all rights reserved).
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Fever-induced Brugada pattern: how common is it and what does it mean?
Heart Rhythm
PUBLISHED: 06-25-2013
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Fever is known to unmask the Brugada pattern on the electrocardiogram (ECG) and trigger ventricular arrhythmias in patients with Brugada syndrome. Genetic studies in selected cases with fever-induced Brugada pattern have identified disease-causing mutations. Thus, "fever-induced Brugada" is a recognized clinical entity. However, its prevalence has not been systematically evaluated.
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Specific neurological phenotypes in autism spectrum disorders are associated with sex representation.
Autism Res
PUBLISHED: 06-17-2013
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Autism spectrum disorder (ASD) is a heritable disorder occurring predominantly in males. The aim of this study was to compare sex differences in the prevalence of specific neurological phenotypes commonly described in ASD. The study included 663 participants, aged 18 months to 15 years, diagnosed with ASD. Neurological and behavioral assessments were performed using standardized tests, and obtaining medical, developmental, and familial histories from the parents. Phenotypes under investigation were macro- and microcephaly, developmental regression, minor neurological and musculoskeletal deficits (MNMD), and seizures. Male?:?female ratio in the ASD group was 6.7:1. No sex differences in autism severity, cognitive ability, and adaptive functioning were noted. Mean head circumference percentile for males (50.1?±?25.6) was significantly larger than females (43.4?±?30.2). Micro- and macrocephaly were more frequent in ASD than expected (5.9%; 18.1%, respectively). Microcephaly in females (15.1%) was significantly more prevalent than in males (4.5%). The prevalence of macrocephaly in both sexes did not differ significantly. Regression was noted in 30.2% of the females with ASD, significantly higher than in males (18.9%). MNMD was documented in 73.8% of the females, significantly higher than in males (57.1%). M:F ratio decreased in a group with two or more phenotypes (3.6:1), while male predominance was more significant in the group without phenotypes (13.6:1). Neurological phenotypes associated with ASD are more prevalent in females than in males, resulting in more complex clinical and neurological manifestations in females. Therefore, involvement of different etiologies is suggested in ASD in females. Autism Res 2013, 6: 596-604. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.
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Ube3a/E6AP is involved in a subset of MeCP2 functions.
Biochem. Biophys. Res. Commun.
PUBLISHED: 06-03-2013
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Rett syndrome (RTT) and Angelman syndrome (AS) are devastating neurological disorders that share many clinical features. The disease-causing mutations have been identified for both syndromes. Mutations in Methyl-CpG Binding Protein 2 (MECP2) are found in a majority of patients with classical RTT while absence of maternal allele or intragenic mutation in the maternal copy of UBE3A gene encoding the human papilloma virus E6-associated protein (E6AP) cause most cases of AS. Extensive studies have been performed to determine the cause of the neurological problems in each disease. However, the genetic and molecular basis of the overlap in phenotypes between RTT and AS remains largely unknown. Here we present evidence that the phenotypic similarities between the two syndromes might be due to the shared molecular functions between MeCP2 and E6AP in gene expression. Our genetic and biochemical studies suggest that E6AP acts as an essential cofactor for a subset of MeCP2 functions. Specifically, decreased expression of Ube3a was able to rescue the cellular phenotypes induced by MECP2-overexpression in Drosophila. And biochemical assays using mice and cell culture systems show that MeCP2 and E6AP physically interact and regulate the expression of shared target genes. Together these data suggest that MeCP2 and E6AP play a role in the transcriptional control of common target gene expression and provide some insight into why RTT and AS share several neurological phenotypes.
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Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.
J. Cell Biol.
PUBLISHED: 05-20-2013
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The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.
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ELOPER: elongation of paired-end reads as a pre-processing tool for improved de novo genome assembly.
Bioinformatics
PUBLISHED: 04-19-2013
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Paired-end sequencing resulting in gapped short reads is commonly used for de novo genome assembly. Assembly methods use paired-end sequences in a two-step process, first treating each read-end independently, only later invoking the pairing to join the contiguous assemblies (contigs) into gapped scaffolds. Here, we present ELOPER, a pre-processing tool for pair-end sequences that produces a better read library for assembly programs.
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Spatial localization of co-regulated genes exceeds genomic gene clustering in the Saccharomyces cerevisiae genome.
Nucleic Acids Res.
PUBLISHED: 01-08-2013
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While it has been long recognized that genes are not randomly positioned along the genome, the degree to which its 3D structure influences the arrangement of genes has remained elusive. In particular, several lines of evidence suggest that actively transcribed genes are spatially co-localized, forming transcription factories; however, a generalized systematic test has hitherto not been described. Here we reveal transcription factories using a rigorous definition of genomic structure based on Saccharomyces cerevisiae chromosome conformation capture data, coupled with an experimental design controlling for the primary gene order. We develop a data-driven method for the interpolation and the embedding of such datasets and introduce statistics that enable the comparison of the spatial and genomic densities of genes. Combining these, we report evidence that co-regulated genes are clustered in space, beyond their observed clustering in the context of gene order along the genome and show this phenomenon is significant for 64 out of 117 transcription factors. Furthermore, we show that those transcription factors with high spatially co-localized targets are expressed higher than those whose targets are not spatially clustered. Collectively, our results support the notion that, at a given time, the physical density of genes is intimately related to regulatory activity.
