Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining
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{"title"=>"Highly precise and developmentally programmed genome assembly in paramecium requires ligase IV-dependent end joining", "type"=>"journal", "authors"=>[{"first_name"=>"Aurélie", "last_name"=>"Kapusta", "scopus_author_id"=>"35226535600"}, {"first_name"=>"Atsushi", "last_name"=>"Matsuda", "scopus_author_id"=>"7401614580"}, {"first_name"=>"Antoine", "last_name"=>"Marmignon", "scopus_author_id"=>"38661609800"}, {"first_name"=>"Michael", "last_name"=>"Ku", "scopus_author_id"=>"7004937929"}, {"first_name"=>"Aude", "last_name"=>"Silve", "scopus_author_id"=>"57201197256"}, {"first_name"=>"Eric", "last_name"=>"Meyer", "scopus_author_id"=>"7401539071"}, {"first_name"=>"James D.", "last_name"=>"Forney", "scopus_author_id"=>"7005791449"}, {"first_name"=>"Sophie", "last_name"=>"Malinsky", "scopus_author_id"=>"35311254800"}, {"first_name"=>"Mireille", "last_name"=>"Bétermier", "scopus_author_id"=>"6701610390"}], "year"=>2011, "source"=>"PLoS Genetics", "identifiers"=>{"pui"=>"361703347", "sgr"=>"79955624002", "issn"=>"15537390", "pmid"=>"21533177", "scopus"=>"2-s2.0-79955624002", "doi"=>"10.1371/journal.pgen.1002049", "isbn"=>"1553-7404 (Electronic)\\n1553-7390 (Linking)"}, "id"=>"1945bfe3-8032-39c3-882c-d3fae6d5e5b3", "abstract"=>"During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a \"cut-and-close\" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.", "link"=>"http://www.mendeley.com/research/highly-precise-developmentally-programmed-genome-assembly-paramecium-requires-ligase-ivdependent-end", "reader_count"=>42, "reader_count_by_academic_status"=>{"Researcher"=>17, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>1, "Student > Master"=>6, "Student > Bachelor"=>8}, "reader_count_by_user_role"=>{"Researcher"=>17, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>1, "Student > Master"=>6, "Student > Bachelor"=>8}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>30, "Chemistry"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>30}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}}, "reader_count_by_country"=>{"Poland"=>1, "France"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/783587"], "description"=>"<p>Ligases produced from ohnologous genes are designated as “a” and “b”. The tree is based on protein sequences using the following parameters: bootstrap 1000, pairwise deletion of gaps, Poisson correction, uniform rates among sites. Accession numbers in ParameciumDB: <i>LIG101a</i> (<i>GSPATG00024948001</i>), <i>LIG101b</i> (<i>PTETG9500001001</i>), <i>LIG102</i> (<i>GSPATG00030449001</i>), <i>LIG4a</i> (<i>PTETG5400008001</i>), <i>LIG4b</i> (<i>PTETG7200002001</i>), <i>LIGK01a</i> (<i>GSPATG00034046001</i>), <i>LIGK01b</i> (<i>GSPATG00037262001</i>), <i>LIGK02</i> (<i>PTETG100004001</i>), <i>LIGK03</i> (<i>GSPATG00025612001</i>). The scale indicates the number of amino acid substitutions at each site between related proteins.</p>", "links"=>[], "tags"=>["atp-dependent", "dna"], "article_id"=>453952, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g001", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Neighbor_joining_tree_of_P_tetraurelia_ATP_dependent_DNA_ligases_/453952", "title"=>"Neighbor-joining tree of <i>P. tetraurelia</i> ATP-dependent DNA ligases.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:05:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/783905"], "description"=>"<p>A. DAPI staining of single cells fixed during autogamy in the macronuclear variant 51Δ<i>A</i>. Cells were not treated with RNase prior to staining. For <i>ND7</i> & <i>LIG4</i> silencing, cells were fixed 3 days following transfer to RNAi medium. For <i>XRCC4,</i> samples were treated at day 4. White arrows: new MAC. Other stained nuclei are old MAC fragments. B. Quantification of DNA content in propidium iodide-stained exconjugants following RNAi against <i>LIG4</i> (pLIG4b-L). Fixed cells were treated with RNase A prior to staining. For each time-point, the average DNA amount (in arbitrary units) contained in the new MACs is displayed in the curve. C. DNA under-amplification in the new developing MACs of <i>XRCC4</i>-silenced cells depends on the presence of wild-type levels of PiggyMac transposase. <i>P. tetraurelia</i> strain 51 was silenced for the expression of <i>ND7</i> (Control), <i>XRCC4</i> or <i>PiggyMac</i> (<i>PGM</i>) individual genes, or cosilenced for <i>XRCC4</i> and <i>PGM</i>. During autogamy, cells were stained with DAPI and observed using a Zeiss fluorescence microscope (magnification: 630X). Except for the <i>ND7</i> control, no viable sexual progeny was recovered from RNAi-treated cells.