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BACKGROUND: The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single-insertion transgenic lines and the need to complete experiments within a reasonable timeframe. RESULTS: In this article, we introduce a simple competitive dilution strategy for rapid generation of single-insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single-insertion F1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single-insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects. CONCLUSIONS: Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research.
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Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The â¼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.
Assuntos
Duplicação Cromossômica , Cromossomos Humanos Par 10/química , Variações do Número de Cópias de DNA , Fator 8 de Crescimento de Fibroblasto/genética , Deformidades Congênitas das Extremidades Inferiores/genética , Adolescente , Alelos , Animais , Sistemas CRISPR-Cas , Pré-Escolar , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 10/metabolismo , Elementos Facilitadores Genéticos , Família , Feminino , Fêmur/anormalidades , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Edição de Genes , Heterozigoto , Humanos , Lactente , Deformidades Congênitas das Extremidades Inferiores/diagnóstico por imagem , Deformidades Congênitas das Extremidades Inferiores/metabolismo , Deformidades Congênitas das Extremidades Inferiores/patologia , Masculino , Camundongos , Camundongos Transgênicos , Linhagem , FenótipoRESUMO
OBJECTIVES: The present study examined if disruption of serial position effects in list recall could serve as an early marker of Alzheimer's disease (AD) in Spanish-English bilinguals. METHODS: We tested 20 participants initially diagnosed as cognitively normal or with mild cognitive impairment who declined and eventually received a diagnosis of AD (decliners), and 37 who remained cognitively stable (controls) over at least 2 years. Participants were tested on the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List Learning Test in English or Spanish as part of an annual neuropsychological evaluation. RESULTS: Compared to controls, decliners exhibited significantly reduced recall including reduced primacy scores (i.e., items recalled from the first three list items on Trial 1), whereas recency scores (i.e., items recalled from the last 3 list items on Trial 1) were equivalent in decliners and controls. Further analyses suggested that the sensitivity of the primacy effect to preclinical AD was initially stronger in participants tested in Spanish, a surprising finding given that the CERAD was developed for English speakers. However, in the subsequent year of testing, primacy scores declined to the same level regardless of language of testing. CONCLUSIONS: Several list learning measures may facilitate early diagnosis of AD in Spanish-English bilinguals, possibly including the relatively understudied primacy effect. Additional studies are needed to investigate the possibility that linguistic or demographic variables might modulate sensitivity of list learning tests to preclinical AD, which could lead to broader improvements in their utility for early diagnosis of AD in all populations.
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Doença de Alzheimer , Disfunção Cognitiva , Humanos , Doença de Alzheimer/diagnóstico , Idioma , Linguística , Disfunção Cognitiva/diagnóstico , AprendizagemRESUMO
The question of how features are bound together in working memory has become a topic of much research in recent years. However, this is typically focused on visual and/or auditory stimuli. The purpose of this study is to apply established feature binding procedures to investigate odour binding in working memory. Across three experiments, memory for intentionally and incidentally formed odour-colour pairings was tested. Experiment 1 showed that following explicit instruction to remember the odour-colour combinations, young adults can recall lists of 3-pairings at levels above that of chance and exhibit a recency advantage for the last pairing. In Experiment 2 participants were asked to prioritise the first pairing in the list or treat all pairings equally. We observed only limited evidence of prioritisation affecting the serial position function. Experiment 3 explored incidental odour-colour binding. Using a yes/no recognition procedure, accuracy did not differ for positive test probes presented in the same (bound) or different (unbound) colour to encoding. This study is one of the first to examine odour-colour binding in working memory and, taking the evidence together, suggests that odour-colour bindings can be formed in working memory; however, functionality may be limited compared to that of visual feature binding.
