Dissection of the Beta-Globin Replication-Initiation Region Reveals Specific Requirements for Replicator Elements during Gene Amplification
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{"title"=>"Dissection of the Beta-Globin Replication-Initiation Region Reveals Specific Requirements for Replicator Elements during Gene Amplification", "type"=>"journal", "authors"=>[{"first_name"=>"Naoya", "last_name"=>"Okada", "scopus_author_id"=>"55874130700"}, {"first_name"=>"Noriaki", "last_name"=>"Shimizu", "scopus_author_id"=>"7403575447"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pui"=>"369964628", "issn"=>"19326203", "pmid"=>"24124615", "doi"=>"10.1371/journal.pone.0077350", "sgr"=>"84885081127", "scopus"=>"2-s2.0-84885081127"}, "id"=>"200d9c56-1e52-31a9-9c76-781dc7650670", "abstract"=>"Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR). Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.", "link"=>"http://www.mendeley.com/research/dissection-betaglobin-replicationinitiation-region-reveals-specific-requirements-replicator-elements", "reader_count"=>8, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Librarian"=>1, "Student > Ph. D. Student"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Student > Master"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Librarian"=>1, "Student > Ph. D. Student"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Student > Master"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>2, "Medicine and Dentistry"=>3, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1226176"], "description"=>"<p>The regions indicated by the orange bars in the left panel were subjected to the plasmid-stability assay in COLO 320DM cells, as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g002\" target=\"_blank\">Figure 2</a>. The results are shown in the right panel. The data suggest that the gray rectangular region drawn in the left panel has HSR-generation activity.</p>", "links"=>[], "tags"=>["replicator", "plasmid-stability", "assay"], "article_id"=>815853, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g004", "stats"=>{"downloads"=>0, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dissection_of_223_globin_replicator_using_a_plasmid_stability_assay_2_/815853", "title"=>"Dissection of ß-globin replicator using a plasmid-stability assay (2).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226175"], "description"=>"<p>Representative images obtained in this study are shown. Plasmid pTV-Rep-P bearing full-length Rep-P sequence was transfected to COLO 320DM cells (A to C) or CHO-DG44 cells (D). Metaphase chromosome spreads were prepared from the stable transfectants, and the plasmid sequence was detected by FISH. Long HSR appeared in the metaphase cells (A and B) or a interphase cell (A). The DMs with plasmid sequence appear in the metaphase cells in panel B and C as well as an interphase cell in panel C. In CHO-DG44 cells, the same plasmid did not generate long HSR, instead it remained as the short HSRs that were arrowed (D), as explained in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g001\" target=\"_blank\">Figure 1</a>.</p>", "links"=>[], "tags"=>["amplified", "plasmid"], "article_id"=>815852, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g003", "stats"=>{"downloads"=>0, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_the_amplified_plasmid_sequence_by_FISH_/815852", "title"=>"Detection of the amplified plasmid sequence by FISH.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226170"], "description"=>"<p><b>(1)</b>. The ß-globin locus and the positions of two reported replicators, Rep-P and Rep-I [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B20\" target=\"_blank\">20</a>], are drawn in A. We inserted wild type Rep-P as well as fragments bearing four deletions or an AG1 point mutation (orange bars in C) into the <i>Not</i>I site of the vector plasmid pTV-MCS (B). In panel C, RepP-1 and RepP-2 regions are indicated as gray rectangles. The plasmids were transfected into COLO 320DM cells, and polyclonal transfectants were selected in blasticidine for about 1 month. Metaphase spreads from these cells were analyzed by FISH using a pTV-MCS plasmid-derived probe. The frequencies of cells bearing HSRs of plasmid repeats were scored in both metaphase and the interphase cells, and the data are plotted in panel E.</p>", "links"=>[], "tags"=>["replicator", "plasmid-stability"], "article_id"=>815847, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g002", "stats"=>{"downloads"=>11, "page_views"=>134, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dissection_of_the_223_globin_replicator_using_a_plasmid_stability_assay_/815847", "title"=>"Dissection of the ß-globin replicator using a plasmid-stability assay", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226169"], "description"=>"<p>After transfection, a conventional plasmid without an IR/MAR is integrated into the chromosome arm at low copy number in a stable transfectant. Such a sequence is barely detectable by FISH (noted as “0”). By contrast, an IR/MAR-bearing plasmid is amplified at an extrachromosomal location as a large circular molecule of tandem plasmid repeats (step 1; [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B2\" target=\"_blank\">2</a>]). If such a molecule suffers from a DNA break (step 2a), it is eliminated from cells (step 2c; [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B5\" target=\"_blank\">5</a>]) or integrated to a chromosome arm, generating a small HSR that is visible by FISH as paired dots or a line (step 2b). There, the plasmid repeat is elongated to generate a large HSR (step 3; [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B13\" target=\"_blank\">13</a>,<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B14\" target=\"_blank\">14</a>]). These three steps occur spontaneously in human COLO 320DM cells, whereas step 3 does not occur spontaneously in CHO DG44 cells [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B11\" target=\"_blank\">11</a>].