Transposon Excision from an Atypical Site: A Mechanism of Evolution of Novel Transposable Elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3′ to 5′ trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis

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

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

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

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

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

Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2.

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2