The Experts below are selected from a list of 96 Experts worldwide ranked by ideXlab platform
Noriaki Shimizu - One of the best experts on this subject based on the ideXlab platform.
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Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells.
Nucleic acids research, 2019Co-Authors: Manami Asoshina, Genki Myo, Natsuko Tada, Koji Tajino, Noriaki ShimizuAbstract:A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a Breakage-Fusion-Bridge Cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.
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Replication and Transcription Initiation Region Is Mediated by Controllable Conflict between Amplification of Plasmids Containing a Mammalian Replication
2013Co-Authors: Noriaki Shimizu, Toshihiko Hashizume, Kenta ShingakiAbstract:ABSTRACT We previously showed that plasmids containing both a mammalianreplication initiation region and a matrix attachment region were effi-ciently amplified in human cancer cells and that they were either inte-grated into preexisting extrachromosomal double minutes (DMs) or in-duced the generation of a chromosomal homogeneously staining region(HSR). In this article, we elucidated the mechanism by which such plas-mids mimic gene amplification. Hybridization experiments using chroma-tin fiber, metaphase spread, and genomic Southern blot analysis suggestedthat a circular molecule comprising a plasmid direct repeat was generatedinitially. Recombination between this molecule and the preexisting DMsled to the apparent stabilization of the plasmid repeat. If the plasmidrepeat was integrated into the chromosome, it initiated the Breakage-Fusion-Bridge Cycle, which generated HSR. Importantly, we found thatHSR formation was blocked by inserting a poly(A) signal or the orienta-tion-specific replication fork barrier downstream of the drug-resistancegene, where the transcription would meet head to head with the supposedreplication fork from the initiation region. The matrix attachment regionenhanced HSR formation if it was inserted at the same site. These datasuggested that strand breakage generated by the conflict between repli-cation and transcription might trigger the Breakage-Fusion-Bridge Cycle.This is the first study suggesting that such a conflict leads to genomicinstability in higher eukaryotes.
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Amplification of plasmids containing a mammalian replication initiation region is mediated by controllable conflict between replication and transcription.
Cancer research, 2003Co-Authors: Noriaki Shimizu, Toshihiko Hashizume, Kenta Shingaki, June-ko KawamotoAbstract:We previously showed that plasmids containing both a mammalian replication initiation region and a matrix attachment region were efficiently amplified in human cancer cells and that they were either integrated into preexisting extrachromosomal double minutes (DMs) or induced the generation of a chromosomal homogeneously staining region (HSR). In this article, we elucidated the mechanism by which such plasmids mimic gene amplification. Hybridization experiments using chromatin fiber, metaphase spread, and genomic Southern blot analysis suggested that a circular molecule comprising a plasmid direct repeat was generated initially. Recombination between this molecule and the preexisting DMs led to the apparent stabilization of the plasmid repeat. If the plasmid repeat was integrated into the chromosome, it initiated the Breakage-Fusion-Bridge Cycle, which generated HSR. Importantly, we found that HSR formation was blocked by inserting a poly(A) signal or the orientation-specific replication fork barrier downstream of the drug-resistance gene, where the transcription would meet head to head with the supposed replication fork from the initiation region. The matrix attachment region enhanced HSR formation if it was inserted at the same site. These data suggested that strand breakage generated by the conflict between replication and transcription might trigger the Breakage-Fusion-Bridge Cycle. This is the first study suggesting that such a conflict leads to genomic instability in higher eukaryotes.
Jonathan C Strefford - One of the best experts on this subject based on the ideXlab platform.
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intrachromosomal amplification of chromosome 21 iamp21 may arise from a breakage fusion bridge Cycle
Genes Chromosomes and Cancer, 2007Co-Authors: Hazel M Robinson, Christine J Harrison, Anthony V Moorman, Ilse Chudoba, Jonathan C StreffordAbstract:Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B-lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G- and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a Breakage-Fusion-Bridge Cycle: a mechanism previously described in tumors, which we report for the first time in ALL. (c) 2007 Wiley-Liss, Inc
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A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.
Blood, 2005Co-Authors: Hazel M Robinson, Christine J Harrison, Rebecca R. Selzer, Frederik Van Delft, Jonathan C StreffordAbstract:A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the Breakage-Fusion-Bridge (BFB) Cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.
June-ko Kawamoto - One of the best experts on this subject based on the ideXlab platform.
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Amplification of plasmids containing a mammalian replication initiation region is mediated by controllable conflict between replication and transcription.
Cancer research, 2003Co-Authors: Noriaki Shimizu, Toshihiko Hashizume, Kenta Shingaki, June-ko KawamotoAbstract:We previously showed that plasmids containing both a mammalian replication initiation region and a matrix attachment region were efficiently amplified in human cancer cells and that they were either integrated into preexisting extrachromosomal double minutes (DMs) or induced the generation of a chromosomal homogeneously staining region (HSR). In this article, we elucidated the mechanism by which such plasmids mimic gene amplification. Hybridization experiments using chromatin fiber, metaphase spread, and genomic Southern blot analysis suggested that a circular molecule comprising a plasmid direct repeat was generated initially. Recombination between this molecule and the preexisting DMs led to the apparent stabilization of the plasmid repeat. If the plasmid repeat was integrated into the chromosome, it initiated the Breakage-Fusion-Bridge Cycle, which generated HSR. Importantly, we found that HSR formation was blocked by inserting a poly(A) signal or the orientation-specific replication fork barrier downstream of the drug-resistance gene, where the transcription would meet head to head with the supposed replication fork from the initiation region. The matrix attachment region enhanced HSR formation if it was inserted at the same site. These data suggested that strand breakage generated by the conflict between replication and transcription might trigger the Breakage-Fusion-Bridge Cycle. This is the first study suggesting that such a conflict leads to genomic instability in higher eukaryotes.
Hazel M Robinson - One of the best experts on this subject based on the ideXlab platform.
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intrachromosomal amplification of chromosome 21 iamp21 may arise from a breakage fusion bridge Cycle
Genes Chromosomes and Cancer, 2007Co-Authors: Hazel M Robinson, Christine J Harrison, Anthony V Moorman, Ilse Chudoba, Jonathan C StreffordAbstract:Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B-lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G- and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a Breakage-Fusion-Bridge Cycle: a mechanism previously described in tumors, which we report for the first time in ALL. (c) 2007 Wiley-Liss, Inc
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A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.
Blood, 2005Co-Authors: Hazel M Robinson, Christine J Harrison, Rebecca R. Selzer, Frederik Van Delft, Jonathan C StreffordAbstract:A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the Breakage-Fusion-Bridge (BFB) Cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.
Charlotte E. Paquin - One of the best experts on this subject based on the ideXlab platform.
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Telomere sequences at the novel joints of four independent amplifications in Saccharomyces cerevisiae.
Environmental and molecular mutagenesis, 2000Co-Authors: Irene K. Moore, Michael P. Martin, Charlotte E. PaquinAbstract:Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression. We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1. We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system. All four novel joints consist of C(1-3) A telomeric repeats joined to short (14- to 16-bp) CA-rich tracts between ADH4 and the telomere of chromosome VII. In three of the four amplifications, the telomeric sequence and the CA-rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII. In the fourth amplification, the CA-rich tract on chromosome VII is joined to telomere sequences from another chromosome. We suggest that formation of these amplifications was initiated by recombination between these CA-rich tracts and a telomere. The resulting dicentric chromosome could start a Breakage-Fusion-Bridge Cycle that could be resolved by the formation of a circular amplification structure.