RESUMO
Alterations of human karyotype caused by chromosomal rearrangements are often associated with considerable phenotypic effects. Studying molecular mechanisms underlying these effects requires an efficient and scalable experimental model. Here, we propose a Cre-LoxP-based approach for the generation of combinatorial diversity of chromosomal rearrangements. We demonstrate that using the developed system, both intra- and inter-chromosomal rearrangements can be induced in the human haploid HAP1 cells, although the latter is significantly less effective. The obtained genetically modified HAP1 cell line can be used to dissect genomic effects associated with intra-chromosomal structural variations.
Assuntos
Cromossomos , Rearranjo Gênico , Recombinação Genética , Humanos , Cromossomos/genética , Cromossomos/metabolismo , Rearranjo Gênico/genética , Rearranjo Gênico/fisiologia , Integrases/genética , Integrases/metabolismo , Recombinação Genética/genética , Recombinação Genética/fisiologia , Linhagem CelularRESUMO
BACKGROUND: Our understanding of plastid transcriptomes is limited to a few model plants whose plastid genomes (plastomes) have a highly conserved gene order. Consequently, little is known about how gene expression changes in response to genomic rearrangements in plastids. This is particularly important in the highly rearranged conifer plastomes. RESULTS: We sequenced and reported the plastomes and plastid transcriptomes of six conifer species, representing all six extant families. Strand-specific RNAseq data show a nearly full transcription of both plastomic strands and detect C-to-U RNA-editing sites at both sense and antisense transcripts. We demonstrate that the expression of plastid coding genes is strongly functionally dependent among conifer species. However, the strength of this association declines as the number of plastomic rearrangements increases. This finding indicates that plastomic rearrangement influences gene expression. CONCLUSIONS: Our data provide the first line of evidence that plastomic rearrangements not only complicate the plastomic architecture but also drive the dynamics of plastid transcriptomes in conifers.
Assuntos
Evolução Molecular , Rearranjo Gênico/fisiologia , Genomas de Plastídeos , Traqueófitas/genética , Traqueófitas/fisiologia , Regulação da Expressão Gênica de Plantas , FilogeniaRESUMO
Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Telômero/fisiologia , Ustilago/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico/fisiologia , Rad51 Recombinase/genética , RecQ Helicases/genética , Recombinação Genética/genética , Recombinação Genética/fisiologia , Telomerase/metabolismo , Telômero/metabolismo , Ustilago/metabolismoRESUMO
Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.
Assuntos
Cromossomos/metabolismo , Rearranjo Gênico/fisiologia , Genoma de Protozoário , Oxytricha/genética , RNA não Traduzido/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Cromossomos/genética , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Microinjeções , RNA de Protozoário/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/genética , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND: The inference of genome rearrangement operations requires complete genome assemblies as input data, since a rearrangement can involve an arbitrarily large proportion of one or more chromosomes. Most genome sequence projects, especially those on non-model organisms for which no physical map exists, produce very fragmented assembles, so that a rearranged fragment may be impossible to identify because its two endpoints are on different scaffolds. However, breakpoints are easily identified, as long as they do not coincide with scaffold ends. For the phylogenetic context, in comparing a fragmented assembly with a number of complete assemblies, certain combinatorial constraints on breakpoints can be derived. We ask to what extent we can use breakpoint data between a fragmented genome and a number of complete genomes to recover all the arrangements in a phylogeny. RESULTS: We simulate genomic evolution via chromosomal inversion, fragmenting one of the genomes into a large number of scaffolds to represent the incompleteness of assembly. We identify all the breakpoints between this genome and the remainder. We devise an algorithm which takes these breakpoints into account in trying to determine on which branch of the phylogeny a rearrangement event occurred. We present an analysis of the dependence of recovery rates on scaffold size and rearrangement rate, and show that the true tree, the one on which the rearrangement simulation was performed, tends to be most parsimonious in estimating the number of true events inferred. CONCLUSIONS: It is somewhat surprising that the breakpoints identified just between the fragmented genome and each of the others suffice to recover most of the rearrangements produced by the simulations. This holds even in parts of the phylogeny disjoint from the lineage of the fragmented genome.
Assuntos
Algoritmos , Classificação/métodos , Rearranjo Gênico/fisiologia , Genoma , Filogenia , Plantas/classificação , Plantas/genéticaRESUMO
B-cell lymphoma 11A (BCL11A) downregulation in human primary adult erythroid progenitors results in elevated expression of fetal γ-globin. Recent reports showed that BCL11A expression is activated by KLF1, leading to γ-globin repression. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11A in an in vivo model, we used mice carrying a human ß-globin locus transgene with combinations of Klf1 knockout, Bcl11a floxed, and EpoR(Cre) knockin alleles. We found a higher percentage of reticulocytes in adult Klf1(wt/ko) mice and a mild compensated anemia in Bcl11a(cko/cko) mice. These phenotypes were more pronounced in compound Klf1(wt/ko)::Bcl11a(cko/cko) mice. Analysis of Klf1(wt/ko), Bcl11a(cko/cko), and Klf1(wt/ko)::Bcl11a(cko/cko) mutant embryos demonstrated increased expression of mouse embryonic globins during fetal development. Expression of human γ-globin remained high in Bcl11a(cko/cko) embryos during fetal development, and this was further augmented in Klf1(wt/ko)::Bcl11a(cko/cko) embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11a(cko/cko) and Klf1(wt/ko)::Bcl11a(cko/cko) mice, but the levels remained much higher than those observed in control animals. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression.
Assuntos
Proteínas de Transporte/genética , Eritropoese/genética , Genes de Troca/genética , Globinas/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas Nucleares/genética , Animais , Proteínas de Ligação a DNA , Embrião de Mamíferos , Eritropoese/fisiologia , Desenvolvimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico/genética , Rearranjo Gênico/fisiologia , Genes de Troca/fisiologia , Humanos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Repressoras , Reticulocitose/genética , Reticulocitose/fisiologia , Baço/citologia , Baço/embriologia , Baço/metabolismoRESUMO
AIMS: We examined gene rearrangement and the expression of anaplastic lymphoma kinase (ALK) in urinary bladder inflammatory myofibroblastic tumour (IMT) using fluorescence in-situ hybridization (FISH) and two immunohistochemical antibodies to ALK. We also investigated whether IMT represents an immunoglobulin (Ig)G4-related disease. METHODS AND RESULTS: The performance of the Dako FLEX ALK monoclonal antibody (CD246) and the Cell Signaling Technology ALK (D5F3) XP monoclonal antibody were compared. Overall, 11 of 16 tumours showed ALK expression by immunohistochemistry (69%). Ten demonstrated ALK expression with both stains and one was positive with D5F3 but not CD246 (91% correlation). The D5F3 antibody yielded a stronger staining intensity and a higher sensitivity. Nine tumours demonstrated ALK rearrangements (56%) by FISH. Three were ALK(+) by immunohistochemistry but negative for rearrangement by FISH, whereas one showed rearrangement by FISH but was negative by immunohistochemistry. In total, 12 tumours were positive for ALK abnormalities (75%). Using current criteria, no cases were classified as an IgG4-related disease. CONCLUSIONS: The ALK D5F3 immunohistochemical stain showed superior staining characteristics compared with ALK CD246. Discrepancies in the results between FISH and immunohistochemistry for ALK abnormalities may have causes that are multifactorial. By current criteria, IMT does not represent an IgG4-related disease.
Assuntos
Anticorpos Monoclonais/imunologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Imunoglobulina G/fisiologia , Hibridização in Situ Fluorescente , Miofibroma/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Bexiga Urinária/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Criança , Pré-Escolar , Feminino , Rearranjo Gênico/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Miofibroma/imunologia , Miofibroma/patologia , Receptores Proteína Tirosina Quinases/imunologia , Estudos Retrospectivos , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia , Adulto JovemRESUMO
PURPOSE OF REVIEW: Krüppel-like factor 1 (KLF1) regulates most aspects of erythropoiesis. Many years ago, transgenic mouse studies implicated KLF1 in the control of the human γ-globin to ß-globin switch. In this review, we will integrate these initial studies with recent developments in human genetics to discuss our present understanding of how KLF1 and its target genes direct the switch. RECENT FINDINGS: Recent studies have shown that human mutations in KLF1 are common and mostly asymptomatic, but lead to significant increases in levels of fetal hemoglobin (HbF) (α2γ2) and adult HbA2 (α2δ2). Genome-wide association studies (GWAS) have demonstrated that three primary loci are associated with increased HbF levels in the population: the ß-globin locus itself, the BCL11A locus, and a site between MYB and HBS1L. We discuss evidence that KLF1 directly regulates BCL11A, MYB and other genes, which are involved directly or indirectly in γ-globin silencing, thus providing a link between GWAS and KLF1 in hemoglobin switching. SUMMARY: KLF1 regulates the γ-globin to ß-globin genetic switch by many mechanisms. Firstly, it facilitates formation of an active chromatin hub (ACH) at the ß-globin gene cluster. Specifically, KLF1 conscripts the adult-stage ß-globin gene to replace the γ-globin gene within the ACH in a stage-specific manner. Secondly, KLF1 acts as a direct activator of genes that encode repressors of γ-globin gene expression. Finally, KLF1 is a regulator of many components of the cell cycle machinery. We suggest that dysregulation of these genes leads to cell cycle perturbation and 'erythropoietic stress' leading to indirect upregulation of HbF.
Assuntos
Genes de Troca/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Globinas beta/genética , gama-Globinas/genética , Eritropoese/genética , Eritropoese/fisiologia , Regulação da Expressão Gênica , Rearranjo Gênico/genética , Rearranjo Gênico/fisiologia , Genes de Troca/fisiologia , Estudo de Associação Genômica Ampla , HumanosRESUMO
The origin of antibody diversity has intrigued scientists for nearly a century. We now know that the diversity is achieved through a 2-stage process. Gene rearrangement (catalyzed by the RAG1/2 recombinase) allows the production of a primary repertoire of antibodies; targeted deamination of cytosines within these rearranged antibody genes (catalyzed by the DNA deaminase AID) then allows them to be further diversified and matured by somatic hypermutation, gene conversion, and class-switch recombination. Here we review the history of the uncovering of some of these processes, contrasting the relative importance of hypothesis and methodological developments in driving the research at different periods of the work.
Assuntos
Anticorpos/genética , Diversidade de Anticorpos/imunologia , Animais , Linfócitos B/imunologia , Citidina Desaminase/metabolismo , Rearranjo Gênico/fisiologia , Genes de Imunoglobulinas , Proteínas de Homeodomínio/fisiologia , Humanos , Imunoglobulinas/genética , Modelos Imunológicos , Hipermutação Somática de Imunoglobulina/fisiologiaRESUMO
Genomic disorders are a group of human genetic diseases caused by genomic rearrangements resulting in copy-number variation (CNV) affecting a dosage-sensitive gene or genes critical for normal development or maintenance. These disorders represent a wide range of clinically distinct entities but include many diseases affecting nervous system function. Herein, we review selected neurodevelopmental, neurodegenerative, and psychiatric disorders either known or suggested to be caused by genomic rearrangement and CNV. Further, we emphasize the cause-and-effect relationship between gene CNV and complex disease traits. We also discuss the prevalence and heritability of CNV, the correlation between CNV and higher-order genome architecture, and the heritability of personality, behavioral, and psychiatric traits. We speculate that CNV could underlie a significant proportion of normal human variation including differences in cognitive, behavioral, and psychological features.
Assuntos
Dosagem de Genes/fisiologia , Rearranjo Gênico/fisiologia , Genômica , Doenças do Sistema Nervoso/genética , Animais , Aberrações Cromossômicas , Transtornos Cromossômicos/complicações , Cromossomos Humanos , Modelos Animais de Doenças , Humanos , Doenças do Sistema Nervoso/etiologiaRESUMO
BACKGROUND: Chromosomal rearrangements resulting in an interstitial inverted duplication with concomitant terminal deletion were first described for the short arm of chromosome 8 in 1976. Since then, this type of alteration has been identified and characterised for most chromosome arms. Three mechanisms are commonly proposed to explain the origin of this type of rearrangement. All three mechanisms involve formation of a dicentric chromosome that then breaks in a subsequent meiotic division to produce a monocentric duplicated and deleted chromosome. However, the events leading to the formation of the dicentric chromosome differ between the mechanisms. In one mechanism, either parent carries a paracentric inversion. This results in formation of a loop during meiotic pairing with a recombination event occurring in the loop. In the second mechanism, inverted low copy repeats in the same chromosome arm allow partial folding of one homologue onto itself with a recombination event between the inverted repeats. The third mechanism involves a pre-meiotic double-strand break with subsequent fusion, or U-type exchange, between the sister chromatids. The first two mechanisms require a single copy region to exist between the duplicated and deleted regions on the derivative chromosome, and therefore high resolution analysis of the rearrangement can be used to distinguish between these mechanisms. METHODS AND RESULTS: Using G-banded chromosome analysis, fluorescence in situ hybridisation (FISH) and array comparative genomic hybridisation (CGH), we describe 17 new cases of inverted duplication with terminal deletion of 2q, 4p, 5p, 6q, 8p, 9p, 10q, 13q, 15q, 18p, 18q, and 22q. CONCLUSIONS: These new cases, combined with previously described cases, demonstrate that U-type exchange is the most frequent mechanism for this rearrangement and can be observed on most, or perhaps all, chromosome arms.