</p>", "links"=>[], "tags"=>["macs"], "article_id"=>454274, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g005", "stats"=>{"downloads"=>2, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DNA_content_in_developing_MACs_of_LIG4_or_XRCC4_silenced_cells_/454274", "title"=>"DNA content in developing MACs of <i>LIG4-</i> or <i>XRCC4</i>-silenced cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:11:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/392653", "https://ndownloader.figshare.com/files/392693", "https://ndownloader.figshare.com/files/392714", "https://ndownloader.figshare.com/files/392747", "https://ndownloader.figshare.com/files/392781", "https://ndownloader.figshare.com/files/392801", "https://ndownloader.figshare.com/files/392816", "https://ndownloader.figshare.com/files/392831", "https://ndownloader.figshare.com/files/392860", "https://ndownloader.figshare.com/files/392889"], "description"=>"<div><p>During the sexual cycle of the ciliate <em>Paramecium</em>, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of <em>LIG4</em> and <em>XRCC4</em> is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in <em>LIG4</em>-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair <em>via</em> highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.</p> </div>", "links"=>[], "tags"=>["developmentally", "programmed", "genome", "requires", "ligase", "joining"], "article_id"=>137543, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1002049.s001", "https://dx.doi.org/10.1371/journal.pgen.1002049.s002", "https://dx.doi.org/10.1371/journal.pgen.1002049.s003", "https://dx.doi.org/10.1371/journal.pgen.1002049.s004", "https://dx.doi.org/10.1371/journal.pgen.1002049.s005", "https://dx.doi.org/10.1371/journal.pgen.1002049.s006", "https://dx.doi.org/10.1371/journal.pgen.1002049.s007", "https://dx.doi.org/10.1371/journal.pgen.1002049.s008", "https://dx.doi.org/10.1371/journal.pgen.1002049.s009", "https://dx.doi.org/10.1371/journal.pgen.1002049.s010"], "stats"=>{"downloads"=>30, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Highly_Precise_and_Developmentally_Programmed_Genome_Assembly_in_Paramecium_Requires_Ligase_IV_Dependent_End_Joining/137543", "title"=>"Highly Precise and Developmentally Programmed Genome Assembly in <em>Paramecium</em> Requires Ligase IV–Dependent End Joining", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-04-14 02:05:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/784144"], "description"=>"<p>A. LMPCR detection of 4-base and 3-base 5′ overhangs on the broken MAC ends generated at the left boundary of IES #5 = sm19-576 (66 bp) during autogamy of strain 51Δ<i>A</i> submitted to control or <i>LIG4</i> RNAi. LMPCR products were separated on high resolution polyacrylamide denaturing gels to visualize the doublet of bands. The position of linker ligation to the free 5′ end was confirmed by gel purification of LMPCR products and sequencing using primer sm19-4 specific for the left flanking MAC sequences (the chromatograms show the sequence of the top strand). The structure of the ligation products is diagrammed at the bottom, with the linker represented in purple. Arrows indicate the nucleotides that are ligated to the linker on 4-base or 3-base 5′ extensions. B. Nucleotide addition to broken MAC 3′ ends is impaired in Ligase IV-depleted cells. Terminal transferase-mediated poly(C) tailing of the MAC left 3′ end of IES #6 (51G4404) was performed as indicated in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen.1002049-Gratias1\" target=\"_blank\">[11]</a>. Poly(C)-tailed products were amplified using primers 51G18 and I, then a nested PCR was performed with 51G13 and I before electrophoresis on a 3% Nusieve agarose gel. The position of size standards (in bp) is indicated: PCR products are expected around 122 bp, with some variability due to the length of the poly(C) tail added by the terminal transferase. To determine the position of poly(C) addition, gel-purified PCR products were sequenced using 51G13 (from the MAC left flanking sequences) as a sequencing primer. The chromatograms show the sequence of the top strand and the structure of the broken MAC ends identified in each sample is displayed at the bottom.</p>", "links"=>[], "tags"=>["double-strand", "breaks"], "article_id"=>454504, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g008", "stats"=>{"downloads"=>2, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_5_and_3_end_processing_at_double_strand_breaks_in_LIG4_silenced_cells_/454504", "title"=>"Analysis of 5′- and 3′-end processing at double-strand breaks in <i>LIG4</i>-silenced cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:15:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/783987"], "description"=>"<p>Three IESs are presented: 1 = 51A1835 (28bp), 3 = 51A4404 (77 bp) from surface antigen <i>A</i> gene and 6 = 51G4404 (222 bp) from surface antigen <i>G</i> gene. Gene names are indicated on the left of panels A and B. IESs are drawn as grey boxes and MAC flanking sequences as black lines. Vertical arrows indicate the position of DNA cleavage in each experiment. All details about the linkers and primers used in this experiment are provided in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen.1002049.s009\" target=\"_blank\">Table S1</a>. In both time-courses, cells were transferred to RNAi medium at day 0 (D0). The T0-time point is the time when 50% of cells have a fragmented old MAC. RNAi against <i>LIG4</i> was obtained using mixed bacterial cultures producing dsRNA from <i>LIG4a</i> (pLIG4a-R) and <i>LIG4b</i> (pLIG4b-R). Histograms show the progression of autogamy. V: vegetative cells. F: fragmented parental MAC. NM: cells with two visible new developing MACs. PA: post-autogamous cells with one MAC and surrounding fragments. A. LMPCR detection of free broken MAC ends at IES excision sites during autogamy of strain 51 submitted to control or <i>LIG4</i> RNAi. B. LMPCR detection of free broken IES ends at the left boundary of IES #6 during the same time-course.</p>", "links"=>[], "tags"=>["detection", "ends", "ies", "boundaries"], "article_id"=>454347, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g006", "stats"=>{"downloads"=>2, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_LMPCR_detection_of_free_broken_ends_at_IES_boundaries_in_LIG4_silenced_cells_/454347", "title"=>"LMPCR detection of free broken ends at IES boundaries in <i>LIG4</i>-silenced cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:12:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/783842"], "description"=>"<p>A. Maps of <i>LIG4</i> and <i>XRCC4</i> genes. <i>XRCC4</i> accession number in ParameciumDB is <i>GSPATG00029540001</i>. The fragments used in the construction of RNAi plasmids are shown in hatched boxes and designated in italics (<i>L</i> or <i>R</i>). Probes used for Northern blot hybridization are displayed as black boxes: we used the first 510 bp of <i>LIG4a</i> open reading frame and a 302-bp fragment encompassing bp 508–809 for <i>XRCC4</i>. Restriction sites: P  =  <i>Pst</i>I, S  =  <i>Spe</i>I, K  =  <i>Kpn</i>I, H  =  <i>Hind</i>III. B. Survival and genetic analysis of F1 progeny from a cross between a3093 (mt7 <i>pwB</i>) and d4-502 (mt8 <i>pwA</i>). The histogram shows the number of cells surviving conjugation and the genotype of their MAC, out of 18 cells for the control experiment and 17 for the <i>LIG4</i> knock-down. NC: cells resulting from a conjugation failure within a mating pair (mutant phenotype in F1 and F2). Zyg. MAC: cells containing a functional zygotic nucleus. Regen: MAC regenerants in F1 (mutant phenotype in F1 and recovery of wild-type clones in post-autogamous F2). Control RNAi was carried out with an empty vector (no insert). The segregation of <i>pwA</i> (A-) and <i>pwB</i> (B-) markers in F1 MICs is shown below the histogram. The cell phenotype determined by the MAC is indicated between square brackets. C. MAC regeneration in post-autogamous progeny of 51 cells injected with a GFP transgene. Cells were transferred at day 0 in control (no RNAi or RNAi against <i>ND7</i>) or <i>LIG4</i>-silencing medium and, following starvation, 100% autogamy was observed at day 2. Survival tests were performed on 12-cell samples at day 2 (I) or day 4 (II). Regen: MAC regenerants, as judged by GFP fluorescence. D. Survival of post-autogamous progeny following <i>LIG4</i> or <i>XRCC4</i> silencing (experiments are independent of the one presented in B). Regen: MAC regenerants. The inserts cloned in plasmids used for feeding experiments are displayed in A. The histogram represents a compilation of several independent experiments, in each of which the progeny of 30 individual autogamous cells was assayed for survival.</p>", "links"=>[], "tags"=>["progeny"], "article_id"=>454210, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g004", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_sexual_progeny_of_LIG4_silenced_cells_/454210", "title"=>"Analysis of sexual progeny of <i>LIG4</i>-silenced cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:10:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/784237"], "description"=>"<p>A. Model for initial cleavage at IES boundaries by the PiggyMac-associated complex. Nicking of the first strand would liberate a reactive 3′OH residue at IES ends. The putative nucleophilic attack of the top strand is represented by arrows with question marks. B. DNA intermediates and end joining factors involved in IES excision are shown. In wild-type conditions, repair of the chromosomal excision sites (left) is probably mediated by alignment of 4-base 5′ overhangs <i>via</i> the pairing of their central conserved TA. The formation of IES circles (right) may require the resolution of putative hairpins at IES ends, prior to the formation of intramolecular paired-end intermediates. The actors involved in the controlled processing of ends are still unknown. At least for the MAC chromosome junctions, the removal of the 5′ terminal nucleotide does not require Lig4p/Xrcc4p, in contrast to the gap filling step, which is strongly inhibited in <i>LIG4</i> knock-downs. The final ligation is totally Lig4p/Xrcc4p-dependent. In <i>LIG4</i> or <i>XRCC4</i> silencing, no MAC junctions are formed and DSBs accumulate. No circle junctions could be detected.</p>", "links"=>[], "tags"=>["ies", "excision"], "article_id"=>454606, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g009", "stats"=>{"downloads"=>4, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Model_for_IES_excision_in_P_tetraurelia_/454606", "title"=>"Model for IES excision in <i>P. tetraurelia.</i>", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:16:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/783657"], "description"=>"<p>A. Northern blot hybridization of total RNA extracted from strain 51 during an autogamy time-course in standard growth medium. The blot was hybridized successively with <i>LIG4</i>, <i>XRCC4</i> (see maps in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen-1002049-g004\" target=\"_blank\">Figure 4A</a>) and 17S rDNA probes. The histograms show the progression of autogamy, with the different cellular stages diagrammed on top. Bottom panel shows the LMPCR-mediated detection of DSBs at the left boundary of IES sm19-576 (broken MAC ends). V: vegetative cells. The following time-points are indicated in hrs. Time 0 corresponds to 50% of the cells harboring a fragmented old MAC. B. Quantification of mRNA levels from the blots shown in A. For <i>PiggyMac,</i> values were calculated from a previous hybridization of the same blot <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen.1002049-Baudry1\" target=\"_blank\">[12]</a>. 17S rRNA signal was used for normalization. Y-axes are in arbitrary units.</p>", "links"=>[], "tags"=>["transcription"], "article_id"=>454029, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g002", "stats"=>{"downloads"=>11, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Induction_of_LIG4_and_XRCC4_transcription_during_autogamy_/454029", "title"=>"Induction of <i>LIG4</i> and <i>XRCC4</i> transcription during autogamy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:07:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/783759"], "description"=>"<p>A. Northern blot hybridization of total RNA extracted during conjugation of d4-110 mt7 and mt8. <i>LIG4</i> and <i>XRCC4</i> probes (<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen-1002049-g004\" target=\"_blank\">Figure 4A</a>) were used successively. Mac. Dev.: MAC development. B. Localization of a Lig4a-GFP fusion during conjugation. Reactive mt7 cells transformed with plasmid pLIG401GC, and exhibiting a wild-type swimming behavior (see <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#s4\" target=\"_blank\">Materials and Methods</a>), were mated with non injected d4-502 (mt8 <i>pwA</i>) at t = 0 hrs. At indicated time-points, cells were fixed and stained with DAPI before observation with a standard fluorescence microscope (GFP filter: a–f; DAPI filter: a’–f’). Developing new MACs are indicated with arrows. C. Quantification of intracellular GFP fluorescence. White circles represent the signal from strongly fluorescent cells originating from the transformed mt7 parent, black triangles represent exconjugants produced by the non-transformed mt8 partner, into which fluorescence has diffused during mating. The calculated total amount of Lig4a-GFP produced by a single transformant is represented as the sum of the two signals (dotted line). Bar  =  standard deviation.