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Memória de Curto Prazo , Odorantes , Adulto Jovem , Humanos , Cor , Rememoração Mental , Reconhecimento PsicológicoRESUMO
The forward testing effect (FTE) refers to the finding that retrieval practice of previously studied material can facilitate recall of newly studied (critical) material. Such interim retrieval practice can also lead to a differential FTE, i.e., a more pronounced FTE for items at early than later serial positions in the critical material. The present study examined whether this differential FTE also holds with interim semantic generation of extra-list items, and whether it is influenced by study material. Consistent with prior work, the results of two experiments showed that both interim retrieval practice and interim semantic generation induce the general (list-level) FTE when unrelated study lists are applied, whereas retrieval practice only creates the effect with categorised study lists. Critically, however, the differential FTE was present in response to retrieval practice but absent in response to semantic generation. This pattern held regardless of which material was studied, thus experimentally dissociating the general (list-level) from the differential (item-level) FTE. The findings may suggest that retrieval practice, but not semantic generation, induces a reset of the encoding process which promotes attentional encoding such that a more pronounced FTE arises for early than middle and late serial positions in the critical list.
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Rememoração Mental , Semântica , Humanos , Rememoração Mental/fisiologia , AtençãoRESUMO
The activity of a gene newly integrated into a chromosome depends on the genomic context of the integration site. This "position effect" has been widely reported, although the other side of the coin, that is, how integration affects the local chromosomal environment, has remained largely unexplored, as have the mechanism and phenotypic consequences of this "externality" of the position effect. Here, we examined the transcriptome profiles of approximately 250 Saccharomyces cerevisiae strains, each with GFP integrated into a different locus of the wild-type strain. We found that in genomic regions enriched in essential genes, GFP expression tended to be lower, and the genes near the integration site tended to show greater expression reduction. Further joint analysis with public genome-wide histone modification profiles indicated that this effect was associated with H3K4me2. More importantly, we found that changes in the expression of neighboring genes, but not GFP expression, significantly altered the cellular growth rate. As a result, genomic loci that showed high GFP expression immediately after integration were associated with growth disadvantages caused by elevated expression of neighboring genes, ultimately leading to a low total yield of GFP in the long run. Our results were consistent with competition for transcriptional resources among neighboring genes and revealed a previously unappreciated facet of position effects. This study highlights the impact of position effects on the fate of exogenous gene integration and has significant implications for biological engineering and the pathology of viral integration into the host genome.
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Efeitos da Posição Cromossômica , Mutagênese Insercional , Transcriptoma , Aptidão Genética , Código das Histonas , Saccharomyces cerevisiaeRESUMO
Serial position effects involve the differential recall of information based on its temporal order at encoding. Previous research indicates that learners may be aware of these effects under certain encoding conditions, but it is unclear whether metacognitive control is sensitive to serial position effects. The current study examined whether there are serial position effects in participants' study time and whether they can learn about serial position effects under fixed encoding conditions and then transfer what they have learned to self-paced study conditions. Specifically, participants were given lists of to-be-remembered words and studied each word for a fixed duration on initial lists, but self-paced their study time on later lists. Results revealed that self-paced study times oppositely mirrored serial position effects (i.e., briefer study times in the beginning and end of each list), and serial position effects were reduced in self-paced study conditions, particularly in participants initially studying under fixed conditions before self-pacing their study time. Specifically, participants may have monitored their output and, based on observations of forgetting middle items, transferred their learning of serial position effects from prior lists. Thus, participants may use forgetting and serial position information to guide encoding, indicating that fundamental properties of the memory system can be incorporated into the processes that guide metacognitive control.
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Metacognição , Humanos , Aprendizagem , Rememoração Mental , Aprendizagem Seriada , TempoRESUMO
In four experiments pupillary responses were used to examine attention allocation and encoding dynamics in free recall. In Experiment 1, pupillary responses increased (and then decreased) across serial position suggesting that attention was increasingly allocated to items during learning until working memory was overloaded. In Experiment 2, manipulating presentation duration resulted in larger and more sustained pupillary responses with increased presentation duration, suggesting that participants were likely engaging in more elaborative and attention-demanding processes. In Experiment 3a, manipulating list-length resulted in decreased pupillary responses across serial position suggesting that participants were prioritizing early list items and less attention was allocated to later items. In Experiment 3b, when list-length was known, pupillary responses in the long-list length condition tended to decrease across serial position whereas pupillary responses in the short list-length condition tended to increase and decrease across serial positon. These results suggest that participants flexibly allocate attention to items during encoding depending on the nature of the task and the types of processes that are engaged in. These results further suggest the potential of utilizing pupillary responses to track attention allocation during learning.
