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Transposable elements amplify in genomes as selfish DNA elements and challenge host fitness because their intrinsic integration steps during mobilization can compromise genome integrity. In gene-dense genomes, transposable elements are notably under selection to avoid insertional mutagenesis of host protein-coding genes. We describe an example of convergent evolution in the distantly related amoebozoan Dictyostelium discoideum and the yeast Saccharomyces cerevisiae, in which the D. discoideum retrotransposon DGLT-A and the yeast Ty3 element developed different mechanisms to facilitate position-specific integration at similar sites upstream of tRNA genes. Transcription of tRNA genes by RNA polymerase III requires the transcription factor complexes TFIIIB and TFIIIC. Whereas Ty3 recognizes tRNA genes mainly through interactions of its integrase with TFIIIB subunits, the DGLT-A-encoded ribonuclease H contacts TFIIIC subunit Tfc4 at an interface that covers tetratricopeptide repeats (TPRs) 7 and 8. A major function of this interface is to connect TFIIIC subcomplexes τA and τB and to facilitate TFIIIB assembly. During the initiation of tRNA gene transcription τB is displaced from τA, which transiently exposes the TPR 7/8 surface of Tfc4 on τA. We propose that the DGLT-A intasome uses this binding site to obtain access to genomic DNA for integration during tRNA gene transcription.

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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.

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Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drug's action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

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Retrotransposons contribute significantly to the evolution of eukaryotic genomes. They replicate by producing DNA copies of their own RNA, which are integrated at new locations in the host cell genome. In the gene-dense genome of the social amoeba Dictyostelium discoideum, retrotransposon TRE5-A avoids insertional mutagenesis by targeting the transcription factor (TF) IIIC/IIIB complex and integrating ∼ 50 bp upstream of tRNA genes. We generated synthetic TRE5-A retrotransposons (TRE5-A(bsr)) that were tagged with a selection marker that conferred resistance to blasticidin after a complete retrotransposition cycle. We found that the TRE5-A(bsr) elements were efficiently mobilized in trans by proteins expressed from the endogenous TRE5-A population found in D. discoideum cells. ORF1 protein translated from TRE5-A(bsr) elements significantly enhanced retrotransposition. We observed that the 5' untranslated region of TRE5-A could be replaced by an unrelated promoter, whereas the 3' untranslated region of TRE5-A was essential for retrotransposition. A predicted secondary structure in the RNA of the 3' untranslated region of TRE5-A may be involved in the retrotransposition process. The TRE5-A(bsr) elements were capable of identifying authentic integration targets in vivo, including formerly unnoticed, putative binding sites for TFIIIC on the extrachromosomal DNA element that carries the ribosomal RNA genes.

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Approximately 30% of chronic myeloid leukemia patients show initially no response to Imatinib, a potent inhibitor of BCR-ABL. This intrinsic resistance may be due to BCR-ABL-independent cell growth. Here we analysed the cytogenetic anomalies and the proteomic profiling in KCL22-S and KCL22-R, two Imatinib-sensitive and -resistant derivative cell lines of KCL22. A tetrasomy 8 and a non-reciprocal translocation +der(6)t(6;13)(p11.1;q12) were found only in KCL22-R as new evolved anomalies. Chromosome der(6)t(6;13) showed four variants differing in the chromatin content of 13q14-13qter including the retinoblastoma gene. Due to these sub-clones, approximately 65-79% of the Imatinib-treated KCL22-R cells showed a disomy and 21-35% a monosomy for 13q14. Imatinib removal reduced the main clone to approximately 20% in the benefit of the monosomic sub-clones. This was accompanied by an increased apoptosis rate and was revertible by Imatinib re-treatment. This effect may be connected with genes located in 13q14-qter. Proteomic profiling of the cell lines performed with ProteinChip technology (SELDI) revealed several differentially expressed proteins (n=45). In summary, we demonstrate here the complex changes on the cytogenetic and proteomic level which could be caused by Imatinib and the resistance resulting from it.

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