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1.
Nature ; 609(7925): 183-190, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35922507

RESUMO

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. Although condensin confers mechanical resistance to spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.


Assuntos
Cromatina , Microtúbulos , Mitose , Acetilação , Cromatina/metabolismo , Segregação de Cromossomos , DNA/metabolismo , Histonas/metabolismo , Microtúbulos/metabolismo , Transição de Fase , Fuso Acromático/metabolismo
2.
EMBO J ; 42(16): e113475, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37357575

RESUMO

Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.


Assuntos
Cromátides , Proteínas Cromossômicas não Histona , Humanos , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mitose , DNA , Fase G2 , Coesinas
3.
Nature ; 587(7833): 285-290, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32879492

RESUMO

Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.


Assuntos
Posicionamento Cromossômico , Cromossomos Humanos/metabolismo , Citoplasma/metabolismo , Antígeno Ki-67/metabolismo , Membrana Nuclear/metabolismo , Transporte Biológico , Células HeLa , Humanos , Mitose , Ribossomos/metabolismo , Fuso Acromático
4.
Proc Natl Acad Sci U S A ; 120(18): e2218085120, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37094140

RESUMO

Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al., Cell 179, 470-484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14, 50 (2021); H. Strickfaden et al., Cell 183, 1772-1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.


Assuntos
Cromatina , Histonas , Histonas/metabolismo , Nucleossomos , DNA/metabolismo , Montagem e Desmontagem da Cromatina
5.
Nature ; 535(7611): 308-12, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27362226

RESUMO

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.


Assuntos
Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Modelos Biológicos , Tensoativos/química , Fenômenos Biomecânicos , Compartimento Celular , Cromatina/metabolismo , Cromossomos Humanos/química , Humanos , Antígeno Ki-67/química , Antígeno Ki-67/genética , Membrana Nuclear/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Solventes/química , Fuso Acromático/metabolismo , Eletricidade Estática
6.
Nat Methods ; 11(7): 731-3, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24859753

RESUMO

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.


Assuntos
Actinas/química , Citoesqueleto/ultraestrutura , Corantes Fluorescentes , Microscopia Confocal/métodos , Tubulina (Proteína)/química , Animais , Axônios/química , Células Cultivadas , Eritrócitos/ultraestrutura , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Neurônios/citologia , Ratos , Rodaminas/química , Silício/química
7.
J Pharmacol Exp Ther ; 352(3): 579-89, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25576074

RESUMO

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/uso terapêutico , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacologia , Pteridinas/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinase 1 Polo-Like
8.
Plant Cell ; 21(12): 3850-67, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20023195

RESUMO

In eukaryotes, class I alpha-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type alpha-mannosidase I (MNS3) and the two Golgi-alpha-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected alpha1,2-mannose residue from Man(9)GlcNAc(2), MNS1/2 readily removed three alpha1,2-mannose residues from Man(8)GlcNAc(2). Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man(8)GlcNAc(2) accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man(9)GlcNAc(2), demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I alpha-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I alpha-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Polissacarídeos/metabolismo , alfa-Manosidase/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Linhagem Celular , Parede Celular/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Retículo Endoplasmático/enzimologia , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/enzimologia , Mutagênese Insercional , Mutação , Filogenia , Raízes de Plantas/enzimologia , RNA de Plantas/genética , Spodoptera/citologia , Especificidade por Substrato , alfa-Manosidase/genética
9.
New Phytol ; 184(2): 457-472, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19691674

RESUMO

* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Genes de Plantas , Células Gigantes/enzimologia , Inositol Oxigenase/genética , Nematoides , Animais , Arabidopsis/citologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Feminino , Perfilação da Expressão Gênica , Glucuronidase , Inositol Oxigenase/metabolismo , Mutação , Raízes de Plantas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
10.
J Cell Biol ; 217(6): 1985-2004, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29695489

RESUMO

Faithful genome transmission in dividing cells requires that the two copies of each chromosome's DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Furthermore, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.


Assuntos
Ciclo Celular , Cromátides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sobrevivência Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Período de Replicação do DNA , Corantes Fluorescentes/metabolismo , Loci Gênicos , Genoma Humano , Humanos , Interfase , Modelos Biológicos , Coesinas
12.
Nat Commun ; 6: 8497, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26423723

RESUMO

Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR-Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging.


Assuntos
DNA/química , Corantes Fluorescentes/química , Animais , Linhagem Celular , Drosophila , Humanos , Microscopia/métodos , Microscopia de Fluorescência , Imagem Molecular/métodos , Estrutura Molecular , Coloração e Rotulagem/métodos
13.
Methods Mol Biol ; 715: 221-44, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21222088

RESUMO

Undoubtedly, the function of the plant cell wall in the control of cell growth far exceeds its mechanical role. The plant's monitoring of cell wall function and integrity comprises a central checkpoint to integrate cues for survival and division, expansion and differentiation, as well as fluctuations in the biotic and abiotic environment (Somerville et al., Science 306:2206-2211, 2004). With their biochemical nature yet unknown, the identification of molecular constituents of cell wall performance, and integrity control initially depends on a combination of genetic and physiological approaches.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Parede Celular/metabolismo , Mucoproteínas/metabolismo , Apoptose , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Mapeamento Cromossômico , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Genótipo , Glucosídeos/síntese química , Glucosídeos/metabolismo , Glicosilação , Imunoquímica , Endogamia , Indicadores e Reagentes , Mucoproteínas/genética , Mutagênese , Floroglucinol/análogos & derivados , Floroglucinol/síntese química , Floroglucinol/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Polinização , Ligação Proteica , Análise de Sequência de DNA
14.
Plant Physiol ; 148(3): 1238-53, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18768911

RESUMO

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Glucosiltransferases/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Sequência de Bases , Citoesqueleto/ultraestrutura , Primers do DNA , Genes de Plantas , Germinação , Microscopia Eletrônica de Transmissão , Raízes de Plantas/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/enzimologia
15.
Plant J ; 50(6): 1118-28, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17565618

RESUMO

We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from multiple organs or tissues with the generation of microarrays, which are probed with monoclonal antibodies (mAbs) or carbohydrate-binding modules (CBMs) with specificities for cell-wall components. The profiles generated provide a global snapshot of cell-wall composition, and also allow comparative analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant cell walls, and demonstrate the potential of CoMPP as a component of systems-based approaches to cell-wall biology.


Assuntos
Arabidopsis/metabolismo , Bryopsida/metabolismo , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Análise Serial de Tecidos/métodos , Arabidopsis/genética , Configuração de Carboidratos , Mutação
16.
Plant J ; 52(5): 791-802, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17892446

RESUMO

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glucosiltransferases/metabolismo , Pentosiltransferases/metabolismo , Xilanos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Benzamidas/farmacologia , Glucosiltransferases/análise , Glucosiltransferases/genética , Glucuronidase/análise , Pectinas/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/genética
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