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Rememoração Mental , Humanos , Aprendizagem , Memória de Curto PrazoRESUMO
Models of free recall describe free recall initiation as a decision-making process in which items compete to be retrieved. Recently, Osth and Farrell (Psychological Review, 126, 578-609, 2019) applied evidence accumulation models to complete RT distributions and serial positions of participants' first recalls in free recall, which resulted in some novel conclusions about primacy and recency effects. Specifically, the results of the modeling favored an account in which primacy was due to reinstatement of the start-of-the-list, and recency was found to be exponential in shape. In this work, we examine what happens when participants are given alternative recall instructions. Prior work has demonstrated weaker primacy and greater recency when fewer items are required to report (Ward & Tan, Memory & Cognition, 2019), and a key question is whether this change in instructions qualitatively changes the nature of the recall process, or merely changes the parameters of the recall competition. We conducted an experiment where participants studied six- or 12-item lists and were post-cued as to whether to retrieve a single item, or as many items as possible. Subsequently, we applied LBA models with various assumptions about primacy and recency, implemented using hierarchical Bayesian techniques. While greater recency was observed when only one item was required for output, the model selection did not suggest that there were qualitative differences between the two conditions. Specifically, start-of-list reinstatement and exponential recency functions were favored in both conditions.
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Rememoração Mental , Teorema de Bayes , Cognição , Sinais (Psicologia) , Humanos , Aprendizagem SeriadaRESUMO
ABSTRACTSerial position effects are often observed within the free recall of unassociated words but also when recalling items from a semantic category like U.S. presidents. We investigated the dynamics of recall for U.S. presidents in younger and older adults to examine potential age-related differences in the organisation of retrieval from semantic long-term memory. Older adults recalled more presidents than younger adults and also demonstrated dual serial position effects such that, in addition to overall serial position effects, primacy (e.g., Eisenhower) and recency presidents (e.g., Obama) within older adults' lifetime were better recalled than presidents from the middle of their lives (e.g., Ford). Additionally, participants initiated recall with the most distinct presidents (highly familiar or memorable presidents like Washington, Obama, Trump), and conditional-response probabilities revealed that presidents from similar eras were recalled in close proximity, indicating that the retrieval of distinct presidents can facilitate memory for presidents from a similar era. Collectively, we demonstrate the potential interplay of the mechanisms that influence the organisation of retrieval such that distinctiveness and temporal contiguity effects may simultaneously impact recall. Specifically, semantic and temporal-contextual associations can drive semantic autobiographical memory and people likely organise retrieval from long-term memory according to familiarity and distinctiveness.
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Memória Episódica , Rememoração Mental , Idoso , Humanos , Memória de Longo Prazo , Rememoração Mental/fisiologia , Reconhecimento Psicológico , Semântica , Estados UnidosRESUMO
Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.