</p>", "links"=>[], "tags"=>["plasmid", "transfected"], "article_id"=>815846, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g001", "stats"=>{"downloads"=>4, "page_views"=>183, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Amplification_of_the_IR_MAR_plasmid_in_transfected_cells_/815846", "title"=>"Amplification of the IR/MAR plasmid in transfected cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226180"], "description"=>"<p>The replication initiation at the chromosomal context, the gene amplification from the endogenous chromosomal sequence and the gene amplification from the plasmid sequence require the same MAR-like element and the same AT-rich element, whereas the former one and the latter two events require similar but different AG-rich elements. The MAR elements is also required for these three events, and it is included in the plasmid (AR1) or it may be located at the chromosomal flanking region.</p>", "links"=>[], "tags"=>[], "article_id"=>815857, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g008", "stats"=>{"downloads"=>0, "page_views"=>34, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_model_from_this_study_/815857", "title"=>"A model from this study.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226178"], "description"=>"<p><b>(4)</b>. The regions indicated by orange bars in panel A were subjected to the plasmid-stability assay in COLO 320DM cells, as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g002\" target=\"_blank\">Figure 2</a>. pG20 contains a direct repeat of fragment G24 (765-1310). The results from both cell lines are shown in panel B. The data suggest that the gray rectangular regions drawn in the left panel have HSR-generation activity.</p>", "links"=>[], "tags"=>["replicator", "plasmid"], "article_id"=>815855, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g006", "stats"=>{"downloads"=>1, "page_views"=>36, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dissection_of_the_223_globin_replicator_using_a_plasmid_stability_assay_/815855", "title"=>"Dissection of the ß-globin replicator using a plasmid stability assay", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226179"], "description"=>"<p>A. To examine the effect of the AR1 MAR sequence of the vector plasmid, we removed it and constructed three new plasmids, pTV-Rep-P∆AR1, pTV-MCS∆AR1, and pG5∆AR1. We transfected plasmids with or without the AR1 MAR into COLO 320DM cells, selected transfectants, and analyzed them by FISH. Frequency of HSR is plotted in the graph. B–D. The IR sequences from ß-globin Rep-P (B), c-myc (C), and <i>DHFR</i> (D) were analyzed using MAR-Wiz program, and the results (blue graph area) are shown. In each graph, regions with HSR-generation activity revealed by our previous study (<i>DHFR</i> and c-myc; [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#B18\" target=\"_blank\">18</a>]) and this study (ß-globin) are shown as gray rectangle(s). AT-rich and AG-rich tracts are also indicated.</p>", "links"=>[], "tags"=>["mar", "hsr"], "article_id"=>815856, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g007", "stats"=>{"downloads"=>0, "page_views"=>43, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Requirement_of_MAR_for_HSR_generation_/815856", "title"=>"Requirement of MAR for HSR generation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/1226177"], "description"=>"<p><b>(3)</b>. The G5 fragment, which exhibited the highest HSR-generation activity in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g003\" target=\"_blank\">Figure 3</a>, was further dissected. The positions of AT-rich, AG-rich, palindrome, topo II binding-site, and CAAT-box regions are indicated. “MAR?” indicates the position of a MAR-like element predicted by the MAR-Wiz program. The regions indicated by orange bars in panel A were subjected to the plasmid-stability assay as in Figure 2. In this case, we examined HSR generation in both COLO 320DM cells and CHO-DG44 cells, in which gene amplification progresses differently, as shown in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077350#pone-0077350-g001\" target=\"_blank\">Figure 1</a>. The results from both cell lines are shown in panels B and C. The data suggest that the gray rectangular regions drawn in the left panel have HSR-generation activity.</p>", "links"=>[], "tags"=>["replicator", "plasmid"], "article_id"=>815854, "categories"=>["Biological Sciences"], "users"=>["Naoya Okada", "Noriaki Shimizu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0077350.g005", "stats"=>{"downloads"=>0, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dissection_of_223_globin_replicator_using_a_plasmid_stability_assay_/815854", "title"=>"Dissection of ß-globin replicator using a plasmid stability assay", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-10-04 03:02:46"}

PMC Usage Stats | Further Information

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Relative Metric

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