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Genome-wide array-based copy number profiling in human placentas from unexplained stillbirths.
Prenat. Diagn.
PUBLISHED: 03-07-2011
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Accumulating evidence suggests that genomic structural variations, particularly copy number variations (CNV), are a common occurrence in humans that may bear phenotypic consequences for living individuals possessing the variant. While precise estimates vary, large-scale karyotypic abnormalities are present in 6-12% of stillbirths (SB). However, due to inherent limitations of conventional cytogenetics, the contribution of genomic aberrations to stillbirth is likely underrepresented. High-resolution copy number variant analysis by genomic array-based profiling may overcome such limitations.
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Large-scale population screening for spinal muscular atrophy: clinical implications.
Genet. Med.
PUBLISHED: 01-15-2011
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To determine the frequency of SMN1 deletion carriers in the Israeli population and to assess the feasibility of population screening for spinal muscular atrophy.
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The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components.
PLoS Genet.
PUBLISHED: 01-11-2011
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Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ? 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.
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Subtle alterations in PCNA-partner interactions severely impair DNA replication and repair.
PLoS Biol.
PUBLISHED: 06-09-2010
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The robustness of complex biological processes in the face of environmental and genetic perturbations is a key biological trait. However, while robustness has been extensively studied, little is known regarding the fragility of biological processes. Here, we have examined the susceptibility of DNA replication and repair processes mediated by the proliferating cell nuclear antigen (PCNA). Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. We found that increases in PCNA-partner interaction affinities led to severe in vivo phenotypic defects. Surprisingly, such defects are much more severe than those induced by complete abolishment of the respective interactions. Thus, the subtle and tunable nature of these affinity perturbations produced different phenotypic effects than realized with traditional "on-off" analysis using gene knockouts. Our findings indicate that biological systems can be robust to one set of perturbations yet fragile to others.
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Making temperature-sensitive mutants.
Meth. Enzymol.
PUBLISHED: 03-01-2010
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The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.
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Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA.
EMBO J.
PUBLISHED: 02-04-2010
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Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.
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Proteasome nuclear activity affects chromosome stability by controlling the turnover of Mms22, a protein important for DNA repair.
PLoS Genet.
PUBLISHED: 01-20-2010
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To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G(2)/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair.
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Mouse models of MeCP2 disorders share gene expression changes in the cerebellum and hypothalamus.
Hum. Mol. Genet.
PUBLISHED: 04-15-2009
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A group of post-natal neurodevelopmental disorders collectively referred to as MeCP2 disorders are caused by aberrations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Loss of MeCP2 function causes Rett syndrome (RTT), whereas increased copy number of the gene causes MECP2 duplication or triplication syndromes. MeCP2 acts as a transcriptional repressor, however the gene expression changes observed in the hypothalamus of MeCP2 disorder mouse models suggest that MeCP2 can also upregulate gene expression, given that the majority of genes are downregulated upon loss of MeCP2 and upregulated in its presence. To determine if this dual role of MeCP2 extends beyond the hypothalamus, we studied gene expression patterns in the cerebellum of Mecp2-null and MECP2-Tg mice, modeling RTT and MECP2 duplication syndrome, respectively. We found that abnormal MeCP2 dosage causes alterations in the expression of hundreds of genes in the cerebellum. The majority of genes were upregulated in MECP2-Tg mice and downregulated in Mecp2-null mice, consistent with a role for MeCP2 as a modulator that can both increase and decrease gene expression. Interestingly, many of the genes altered in the cerebellum, particularly those increased by the presence of MeCP2 and decreased in its absence, were similarly altered in the hypothalamus. Our data suggest that either gain or loss of MeCP2 results in gene expression changes in multiple brain regions and that some of these changes are global. Further delineation of the expression pattern of MeCP2 target genes throughout the brain might identify subsets of genes that are more amenable to manipulation, and can thus be used to modulate some of the disease phenotypes.
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The ELG1 clamp loader plays a role in sister chromatid cohesion.
PLoS ONE
PUBLISHED: 03-03-2009
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Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.
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Coexistence of an unbalanced chromosomal rearrangement and spinal muscular atrophy in an infant with multiple congenital anomalies.
