Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria

Isolation of a transcriptionally active element of high copy number retrotransposons in sweetpotato genome

Many plant retrotransposons have been characterized, but only three families (Tnt1, Tto1 and Tos17) have been demonstrated to be transpositionally competent. We followed a novel approach that enabled us to identify an active element of the Ty1-copia retrotransposon family with estimated 400 copies in the sweetpotato genome. DNA sequences of Ty1 -copia reverse transcriptase (RTase) from the sweetpotato genome were analyzed, and a group of retrotransposon copies probably formed by recent transposition events was further analyzed. 3’RACE on callus cDNA amplified transcripts containing long terminal repeats (LTR) of this group. The sequence -specific amplification polymorphism (S-SAP) patterns of the LTR sequence in the genomic DNA were compared between a normal plant and callus lines derived from it. A callus -specific S-SAP product was found into which the retrotransposon detected by the 3’RACE had been transposed apparently during cell culture. We conclude that our approach provides an effective way to identify active elements of retrotransposons with high copy numbers.</p

Evolutionary Dynamics of Multigene Families in Triportheus (Characiformes, Triportheidae): A Transposon Mediated Mechanism?

Triportheus (Characiformes, Triportheidae) is a freshwater fish genus with 18 valid species. These fishes are widely distributed in the major river drainages of South America, having commercial importance in the fishing market, mainly in the Amazon basin. This genus has diverged recently in a complex process of speciation carried out in different river basins. The use of repetitive sequences is suitable to trace the genomic reorganizations occured along the speciation process. In this work, the 5S rDNA multigene family has been characterized at molecular and phylogenetic level. The results showed that other multigene family has been found within the non-transcribed spacer (NTS): the U1 snRNA gene. Double-FISH with 5S and U1 probes were also performed, confirming the close linkage between these two multigene families. Moreover, evidences of different transposable elements (TE) were detected within the spacer, thus suggesting a transposon-mediated mechanism of 5S-U1 evolutionary pathway in this genus. Phylogenetic analysis demonstrated a species-specific grouping, except for Triportheus pantanensis, Triportheus aff. rotundatus and Triportheus trifurcatus. The evolutionary model of the 5S rDNA in Triportheus species has been discussed. In addition, the results suggest new clues for the speciation and evolutionary trend in these species, which could be suitable to use in other Characiformes species

Calling Cards For DNA-Binding Proteins

Organisms respond to their environment by altering patterns of gene expression. This process is orchestrated by transcription factors, which bind to specific DNA sequences near genes. In order to understand the regulatory networks that control transcription, the genomic targets of all transcription factors under various conditions and in different cell types must be identified. This remains a distant goal, mainly due to the lack of a high-throughput, in vivo method to study protein-DNA interactions. To fill this gap, I developed transposon Calling Cards for DNA-binding proteins. I endowed DNA binding proteins with the ability to direct the insertion of a transposon into the genome near to where they bind. The transposon becomes a Calling Card that marks the visit of a DNA-binding protein to the genome. I demonstrated that the Calling Card method is accurate and robust. I combined Calling Cards with next generation DNA sequencing technology to increase the sensitivity, specificity, and resolution of the method. This improved method: Calling Card-Seq ) allows for multiple transcription factors to be analyzed in a single experiment, greatly increasing sample throughput. I used Calling Card-Seq to study transcription factors of the yeast S. cerevisiae that have not been well-characterized, and I successfully identified DNA sequence recognition motifs and target genes for many of them. Calling Card-Seq will enable a systematic exploration of transcription factor binding under many different environments and growth conditions in a way that has heretofore not been possible. This dissertation describes my work developing this method, as well as several interesting results obtained using this method to study the gene regulatory networks of the yeast S. cerevisiae

Temporal Difference Learning in Complex Domains

PhDThis thesis adapts and improves on the methods of TD(k) (Sutton 1988) that were successfully used for backgammon (Tesauro 1994) and applies them to other complex games that are less amenable to simple pattem-matching approaches. The games investigated are chess and shogi, both of which (unlike backgammon) require significant amounts of computational effort to be expended on search in order to achieve expert play. The improved methods are also tested in a non-game domain. In the chess domain, the adapted TD(k) method is shown to successfully learn the relative values of the pieces, and matches using these learnt piece values indicate that they perform at least as well as piece values widely quoted in elementary chess books. The adapted TD(X) method is also shown to work well in shogi, considered by many researchers to be the next challenge for computer game-playing, and for which there is no standardised set of piece values. An original method to automatically set and adjust the major control parameters used by TD(k) is presented. The main performance advantage comes from the learning rate adjustment, which is based on a new concept called temporal coherence. Experiments in both chess and a random-walk domain show that the temporal coherence algorithm produces both faster learning and more stable values than both human-chosen parameters and an earlier method for learning rate adjustment. The methods presented in this thesis allow programs to learn with as little input of external knowledge as possible, exploring the domain on their own rather than by being taught. Further experiments show that the method is capable of handling many hundreds of weights, and that it is not necessary to perform deep searches during the leaming phase in order to learn effective weight

Systematic Identification of Balanced Transposition Polymorphisms in Saccharomyces cerevisiae

High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH) to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90) and a polymorphic wild isolate (Y101) through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism