Repair footprints following <i>Ds</i> excision

<p>Repair footprints following <i>Ds</i> excision</p

A model proposing the formation and movement of novel inverted repeat elements.

<p>(A) TIRs of a transposable element are shown in filled boxes. The boxed sequences are the 7bp duplicated half-sites. Transposase molecules bind at the TIRs, and (B) make pairs of staggered nicks on each flank (triangles). Occasionally, the DNA may slip (C) or the transposase may bind to cryptic sites on adjacent host DNA, and make a paired nick, which leads to excision (D). (E) Short repair synthesis followed by trans-esterification leads to a hairpin at the host segment attached to the TIR, which is nicked again near the TIR. Helix unwinding, followed by microhomology-mediated ligation to a new host site inserts the transposon, and single strand gap filling by repair synthesis forms a novel inverted repeat derived from host sequences adjacent to the original location (F). The transposon may repeat the cycle of precise (B) or imprecise (C) excision, and may be dissociated from novel host-derived inverted repeat (G). If the host-derived sequence had a cryptic transposase-binding site, the site is now duplicated on the novel inverted repeat. This may lead to independent movement of the inverted repeat sequence as a novel mobile element so long as a transposase is available.</p

Excision of <i>Ds</i> with atypical flanking DNA.

<p>(A) Configuration of <i>Ds</i> in ET500 parental <i>Ds</i> element with <i>NPTII</i>, <i>GUS</i>, 5′ and 3′ TIRs. The 598bp inverted repeat is immediately 3′ of the <i>Ds</i> 3′ TIR. The duplicated target half sites (gray boxes) flank the 5′ TIR (typical) and the 598bp inverted repeat (atypical). (B) Excision of <i>Ds</i> yields broken ends that are adjacent to the leftward duplicated target at the 5′ end of <i>Ds</i> and to the 598bp inverted repeat at the 3′ end of <i>Ds</i>, and is predicted to destroy a <i>Sac</i>I site. (C) Southern blot analysis of plant DNA digested with <i>Sac</i>I, confirming <i>Ds</i> excision. Upper panel (labelled GUS) was probed with GUS sequences, stripped and reprobed with one-half of the 598bp inverted repeat sequence (middle panel, labelled 598bp). Lower portion of blot was cut out and probed with <i>DCL1</i> cDNA sequences as a loading control (lower panel, labelled DCL1). Lanes 1–5 contained DNA from F2 lines with empty donor sites; lane 6, ET500 parent. The up-shift in band size between ET500 parent and F2 progeny plants in the middle panel is because of destruction of a <i>Sac</i>1 site due to excision. Lanes 1, 2, 4, and 5; F2 progeny had lost <i>Ds</i> from the genome, but retained the 598bp fragment. Lane 3; F2 progeny had a reinsertion of <i>Ds</i> but retained the 598bp fragment at the original locus. (D) Southern blot analysis of <i>Sac</i>I digested DNA showing movement of the 598bp segment. Lanes 1 and 2 had F2 DNA from two independent F1 lines, and lane 3 had ET500 parental DNA. Probes were as labelled for panel (C). Lane 1 shows an F1 line in which the <i>Ds</i> and the 598bp inverted repeat had a change of position. Lane 2 shows a line that had lost <i>Ds</i> but not the 598bp inverted repeat sequence from the original location. Hybridization signals expected from the original location are identified with triangles; signals due to sequence alteration are identified with circles (loss of <i>Sac</i>I site adjacent to 598 bp repeat), asterisks denote movement of GUS and the 598bp repeat to another location.</p

<i>Ds</i> excision does not influence crossover recombination

<p><i>Ds</i> excision does not influence crossover recombination</p

Structure and position of the <i>Ds</i> element in strain ET500.

