RESUMO
The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.
Assuntos
Cromossomos Artificiais Bacterianos , Cromossomos Artificiais Humanos , Antígenos HLA-DR/genética , Camundongos Transgênicos/genética , Transgenes , Animais , Células CHO , Cricetulus , Regulação da Expressão Gênica , Genoma , Humanos , Camundongos , Especificidade de ÓrgãosRESUMO
RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. The biological function of the RNAi-related pathway in vertebrate cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken-human hybrid DT40 cell line that contains human chromosome 21. We show that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from alpha-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.
Assuntos
Endorribonucleases/genética , Endorribonucleases/fisiologia , Heterocromatina/química , Heterocromatina/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Linhagem Celular , Sobrevivência Celular , Centrômero/química , Galinhas , Cromossomos Humanos Par 21 , Proteínas de Ligação a DNA , Endorribonucleases/deficiência , Inativação Gênica , Heterocromatina/genética , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Interferência de RNA , Mapeamento por Restrição , TransgenesRESUMO
CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.
Assuntos
Autoantígenos , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Satélite/metabolismo , Proteínas de Ligação a DNA , Cinetocoros/química , Cinetocoros/metabolismo , Anticorpos/imunologia , Sequência de Bases , Sítios de Ligação , Western Blotting , Centrifugação com Gradiente de Concentração , Proteína Centromérica A , Proteína B de Centrômero , Cromatina/genética , Proteínas Cromossômicas não Histona/imunologia , Cromossomos Humanos/química , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA Satélite/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Nuclease do Micrococo/metabolismo , Peso Molecular , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Testes de Precipitina , SolubilidadeRESUMO
Human artificial chromosomes (HACs) are alternative vectors that promise to overcome problematic transgene expression often occurring with conventional vectors in mammalian cells and bodies. We have successfully generated HACs by multimerization of a cloned long alphoid stretch in a human cell line, HT1080. Furthermore, we developed technologies for cloning large genomic regions into HACs by means of co-transfection of clones with the alphoid array and clones encoding the genomic region of interest. The purpose of this study was to investigate the mitotic and meiotic stability of such HACs in mouse cells and bodies. We transferred a circular HAC containing the guanosine triphosphate cyclohydrolase I gene (GCH1-HAC) and a linear HAC containing the human globin gene cluster (globin-HAC) from HT1080 cells into mouse embryonic stem (ES) cells by microcell-mediated chromosome transfer. The HACs were stably maintained in mouse ES cells for 3 months. GCH1-HACs in every ES cell line and globin-HACs in most ES cell lines maintained their structures without detectable rearrangement or acquisition of mouse genomic DNA except one globin-HAC in an ES cell line rearranged and acquired mouse-type centromeric sequences and long telomeres. Creation of chimeric mice using ES cells containing HAC and subsequent crossing showed that both the globin-HAC that had rearranged and acquired mouse type centromeric sequences/long telomeres and GCH1-HACs were retained in tissues of mice and transmitted to progeny. These results indicate that human artificial chromosomes constructed using the bottom-up strategy based on alphoid DNA are stable in mouse bodies and are transmissible.
Assuntos
Cromossomos Artificiais Humanos/metabolismo , Clonagem Molecular/métodos , Técnicas de Transferência de Genes , Mitose/fisiologia , Animais , Encéfalo/citologia , Linhagem Celular , Quimera , DNA Circular/genética , DNA Circular/metabolismo , Células-Tronco Embrionárias , GTP Fosfo-Hidrolases/genética , Globinas/genética , Humanos , Hibridização in Situ Fluorescente , Rim/citologia , Camundongos , Baço/citologiaRESUMO
CENP-A, a centromere-specific histone H3, is conserved throughout eukaryotes, and formation of CENP-A chromatin defines the active centromere region. Here, we report the isolation of CENP-A chromatin from HeLa interphase nuclei by chromatin immunoprecipitation using anti-CENP-A monoclonal antibody, and systematic identification of its components by mass spectrometric analyses. The isolated chromatin contained CENP-B, CENP-C, CENP-H, CENP-I/hMis 6 and hMis 12 as well as CENP-A, suggesting that the isolated chromatin may represent the centromere complex (CEN-complex). Mass spectrometric analyses of the CEN-complex identified approximately 40 proteins, including the previously reported centromere proteins and the proteins of unknown function. In addition, we unexpectedly identified a series of proteins previously reported to be related to functions other than chromosome segregation, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, polycomb group proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), KNL5 and racGAP. We found that uvDDB-1 was actually localized to the centromeric region throughout cell cycle, while BMI-1 was transiently co-localized with the centromeres in interphase. These results give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation.
Assuntos
Autoantígenos/análise , Centrômero/química , Proteínas Cromossômicas não Histona/análise , Proteínas de Ligação a DNA/análise , Interfase , Proteínas Nucleares/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Repressoras/análise , Autoantígenos/isolamento & purificação , Proteína Centromérica A , Cromatina/química , Cromatina/isolamento & purificação , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas de Ligação a DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Histonas/química , Humanos , Espectrometria de Massas , Complexo Repressor Polycomb 1 , Testes de Precipitina , Proteômica/métodos , RNA Interferente Pequeno/farmacologiaRESUMO
BACKGROUND: Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing alpha-satellite DNA with CENP-B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthase. RESULTS: We constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co-transfection with the alpha-satellite DNA-containing BAC to a human fibroblast cell line. Two cell lines carrying a HAC with GCH1 genes were obtained. Both HACs were composed of multiple copies of precursor BACs and were maintained stably in human and mouse cell lines. The GCH1 activities of the HAC-carrying human fibroblast cell lines were elevated but still highly sensitive to IFN-gamma induction, mimicking the response of the gene expression from the authentic chromosomal genes. CONCLUSION: These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.
Assuntos
Cromossomos Artificiais Humanos , GTP Cicloidrolase/genética , Animais , DNA/química , Humanos , Camundongos , Conformação de Ácido NucleicoRESUMO
Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.