Untersuchungen zu positionsspezifisch integrierenden Retrotransposons in sozialen Amöben

Social amoebae have very compact genomes. Therefore, it is likely that tRNA-gene-specific integration in the genome of the social amoeba Dictyostelium discoideum is an adaption to avoid insertional mutagenesis. In this study, a survey of mobile genetic elements was performed in the recently sequenced species D. purpureum, Polysphondylium pallidum and D. fasciculatum with emphasis on tRNA gene-targeting retrotransposons. Considering phylogenetic relations between different species, the detection of tRNA gene-specific elements in all species indicates even the last common ancestor of social amoebae possessed such elements within their genomes. The generation of a codon-adapted, tagged TRE5-A element enabled the discrimination of these tagged retrotransposons from the endogenous TRE5-A population. It was found that most tRNA genes of D. discoideum are principle targets for TRE5-A integration. New TRE5-A integrations were observed to occur into the extrachromosomal rDNA palindrome that carries the ribosomal RNA genes. It was shown that, distinct A- and B-box-motives on the palindrome, which have strong similarities with the features of a tRNA-gene, are recognized by the artificial TRE5-A-retrotransposons. The authentic distances of integrated TRE5-A copies to the targeted B box motifs and the 5S gene indicates an involvement of RNA-polymerase-III-transcription units in integration site recognition. It was shown that the targeted B box loci are actively transcribed, which argues for an involvement of the RNA polymerase III complex in target site selection by TRE5-A. In this work genetically tagged LTR retrotransposons were developed and characterized. It could be shown that a selection marker for retrotransposition events can be introduced into the elements without disruption of transposon activity. Interestingly, it seemed that such tagged retrotransposons were only mobilized in D. discoideum mutants with a defective RNA interference machinery

Компьютерное моделирование стендовых испытаний материалов дорожных покрытий

Материалы IX Междунар. межвуз. науч.-техн. конф. студентов, магистрантов и аспирантов,Гомель, 28–29 апр. 2009 г

Role of PLEXIND1/TGFβ signaling axis in pancreatic ductal adenocarcinoma progression correlates with the mutational status of KRAS

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status

Dictyostelium transfer RNA gene-targeting retrotransposons: Studying mobile element-host interactions in a compact genome

The model species of social amoebae, Dictyostelium discoideum, has a compact genome consisting of about two thirds protein-coding regions, with intergenic regions that are rarely larger than 1,000 bp. We hypothesize that the haploid state of D. discoideum cells provides defense against the amplification of mobile elements whose transposition activities would otherwise lead to the accumulation of heterozygous, potentially lethal mutations in diploid populations. We further speculate that complex transposon clusters found on D. discoideum chromosomes do not a priori result from integration preferences of these transposons, but that the clusters instead result from negative selection against cells harboring insertional mutations in genes. D. discoideum cells contain a fraction of retrotransposons that are found in the close vicinity of tRNA genes. Growing evidence suggests that these retrotransposons use active recognition mechanisms to determine suitable integration sites. However, the question remains whether these retrotransposons also cause insertional mutagenesis of genes, resulting in their enrichment at tRNA genes, which are relatively safe sites in euchromatic regions. Recently developed in vivo retrotransposition assays will allow a detailed, genome-wide analysis of de novo integration events in the D. discoideum genome

TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element

The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotran-sposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions similar to 47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only similar to 24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-A(bsr)) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-A(bsr) targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-A(bsr) retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element

Convergent evolution of tRNA gene targeting preferences in compact genomes

Background: In gene-dense genomes, mobile elements are confronted with highly selective pressure to amplify without causing excessive damage to the host. The targeting of tRNA genes as potentially safe integration sites has been developed by retrotransposons in various organisms such as the social amoeba Dictyostelium discoideum and the yeast Saccharomyces cerevisiae. In D. discoideum, tRNA gene-targeting retrotransposons have expanded to approximately 3 % of the genome. Recently obtained genome sequences of species representing the evolutionary history of social amoebae enabled us to determine whether the targeting of tRNA genes is a generally successful strategy for mobile elements to colonize compact genomes. Results: During the evolution of dictyostelids, different retrotransposon types independently developed the targeting of tRNA genes at least six times. DGLT-A elements are long terminal repeat (LTR) retrotransposons that display integration preferences similar to 15 bp upstream of tRNA gene-coding regions reminiscent of the yeast Ty3 element. Skipper elements are chromoviruses that have developed two subgroups: one has canonical chromo domains that may favor integration in centromeric regions, whereas the other has diverged chromo domains and is found similar to 100 bp downstream of tRNA genes. The integration of D. discoideum non-LTR retrotransposons similar to 50 bp upstream (TRE5 elements) and similar to 100 bp downstream (TRE3 elements) of tRNA genes, respectively, likely emerged at the root of dictyostelid evolution. We identified two novel non-LTR retrotransposons unrelated to TREs: one with a TRE5-like integration behavior and the other with preference similar to 4 bp upstream of tRNA genes. Conclusions: Dictyostelid retrotransposons demonstrate convergent evolution of tRNA gene targeting as a probable means to colonize the compact genomes of their hosts without being excessively mutagenic. However, high copy numbers of tRNA gene-associated retrotransposons, such as those observed in D. discoideum, are an exception, suggesting that the targeting of tRNA genes does not necessarily favor the amplification of position-specific integrating elements to high copy numbers under the repressive conditions that prevail in most host cells

Visualization of TRE5-A^bsr integrations at tRNA genes.

<p>The 42 tRNA gene families and their individual copy numbers are shown as white squares. The colors indicate the association of a distinct tRNA gene copy with a TRE5-A or TRE5-A^bsr element. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175729#pone.0175729.s001" target="_blank">S1 Fig</a> for the distribution of individual tRNA genes on the six chromosomes. Gray squares indicate mitochondrial tRNA genes. (<b>A</b>) Display of tRNA gene loci associated with a TRE5-A element. (<b>B</b>) TRE5-A^bsr-targeted tRNA genes identified in the tDNA primer library prepared after 20 generations (20G) or 100 generations (100G) of cell culture. Light red and dark red squares indicate tRNA genes identified only in the 20G or 100G library, respectively. Bright red squares indicate tRNA genes found in both libraries. (<b>C</b>) TRE5-A^bsr-targeted tRNA genes found in the adapter primer library prepared after 100 generations of cell culture (100G).</p

Summary of Illumina sequence mapping results.

<p>Summary of Illumina sequence mapping results.</p

Outline of TRE5-A^bsr retrotransposition profiling.

<p>Note that TRE5-A always integrates in an orientation-specific manner with the 5’ end of the retrotransposon facing the 5’ end of the targeted tRNA gene. LAM-PCR was performed on genomic DNA prepared from a pool of blasticidin-resistant clones. The 5'-biotinylated primer bound selectively to the codon-adapted ORF1 gene of the TRE5-A^bsr element and did not recognize ORF1 genes of endogenous TRE5-A elements. The resulting linear, single-stranded LAM-PCR products were immobilized on streptavidin beads and washed extensively. To perform profiling of TRE5-A^bsr insertions at tRNA genes, exponential PCRs were performed in parallel reactions with primers specific for selected tRNA gene families (“tDNA primer library”). To profile TRE5-A^bsr insertions at any position in the genome, second-strand synthesis was initiated with a random hexamer primer linked to a unique adapter oligonucleotide (adapter-N₆). Next, exponential PCR was performed with the adapter primer and an ORF1-specific primer to yield an “adapter primer library”. SD, splice donor site; SA, splice acceptor site.</p