<p>(A) Sequence of the <i>Ds</i> element and adjacent sequences showing the important elements. The insert is flanked by 7 bp target site duplication (boxed). DNA outside the duplicated target sequences are identical to the surrounding plant DNA sequence. Sequence of the 598 bp inverted repeat is identical to a fragment of the T-DNA containing 3′-octopine synthase promoter (3′OCS) and the right border (RB) element. Black circle within the boxed sequence shows the site of sequence inversion. (B) Structure of the <i>Ds</i> element. The <i>Ds</i> element contains a promoter-less bacterial <i>GUS</i> gene just inside the leftward TIR, and a bacterial <i>NPTII</i> gene with its own promoter. A plant-specific promoter-enhancer element (stippled oval) 5′ to the <i>Ds</i> insertion site confers constitutive <i>GUS</i> expression. (C) Map of the chromosomal region containing <i>Ds</i>. Numbers denote the first base on chromosome 4 (counting from the top) for the respective site. Roman numerals denote the two genetic intervals assayed for crossover. The transposase source, <i>Ac</i>, was unlinked to chromosome 4, and was present only in the experimental cross. Superscripts identify La-er (L) and No-0 (N) alleles.</p

Excision of <i>Ds</i>.

<p>(A) A leaf of a parental <i>Ds</i> strain homozygous for <i>Ds</i> (ET500), showing uniform GUS⁺ staining. (B) GUS⁻ somatic sectors due to <i>Ds</i> excision in F1 of ET500×Nae<i>Ac</i>. (C) Southern blot analysis showing the excision of <i>Ds</i>. Upper panel was probed with GUS; lower panel represents the same blot cut off between 2kb and 3kb markers, and probed with DCL1 cDNA sequences as a loading control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000965#pone.0000965-Sundaresan1" target="_blank">[19]</a>. Lane 1, ET500 parental (before <i>Ds</i> excision). Lanes 2-3, DNA from two independent F2 derivatives of ET500×Nae<i>Ac</i> showing <i>Ds</i> excision, and lane 4, DNA from an F2 plant showing excision and reinsertion of <i>Ds</i> to another location. (D) Amplification of molecular markers: Landsberg <i>erecta</i> (L); Nossen (N); heterozygous (L/N) Left hand panel, nga8; middle panel, Ga1.1/BsaB1; right hand panel, nga1111.</p

Models explaining <i>Ds</i> excision footprints.

<p>(A–C) A model explaining the formation of the most common footprints. Boxed bases are the 7bp duplicated target sequence. Thin straight arrows show putative transposase nick sites, curved arrows depict <i>trans</i>-esterification reaction between the 3′ OH group of one strand and the 5′ phosphate of the other, and arrowheads show the points of ligation. Gap repair synthesis bases are in bold and direction of synthesis is shown by an arrow; deleted bases are underlined. Note the absence of symmetrical targets flanking the excision site. (D) Model explaining the formation of a less common footprint.</p

A cartoon illustrating relative connectivity of subgraphs.

<p>Successive subgraphs are generated from a ranked degree list, and the relative connectivity <i>f</i> is computed from them. Each node is represented by a black center with a gray ‘halo’ whose size is proportional to the degree of the node. Note that newer nodes have smaller halos (lower degrees). Interactions involving newly added nodes are shown as dotted edges, while previously established interactions are shown as dark edges. Note that all subgraphs upto <i>G₄</i> are completely disconnected in this example.</p

Bimodality of PCC distribution for the HC network.

<p>Inclusion of non-hub nodes into the list of HC hubs leads to reduction in bi-modality of the average PCC distribution. This can be seen as the number of hubs included increases from 40 to 419 in the HC dataset. The panel on the left displays smoothed probability density functions corresponding to the average PCC distribution while the panel on the right displays the cumulative distribution functions. Percentiles refer to the percentages of top high degree nodes included in the hub set, following <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005344#pone.0005344-Batada1" target="_blank">[19]</a>.</p

Essential gene enrichment.

<p>Enrichment for essential genes among hubs relative to non-hubs, as measured by the Jensen-Shannon divergence (upper panels) and the P-value for the Kolmogorov-Smirnov test (lower panels).</p