</p>", "links"=>[], "tags"=>["synchronized"], "article_id"=>454131, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g003", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_LIG4_and_XRCC4_expression_during_synchronized_conjugation_/454131", "title"=>"<i>LIG4</i> and <i>XRCC4</i> expression during synchronized conjugation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:08:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/784056"], "description"=>"<p>IESs #1 ( = 51A1835), 3 ( = 51A4404) and 6 ( = 51G4404) are described in the legend to <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen-1002049-g006\" target=\"_blank\">Figure 6</a>. Two additional IESs from surface antigen <i>A</i> gene are presented: 2 = 51A2591 (370 bp) and 4 = 51A4578 (883 bp). In each diagram, IESs are represented by a grey box and their flanking MAC sequences by a black line. PCR primers are drawn as arrowheads. A. Detection of IES excision junctions in the <i>A</i> gene in the macronuclear variant 51Δ<i>A</i>. Top: gel electrophoresis of PCR products around IESs. Bottom: representation of MAC development stages. In vegetative cells, only the MIC contributes to the PCR signal (IES+). When new MACs develop, <i>de novo</i> chromosomal junctions give an IES- PCR signal. At later stages, this signal disappears due to maternal epigenetic inheritance of the <i>A</i> deletion <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002049#pgen.1002049-Gratias2\" target=\"_blank\">[13]</a>. B. Detection of IES circle junctions by PCR in 51Δ<i>A</i>, using two internal divergent primers for each IES (same time-course as in A).</p>", "links"=>[], "tags"=>["junction", "products", "ies", "excision"], "article_id"=>454421, "categories"=>["Molecular Biology", "Genetics", "Microbiology"], "users"=>["Aurélie Kapusta", "Atsushi Matsuda", "Antoine Marmignon", "Michael Ku", "Aude Silve", "Eric Meyer", "James D. Forney", "Sophie Malinsky", "Mireille Bétermier"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1002049.g007", "stats"=>{"downloads"=>2, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Absence_of_final_junction_products_following_IES_excision_in_LIG4_silenced_cells_/454421", "title"=>"Absence of final junction products following IES excision in <i>LIG4</i>-silenced cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-14 01:13:41"}

PMC Usage Stats | Further Information

  • {"month"=>"5", "scanned-page-browse"=>"0", "cited-by"=>"0", "abstract"=>"1", "full-text"=>"43", "year"=>"2011", "pdf"=>"22", "unique-ip"=>"39", "figure"=>"27", "scanned-summary"=>"0", "supp-data"=>"5"}
  • {"scanned-page-browse"=>"0", "month"=>"6", "cited-by"=>"0", "abstract"=>"0", "full-text"=>"13", "unique-ip"=>"15", "pdf"=>"11", "year"=>"2011", "figure"=>"9", "scanned-summary"=>"0", "supp-data"=>"1"}
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  • {"scanned-page-browse"=>"0", "month"=>"8", "cited-by"=>"0", "abstract"=>"0", "full-text"=>"17", "unique-ip"=>"14", "pdf"=>"2", "year"=>"2011", "figure"=>"1", "scanned-summary"=>"0", "supp-data"=>"10"}
  • {"month"=>"9", "scanned-page-browse"=>"0", "cited-by"=>"0", "abstract"=>"0", "full-text"=>"20", "year"=>"2011", "pdf"=>"10", "unique-ip"=>"21", "figure"=>"5", "scanned-summary"=>"0", "supp-data"=>"0"}
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  • {"unique-ip"=>"9", "full-text"=>"7", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"6", "full-text"=>"4", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"7", "full-text"=>"4", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"2", "cited-by"=>"1", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"6", "full-text"=>"0", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}

Relative Metric

{"start_date"=>"2011-01-01T00:00:00Z", "end_date"=>"2011-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[290, 559, 698, 813, 927, 1028, 1121, 1206, 1289, 1360, 1433, 1501, 1569, 1637, 1707, 1774, 1841, 1911, 1974, 2038, 2103, 2161, 2219, 2280, 2345, 2406, 2466, 2529, 2592, 2654, 2713, 2772, 2835, 2893, 2954, 3020, 3071]}, {"subject_area"=>"/Biology and life sciences/Molecular biology", "average_usage"=>[295, 553, 688, 802, 914, 1017, 1108, 1194, 1278, 1351, 1419, 1500, 1570, 1633, 1712, 1774, 1839, 1903, 1970, 2033, 2101, 2162, 2228, 2291, 2352, 2418, 2486, 2547, 2610, 2680, 2737, 2799, 2859, 2919, 2974, 3029, 3082]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[316, 571, 699, 812, 915, 1008, 1095, 1180, 1255, 1319, 1393, 1461, 1528, 1595, 1662, 1729, 1802, 1868, 1937, 1997, 2062, 2130, 2192, 2254, 2324, 2386, 2450, 2510, 2579, 2642, 2716, 2778, 2842, 2908, 2979, 3042, 3095]}]}
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