RESUMEN
Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method.
Asunto(s)
Proliferación Celular , Transgenes , Humanos , Células HEK293 , Proliferación Celular/genética , Expresión Génica/genética , Dosificación de Gen , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transfección , Regiones Promotoras Genéticas , Vectores Genéticos/genéticaRESUMEN
Objectives: : We aimed to review the most recent articles on the rehabilitation of patients after coronavirus disease 2019 (COVID-19) and to identify the methods and effects of rehabilitation on such patients. Methods: : A literature search was conducted using PubMed and Web of Science from study inception to October 2022 using the following search terms to identify meta-analyses and randomized controlled studies with abstracts written in English: ["COVID-19" or "COVID 19" or "2019-nCoV" or "SARS-CoV" or "novel coronavirus" or "SARS-CoV-2"] and ["rehabilitation"]. Publications investigating the effects of pulmonary and physical rehabilitation on patients with COVID-19 were extracted. Results: The extraction process selected four meta-analyses, two systematic reviews, two literature reviews, and two randomized controlled trials. Pulmonary rehabilitation recovered forced vital capacity (FVC), 6-min walk distance (6MWD), health-related quality of life (HRQOL), and dyspnea. Pulmonary rehabilitation increased predicted FVC, distance in the 6MWD test, and HRQOL score compared with baseline values. Physical rehabilitation, comprising aerobic exercises and resistance training, effectively improved fatigue, functional capacity, and quality of life with no adverse events. Telerehabilitation was an effective tool to provide rehabilitation for patients with COVID-19. Conclusions: Our study suggests that rehabilitation after COVID-19 should be considered an effective therapeutic strategy to improve the functional capacity and quality of life of patients with COVID-19.
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Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.
Asunto(s)
Proteína B del Centrómero , Cromosomas Artificiales Humanos , Proteína A Centromérica/genética , Proteína B del Centrómero/genética , Cromatina , ADN , Heterocromatina , Histonas/metabolismo , HumanosRESUMEN
Objectives: This study aimed to describe the rehabilitation characteristics of patients with acute stage coronavirus disease managed with extracorporeal membrane oxygenation (ECMO) in the intensive care unit. Methods: This retrospective study enrolled coronavirus disease patients who underwent rehabilitation following ECMO between April 21, 2020, and August 20, 2021. The following patient data were evaluated: age, sex, weaning, peak C-reactive protein, lowest albumin level, white blood cell count, use of steroids and muscle relaxants, duration of respiratory management, ECMO management and rehabilitation, Medical Research Council (MRC) score, and Barthel index after sedation and at discharge. Results: ECMO was performed in 20 patients, and 16 were weaned successfully. The median durations of ECMO and respiratory management in survivors were 14.5 and 38 days, respectively. The median MRC scores after sedation and after rehabilitation therapy were 18 and 45, respectively. The median rehabilitation duration after sedation was 14 days. The MRC score after sedation showed significant correlations with the durations of ECMO and intubation. The median Barthel index values after sedation and at discharge were 0 and 30, respectively. Conclusions: Rehabilitation was important for patients with severe coronavirus disease because muscle weakness advanced in proportion with the durations of ECMO and ventilation management in the intensive care unit.
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In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions.
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Centrómero , Cromosomas Artificiales Humanos , Replicación del ADN , ADN Satélite/biosíntesis , Línea Celular , Línea Celular Tumoral , Proteína B del Centrómero/fisiología , Momento de Replicación del ADN , Humanos , Indicadores y Reactivos , Origen de RéplicaRESUMEN
The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells.
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Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Acetilación , Proteína A Centromérica , ADN/metabolismo , Humanos , Cinetocoros/metabolismo , MetilaciónRESUMEN
Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.
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Centrómero/metabolismo , Epigénesis Genética , Histonas/metabolismo , Cinetocoros/fisiología , Acetilación , Autoantígenos/metabolismo , Línea Celular , Proteína A Centromérica , Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Artificiales Humanos , Proteína Vmw65 de Virus del Herpes Simple/genética , Histonas/química , Humanos , Cinetocoros/química , Lisina/metabolismo , Proteínas Recombinantes de Fusión , Factor de Transcripción ReIA/genéticaRESUMEN
Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy, and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of the de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoid(tetO)-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoid(tetO)-HAC that has been formed from a ~50 kb synthetic alphoid(tetO)-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoid(tetO)-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells.
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Centrómero/genética , Cromosomas Artificiales Humanos , ADN/genética , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Cinetocoros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencias Repetidas en TándemRESUMEN
Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.
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Centrómero/genética , Cromosomas Artificiales Humanos/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Animales , Autoantígenos/metabolismo , Células CHO , Proteínas de Ciclo Celular/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Cricetinae , Cricetulus , Expresión Génica , Prueba de Complementación Genética , Genoma Humano/genética , Humanos , Hibridación Fluorescente in Situ , Integrasas/metabolismo , Mutagénesis Insercional/genética , Proteínas Nucleares/genética , Recombinación Genética/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
In 2007-2008, a measles outbreak occurred among children above the age of 10 years in Akita Prefecture, northeastern Japan (population, approximately 1,120,000 at the time). Our group controlled the outbreak by (i) implementing a publically financed urgent vaccination program and (ii) prohibiting non-vaccinated and non-infected students from attending school as per regulations of the school public health law. We encouraged high-risk students to undergo a vaccination program, which resulted in the successful containment of the outbreak without the development of any severe cases. After the outbreak, the Akita Prefectural Government began an annual"Akita measles elimination month" every April, and no measles case found in Akita Prefecture during 2009-2010 subsequently. Our outbreak response initiative can be applied nationally for the complete elimination of measles throughout Japan.