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Catarata/congênito , Catarata/patologia , Pontos de Quebra do Cromossomo , Inversão Cromossômica , Cromossomos Humanos X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Proteínas de Membrana/genética , Anormalidades Dentárias/patologia , Catarata/etiologia , Catarata/metabolismo , Criança , Mapeamento Cromossômico , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/etiologia , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Masculino , Linhagem , Anormalidades Dentárias/etiologia , Anormalidades Dentárias/metabolismoRESUMO
OBJECTIVE: The first (primacy region) and last (recency region) items of a word list are generally better memorized than items from the middle region. The recency effect depends on short-term memory (STM) and the primacy effect on long-term memory (LTM), where verbal information is transferred from STM into LTM by maintenance rehearsal. We compared the serial position effects (SPE) between patients with mild cognitive impairment (MCI) due to Parkinson's disease (PD), i.e., PD-MCI, and patients with MCI due to Alzheimer's disease (AD-MCI), and evaluated the influence of SPE and frontostriatal deficits on verbal memory recall. METHODS: Four similar groups of subjects participated in the study: 26 PD-MCI patients, 26 cognitively normal patients with PD (PD-CN), 26 AD-MCI patients, and 26 normal controls (NC). Verbal episodic memory, verbal span, attentional capacity, executive functions, and verbal working memory performance were assessed. Measures for primacy and recency regions were defined at the first trial of a 16-items word list. Hierarchical regression models were used to investigate the contribution of frontostriatal deficits beyond SPE on verbal memory recall performance ("long-delay free recall") in PD and AD patients. RESULTS: Primacy effects were significantly diminished in both PD-MCI and AD-MCI patients relative to NC and PD-CN (all p < 0.01). Compared to PD-MCI patients, AD-MCI patients exhibited significantly worse "delayed-recall 'savings'." Reduced primacy effect was predictive for decreased recall performance in PD and AD. The conducted hierarchical regression model revealed that in PD, but not in AD patients, performance of attention and executive function significantly increased the prediction of free recalled words. CONCLUSIONS: Reduced recall performance is likely due to impaired transition of newly learned material from STM into LTM in AD and in PD. Whereas AD-MCI patients suffer from a storage deficit, the similarly reduced recall performance found in patients with PD-MCI may additionally be related to deficient attentional and executive capacity.
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Doença de Alzheimer/psicologia , Disfunção Cognitiva/psicologia , Rememoração Mental , Doença de Parkinson/psicologia , Idoso , Atenção , Função Executiva , Feminino , Humanos , Masculino , Memória de Curto Prazo , Testes NeuropsicológicosRESUMO
Cowan, Donnell, and Saults [(2013). A list-length constraint on incidental item-to-item associations. Psychonomic Bulletin & Review, 20, 1253-1258] examined incidental memory of whether two words had occurred in the same list or different lists, after the lists had been presented with an orienting task that did not require memorisation. Performance was superior for 3-word lists compared to 6- or 9-word lists, with memory for the longer lists near chance levels. Here we re-examine this phenomenon with methodological modifications to ensure that learning was incidental: we removed potential clues that a memory test would follow, eliminated trials with special mnemonic cues related to the orienting task, eliminated participants who suspected a memory test according to a post-experimental questionnaire, used signal detection measures to distinguish between memory sensitivity and bias, and tested list length with the relative serial position controlled. Incidental memory formed primarily for the most recent part of each list, an effect that was stronger than that of list length. The new evidence helps to constrain theories about the relation between working memory and incidental learning. A capacity-limited approach to the incidental-learning process still is possible but must be modified compared to Cowan et al., and the evidence is favourable to other theoretical approaches as well.
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Memória de Curto Prazo , Aprendizagem Seriada , Atenção , Feminino , Humanos , MasculinoRESUMO
The activity of any bacterial promoter is generally supposed to be set by its base sequence and the different transcription factors that bind in the local vicinity. Here, we review recent data indicating that the activity of the Escherichia coli lac operon promoter also depends upon its chromosomal location. Factors that affect promoter activity include the binding of nucleoid-associated proteins to neighbouring sequences, supercoiling and the activity of neighbouring promoters. We suggest that many bacterial promoters might be susceptible to similar position-dependent effects and we review recent data showing that the expression of mobile genes encoding antibiotic-resistance determinants is also location-dependent, both when carried on a bacterial chromosome or a conjugative plasmid.
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Efeitos da Posição Cromossômica , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , Cromossomos Bacterianos , Elementos de DNA Transponíveis , Óperon Lac , Plasmídeos , Transcrição GênicaRESUMO
Evidence for false recognition within seconds of encoding suggests that semantic-associative influences are not restricted to long-term memory, consistent with unitary memory accounts but contrary to dual store models. The present study sought further relevant evidence using a modified free recall converging associates task where participants studied 12-item lists composed of 3 semantically distinct quartets (sublists) related to a separate, non-presented theme word (i.e., words 1-4/theme1, 5-8/theme2, and 9-12/theme3). This list construction permits assessment of false recall errors from each sublist, and, particularly, the primacy and recency sublists that have been linked to long- and short-term memory stores. Experiment 1 tested immediate free recall for items. Associative false memories were evident from all sublists, however, significantly less so from the recent sublist, which also showed the highest levels of veridical memory. By inserting a brief (3â s) distractor prior to recall, Experiment 2 selectively reduced veridical memory and increased false memory for the recent sublist while leaving the primacy sublist unaffected. These recall results converge with prior evidence indicating the immediacy of false recognition, and can be understood within a unitary framework where the differential availability of verbatim features and gist-based cues affect memory for primacy and recency sublists.
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Memória de Curto Prazo , Rememoração Mental/fisiologia , Semântica , Adulto , Feminino , Humanos , Masculino , Reconhecimento Psicológico , Adulto JovemRESUMO
Although articulatory suppression abolishes the effect of irrelevant sound (ISE) on serial recall when sequences are presented visually, the effect persists with auditory presentation of list items. Two experiments were designed to test the claim that, when articulation is suppressed, the effect of irrelevant sound on the retention of auditory lists resembles a suffix effect. A suffix is a spoken word that immediately follows the final item in a list. Even though participants are told to ignore it, the suffix impairs serial recall of auditory lists. In Experiment 1, the irrelevant sound consisted of instrumental music. The music generated a significant ISE that was abolished by articulatory suppression. It therefore appears that, when articulation is suppressed, irrelevant sound must contain speech for it to have any effect on recall. This is consistent with what is known about the suffix effect. In Experiment 2, the effect of irrelevant sound under articulatory suppression was greater when the irrelevant sound was spoken by the same voice that presented the list items. This outcome is again consistent with the known characteristics of the suffix effect. It therefore appears that, when rehearsal is suppressed, irrelevant sound disrupts the acoustic-perceptual encoding of auditorily presented list items. There is no evidence that the persistence of the ISE under suppression is a result of interference to the representation of list items in a postcategorical phonological store.
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Percepção Auditiva/fisiologia , Memória de Curto Prazo/fisiologia , Rememoração Mental/fisiologia , Psicolinguística , Adulto , Feminino , Humanos , Masculino , Percepção da Fala/fisiologia , Adulto JovemRESUMO
Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.
Assuntos
Efeitos da Posição Cromossômica/genética , Marcação de Genes , Mutagênese Insercional/genética , Transgenes/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Encéfalo/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Loci Gênicos/genética , Genoma/genética , Integrases/metabolismo , Cristalino/metabolismo , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Xenopus laevis/genéticaRESUMO
The present paper examines the effect of within-sequence item repetitions in tactile order memory. Employing an immediate serial recall procedure, participants reconstructed a six-item sequence tapped upon their fingers by moving those fingers in the order of original stimulation. In Experiment 1a, within-sequence repetition of an item separated by two-intervening items resulted in a significant reduction in recall accuracy for that repeated item (i.e., the Ranschburg effect). In Experiment 1b, within-sequence repetition of an adjacent item resulted in significant recall facilitation for that repeated item. These effects mirror those reported for verbal stimuli (e.g., Henson, 1998a . Item repetition in short-term memory: Ranschburg repeated. Journal of Experimental Psychology: Learning, Memory, and Cognition, 24(5), 1162-1181. doi:doi.org/10.1037/0278-7393.24.5.1162). These data are the first to demonstrate the Ranschburg effect with non-verbal stimuli and suggest further cross-modal similarities in order memory.
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Memória/fisiologia , Percepção do Tato/fisiologia , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Aprendizagem Seriada/fisiologia , Aprendizagem Verbal/fisiologia , Adulto JovemRESUMO
Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.
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Proteínas de Choque Térmico HSP70/genética , Integrases/genética , Oryzias/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Oryzias/metabolismo , Regiões Promotoras Genéticas , Transgenes/genética , Peixe-Zebra/genéticaRESUMO
DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.