Am. J. Med. Genet. A
PUBLISHED: 02-14-2009
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Unbalanced chromosomal abnormalities are frequent and account for about 10% of all chromosomal abnormalities identified in live births. Diagnosis of a coinherited neuromuscular genetic disorder in these individuals is often challenging based on the severity and variability of the phenotype resulting from the genomic imbalance. Herein, we report on a 4-month-old male with multiple congenital anomalies, craniosynostosis, dysmorphic features, and hypotonia. Karyotype analysis revealed an abnormal male karyotype: 46,XY,der(3)(3;7)(p25;q36), with partial monosomy of 3pter and partial trisomy of 7qter. The targeted array-based comparative genomic hybridization (array-CGH) validated the cytogenetic abnormality, with further elucidation of trisomy of the Sonic Hedgehog (SHH) locus on chromosome 7. Based on the severity of hypotonia in this infant, molecular analysis for spinal muscular atrophy (SMA) was performed and the common homozygous deletion of exon 7 in the survival of motor neuron 1 gene (SMN1) was identified. This case demonstrates the challenges in diagnoses of coexisting genetic disorders in infants with neuromuscular disease. A high index of suspicion in such cases is essential for appropriate case management and family risk assessment.
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Biogenesis of RNA polymerases II and III requires the conserved GPN small GTPases in Saccharomyces cerevisiae.
Genetics
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The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1?, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport.
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Increased rate of missense/in-frame mutations in individuals with NF1-related pulmonary stenosis: a novel genotype-phenotype correlation.
Eur. J. Hum. Genet.
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Neurofibromatosis type 1 (NF1) and its related disorders (NF1-Noonan syndrome (NFNS) and Watson syndrome (WS)) are caused by heterozygous mutations in the NF1 gene. Pulmonary stenosis (PS) occurs more commonly in NF1 and its related disorders than in the general population. This study investigated whether PS is associated with specific types of NF1 gene mutations in NF1, NFNS and WS. The frequency of different NF1 mutation types in a cohort of published and unpublished cases with NF1/NFNS/WS and PS was examined. Compared with NF1 in general, NFNS patients had higher rates of PS (9/35=26% vs 25/2322=1.1%, P value<0.001). Stratification according to mutation type showed that the increased PS rate appears to be driven by the NFNS group with non-truncating mutations. Eight of twelve (66.7%) NFNS cases with non-truncating mutations had PS compared with a 1.1% PS frequency in NF1 in general (P<0.001); there was no increase in the frequency of PS in NFNS patients with truncating mutations. Eight out of eleven (73%) individuals with NF1 and PS, were found to have non-truncating mutations, a much higher frequency than the 19% reported in NF1 cohorts (P<0.015). Only three cases of WS have been published with intragenic mutations, two of three had non-truncating mutations. Therefore, PS in NF1 and its related disorders is clearly associated with non-truncating mutations in the NF1 gene providing a new genotype-phenotype correlation. The data indicate a specific role of non-truncating mutations on the NF1 cardiac phenotype.
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Undetected sex chromosome aneuploidy by chromosomal microarray.
Prenat. Diagn.
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We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.
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When emotion does and does not impair performance: a Garner theory of the emotional Stroop effect.
Cogn Emot
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It takes people longer to name the ink colour of emotion or threat words than that of neutral words, the emotional Stroop effect (ESE). In three experiments with normal and patient populations, we show that the ESE is a special case of a generic attention model and effect entailed in Garners speeded classification paradigm. Guided by the Garner model we demonstrate that task-irrelevant dimensions that differ in salience can produce the ESE and mimic it with neutral stimuli. When each word appears in a constant colour, as mandated in the correlation condition of the Garner design, the ESE is eliminated. This important result is consistent with the attention account of the ESE. We conclude that when emotion stimuli appear in a random fashion they interfere with task performance. However, when emotion stimuli are correlated with features of the ongoing task they help task performance not least due to their extreme salience.
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A founder mutation causing a severe methylenetetrahydrofolate reductase (MTHFR) deficiency in Bukharian Jews.
Mol. Genet. Metab.
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Methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessive disorder. A novel homozygous MTHFR c.474A>T (p.G158G) mutation was detected in two unrelated children of Jewish Bukharian origin. This mutation generates an abnormal splicing and early termination codon. A carrier frequency of 1:39 (5/196) was determined among unrelated healthy Bukharian Jews. Given the disease severity and allele frequency, a population screening for individuals of this ancestry is warranted in order to allow prenatal, or preimplantation diagnosis.
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A genomic bias for genotype-environment interactions in C. elegans.
Mol. Syst. Biol.
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The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C. elegans strains (genotype) cultivated in five growth conditions (environment), and find that highly regulated genes, as distinguished by intergenic lengths, motif concentration, and expression levels, are particularly biased toward genotype-environment interactions. Sequencing these strains, we find that genes with expression variation across genotypes are enriched for promoter single-nucleotide polymorphisms (SNPs), as expected. However, genes with genotype-environment interactions do not significantly differ from background in terms of their promoter SNPs. Collectively, these results indicate that the highly regulated nature of particular genes predispose them for exhibiting genotype-environment interaction as a consequence of changes to upstream regulators. This observation may provide a deeper understanding into the origin of the extraordinary gene expression diversity present in even closely related species.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.