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Brotes de Enfermedades/prevención & control , Programas de Inmunización/organización & administración , Vacuna Antisarampión/administración & dosificación , Sarampión/prevención & control , Adolescente , Adulto , Niño , Preescolar , Brotes de Enfermedades/economía , Brotes de Enfermedades/estadística & datos numéricos , Femenino , Humanos , Programas de Inmunización/economía , Lactante , Japón/epidemiología , Masculino , Sarampión/epidemiología , Vacuna Antisarampión/economía , Persona de Mediana Edad , Práctica de Salud Pública/legislación & jurisprudencia , Instituciones Académicas , Estudiantes , Vacunación , Adulto JovenRESUMEN
We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore.
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Cromatina/metabolismo , Cinetocoros/metabolismo , Proteínas Represoras/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular Tumoral , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica , Cromatina/genética , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Artificiales Humanos/genética , Células HeLa , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Represoras/genética , Transfección , Proteína 28 que Contiene Motivos TripartitoAsunto(s)
Centrómero/fisiología , Cinetocoros/fisiología , Animales , Autoantígenos/fisiología , Proteína A Centromérica , Proteína B del Centrómero/fisiología , Cromatina/fisiología , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Cromosomas Artificiales Humanos/genética , ADN , Humanos , RatonesRESUMEN
We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric alpha-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.
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Centrómero/genética , Cromatina/metabolismo , Cromosomas Artificiales Humanos , Epigénesis Genética , Cinetocoros/metabolismo , Animales , Secuencia de Bases , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Artificiales Humanos/genética , Cromosomas Artificiales Humanos/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos Silenciadores TranscripcionalesRESUMEN
The cost of cellulase is still a problem for bioethanol production. As the cellulase of Trichoderma reesei is applicable for producing ethanol from cellulosic materials, the cellulase productivity of this fungus should be increased. Therefore, we attempted to develop a system to isolate the strain with higher degrading ability of a filter paper and superior proliferation characteristics among the conidia treated with the mitotic arrester, colchicine. When green mature conidia of T. reesei RUT C-30 were swollen, autopolyploidized, and incubated in the double-layer selection medium containing Avicel, colonies appeared on the surface earlier than the original strain. When such colonies and the original colony were incubated on the Avicel plates, strain B5, one of the colonies derived from the colchicine-treated conidia, showed superior proliferation characteristics. Moreover, when strain B5 and the original strain were compared in the filter paper degrading ability and the cellulose hydrolyzing activity, strain B5 was also superior to the original strain. It was suspected that superior proliferation characteristics of strain B5 reflects higher filter paper degrading ability. Thus, we concluded that the Trichoderma strain with higher degrading ability of a filter paper and superior proliferation characteristics can be isolated using Avicel plates and the double-layer selection medium.
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Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Celulosa/química , Celulosa/metabolismo , Trichoderma/aislamiento & purificación , Trichoderma/metabolismo , Ultrafiltración/instrumentación , Proliferación Celular , Especificidad de la Especie , Trichoderma/clasificación , Ultrafiltración/métodosRESUMEN
The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.
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Proteína B del Centrómero/metabolismo , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cromosomas Artificiales Humanos/metabolismo , Cromosomas Artificiales de los Mamíferos/metabolismo , ADN Satélite/metabolismo , Fibroblastos/metabolismo , Animales , Autoantígenos/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Proteína A Centromérica , Proteína B del Centrómero/deficiencia , Proteína B del Centrómero/genética , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Islas de CpG , Metilación de ADN , Embrión de Mamíferos , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Lisina/metabolismo , Metilación , Ratones , Ratones Noqueados , Conformación de Ácido Nucleico , Unión Proteica , Factores de Tiempo , TransfecciónRESUMEN
Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.
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Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Satélite/genética , Autoantígenos/genética , Línea Celular Tumoral , Centrómero/fisiología , Proteína A Centromérica , Proteína B del Centrómero/genética , Proteína B del Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Heterocromatina/metabolismo , Humanos , Hibridación Fluorescente in Situ , Cinetocoros/metabolismo , Cinetocoros/fisiología , Modelos Biológicos , Modelos Genéticos , Reacción en Cadena de la PolimerasaRESUMEN
A newly designed axially chiral guanidine is found to function as an effective platform for asymmetric induction at the alpha-carbon of unsymmetrically substituted 1,3-dicarbonyl compounds. Highly efficient and enantioselective electrophilic amination of various 1,3-dicarbonyl compounds with azodicarboxylate was successfully achieved using the present chiral guanidine catalyst, which provides efficient access to the construction of nitrogen-substituted quaternary stereocenters in an optically active form.
Asunto(s)
Electrones , Guanidina/química , Aminación , Catálisis , Estructura Molecular , EstereoisomerismoRESUMEN
Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.
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Anafase/genética , Cromosomas Artificiales Humanos/genética , Metafase/genética , Sitios de Unión , Línea Celular Tumoral , Centrómero/genética , Humanos , Lactosa/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de TiempoRESUMEN
Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by beta geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1alpha and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.
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Ensamble y Desensamble de Cromatina/fisiología , Cromosomas Artificiales Humanos/metabolismo , ADN Satélite/metabolismo , Epigénesis Genética , Heterocromatina/metabolismo , Cinetocoros/metabolismo , Línea Celular Tumoral , Homólogo de la Proteína Chromobox 5 , Cromosomas Artificiales Humanos/genética , Histonas/metabolismo , Humanos , Transcripción Genética/fisiologíaRESUMEN
The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.