Assuntos
Deleção de Genes , Duplicação Gênica , Rearranjo Gênico/fisiologia , Troca de Cromátide Irmã/fisiologia , Bandeamento Cromossômico , Cromossomos Humanos , Hibridização Genômica Comparativa , Humanos , Hibridização in Situ FluorescenteRESUMO
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.
Assuntos
Cromossomos Artificiais Humanos/metabolismo , Rearranjo Gênico/fisiologia , Linhagem Celular Tumoral , Centrômero/metabolismo , Proteína B de Centrômero/genética , Instabilidade Cromossômica , Epigênese Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , HumanosRESUMO
Double-strand breaks (DSBs) represent the most severe type of DNA damage since they can lead to genomic rearrangements, events that can initiate and promote tumorigenic processes. DSBs arise from various exogenous agents that induce two single-strand breaks at opposite locations in the DNA double helix. Such two-ended DSBs are repaired in mammalian cells by one of two conceptually different processes, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ has the potential to form rearrangements while HR is believed to be error-free since it uses a homologous template for repair. DSBs can also arise from single-stranded DNA lesions if they lead to replication fork collapse. Such DSBs, however, have only one end and are repaired by HR and not by NHEJ. In fact, the majority of spontaneously arising DSBs are one-ended and HR has likely evolved to repair one-ended DSBs. HR of such DSBs demands the engagement of a second break end that is generated by an approaching replication fork. This HR process can cause rearrangements if a homologous template other than the sister chromatid is used. Thus, both NHEJ and HR have the potential to form rearrangements and the proper choice between them is governed by various factors, including cell cycle phase and genomic location of the lesion. We propose that the specific requirements for repairing one-ended DSBs have shaped HR in a way which makes NHEJ the better choice for the repair of some but not all two-ended DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Rearranjo Gênico/fisiologia , Recombinação Homóloga/fisiologia , Quebras de DNA de Cadeia Simples , Fase G1/genética , Fase G2/genética , HumanosRESUMO
Heterozygous chromosomal rearrangements can result in failures during the meiotic cycle and the apoptosis of germline, making carrier individuals infertile. The Amazon frog Leptodactylus pentadactylus has a meiotic multivalent, composed of 12 sex chromosomes. The mechanisms by which this multi-chromosome system maintains fertility in males of this species remain undetermined. In this study we investigated the meiotic behavior of this multivalent to understand how synapse, recombination and epigenetic modifications contribute to maintaining fertility and chromosomal sexual determination in this species. Our sample had 2n = 22, with a ring formed by ten chromosomes in meiosis, indicating a new system of sex determination for this species (X1Y1X2Y2X3Y3X4Y4X5Y5). Synapsis occurs in the homologous terminal portion of the chromosomes, while part of the heterologous interstitial regions performed synaptic adjustment. The multivalent center remains asynaptic until the end of pachytene, with interlocks, gaps and rich-chromatin in histone H2A phosphorylation at serine 139 (γH2AX), suggesting transcriptional silence. In late pachytene, paired regions show repair of double strand-breaks (DSBs) with RAD51 homolog 1 (Rad51). These findings suggest that Rad51 persistence creates positive feedback at the pachytene checkpoint, allowing meiosis I to progress normally. Additionally, histone H3 trimethylation at lysine 27 in the pericentromeric heterochromatin of this anuran can suppress recombination in this region, preventing failed chromosomal segregation. Taken together, these results indicate that these meiotic adaptations are required for maintenance of fertility in L. pentadactylus.
Assuntos
Anuros/genética , Animais , Anuros/fisiologia , Análise Citogenética , Epigênese Genética/genética , Epigênese Genética/fisiologia , Fertilidade/genética , Fertilidade/fisiologia , Rearranjo Gênico/genética , Rearranjo Gênico/fisiologia , Masculino , Meiose , Cromossomos Sexuais/genéticaRESUMO
99 DNA samples of organs and tissues of 18 mice were examined using the method of PCR amplification with random primers. Among 27 oligonucleotide primers tested, 4 producing stable, well-reproducible profiles of amplification products were chosen for further analysis. Using 2 of these primers we detected differences in RAPD-profiles in some tissues in several individuals. These differences were associated with the modification of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors may also contribute to this process.
Assuntos
Rearranjo Gênico/fisiologia , Genoma/fisiologia , Mosaicismo , Técnica de Amplificação ao Acaso de DNA Polimórfico , Animais , Primers do DNA/genética , Camundongos , Camundongos Endogâmicos CBA , Especificidade de Órgãos/fisiologia , Especificidade da EspécieRESUMO
Among the various genes that can be rearranged in soft tissue neoplasms associated with nonrandom chromosomal translocations, EWSR1 is the most frequent one to partner with other genes to generate recurrent fusion genes. This leads to a spectrum of clinically and pathologically diverse mesenchymal and nonmesenchymal neoplasms, variably manifesting as small round cell, spindle cell, clear cell or adipocytic tumors, or tumors with distinctive myxoid stroma. This review summarizes the growing list of mesenchymal neoplasms that are associated with EWSR1 gene rearrangements.
Assuntos
Mesenquimoma/genética , Mesenquimoma/patologia , Proteína EWS de Ligação a RNA/genética , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Biomarcadores Tumorais/genética , Rearranjo Gênico/fisiologia , Humanos , Mesenquimoma/classificação , Proteínas de Fusão Oncogênica/genética , Neoplasias de Tecidos Moles/classificação , Translocação GenéticaRESUMO
BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.
Assuntos
Epigênese Genética/fisiologia , Deleção de Genes , Rearranjo Gênico/fisiologia , Macronúcleo/metabolismo , Micronúcleo Germinativo/metabolismo , Paramecium tetraurellia/fisiologia , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Epigênese Genética/genética , Rearranjo Gênico/genética , Proteínas de Fluorescência Verde , Macronúcleo/genética , Micronúcleo Germinativo/genética , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Oligonucleotídeos , Paramecium tetraurellia/genética , Paramecium tetraurellia/metabolismo , Interferência de RNA , Reprodução/genéticaRESUMO
The stability of metazoan genomes during their duplication depends on the spatiotemporal activation of origins and the progression of forks. Human rRNA genes represent a unique challenge to DNA replication since a large proportion of them exist as noncanonical palindromes in addition to canonical tandem repeats. Whether origin usage and/or fork elongation can cope with the variable structure of these genes is unknown. By analyzing single combed DNA molecules from HeLa cells, we studied the rRNA gene replication program according to the organization of canonical versus noncanonical rRNA genes. Origin positioning, spacing, and timing were not affected by the underlying rRNA gene physical structure. Conversely, fork arrest, both temporary and permanent, occurred more frequently when rRNA gene palindromes were encountered. These findings reveal that while initiation mechanisms are flexible enough to adapt to an rRNA gene structure of any arrangement, palindromes represent obstacles to fork progression, which is a likely source of genomic instability.
Assuntos
Replicação do DNA/fisiologia , DNA Intergênico/química , Rearranjo Gênico/fisiologia , RNA Ribossômico/química , Sequências Repetitivas de Ácido Nucleico , Origem de Replicação/fisiologia , Células HeLa , HumanosRESUMO
The t(14;18) chromosomal translocation is the most common translocation in human cancer, and it occurs in all follicular lymphomas. The 150-bp bcl-2 major breakpoint region (Mbr) on chromosome 18 is a fragile site, because it adopts a non-B DNA conformation that can be cleaved by the RAG complex. The non-B DNA structure and the chromosomal translocation can be recapitulated on intracellular human minichromosomes where immunoglobulin 12- and 23-signals are positioned downstream of the bcl-2 Mbr. Here we show that either of the two coding ends in these V(D)J recombination reactions can recombine with either of the two broken ends of the bcl-2 Mbr but that neither signal end can recombine with the Mbr. Moreover, we show that the rejoining is fully dependent on DNA ligase IV, indicating that the rejoining phase relies on the nonhomologous DNA end-joining pathway. These results permit us to formulate a complete model for the order and types of cleavage and rejoining events in the t(14;18) translocation.
Assuntos
Quebra Cromossômica/genética , DNA Ligases/fisiologia , Rearranjo Gênico/fisiologia , Genes bcl-2/fisiologia , Região Variável de Imunoglobulina/genética , Translocação Genética , Sequência de Bases , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , DNA Ligase Dependente de ATP , Humanos , Dados de Sequência Molecular , Recombinação Genética/fisiologiaRESUMO
The RAG proteins were long thought to serve merely as a nuclease, initiating recombination by cleaving DNA. Recent work has shown, however, that these proteins are essential for many steps in the recombination pathway, such as opening hairpins and joining broken DNA ends, and that they can also act as a transposase, targeting distorted DNA structures such as hairpins.