Diss.--Universität Bochum. / Bibliogr. p. 100-118.

Thèse doctorat : Biologie. Bioinformatique : Rennes 1 : 2007. / Bibliogr. p. 171-183.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2007. / Committee Chair: John F. McDonald ; Committee Members: Jung Choi, King Jordan, and Soojin Yi. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems. / text

A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurologic disease. To address this question, we developed methods to amplify genomes of single neurons from human brains, achieving >80% genome coverage of single-cells and allowing study of a wide-range of somatic mutation types.

A two-component transposable element system consisting of a stabilized Activator (Acst) and a chimeric Dissociation (Ds) element has been introduced into the genome of Brassica napus. This Acst/ Ds system incorporates the use of several highly effective screenable and selectable markers. One of these markers is the maize Lc gene, a transcriptional regulator of flavonoid biosynthetic genes. This substrate-independent screenable marker was tested for the first time in B. napus and I show that when overexpressed, there is augmented trichome production and a light-dependent, enhanced accumulation of anthocyanins in B. napus plants. The phenotypes are expressed under a wide range of conditions, are visually distinct, and are observed throughout plant development. When used as a visual marker for the Acst element, Lc B. napus plants were rapidly identified among F2 segregating populations. As part of my goal to develop a very efficient Acst/Ds system for use in B. napus, a conditional negative selectable marker, the E. coli codA gene, was also tested for the first time in B. napus. This was done because use of a substrate-dependent negative selectable marker can facilitate the rapid and reliable identification of stable Ds transposition events when used as a marker for the Acst T-DNA. The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non-toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil. In codA transformed B. napus seedlings, expression of cytosine deaminase results in a severe suppression of growth and this phenotype is dependent on the presence of the 5-FC substrate. Wild-type seedlings, however, lack endogenous cytosine deaminase activity and appear unaffected by the presence of 5-FC in the growth media. These results indicate that codA has the potential to be used effectively in B. napus as a substrate-dependent negative selectable marker for the Acst T-DNA. To determine if Ac transposase cou

Transposons are a diverse collection of mobile genetic elements and are important components of nearly every genome. Because of their mobile and repetitive nature, transposons can have considerable effects on host gene expression, genome organization and evolution. The recent availability of genomic sequence information has expedited the discovery and study of transposons, as exemplified in this thesis by the complete genome analysis of the model plant system Arabidopsis thaliana. Data mining in Arabidopsis has revealed a rich diversity of transposons, of which Basho and Terminal-repeat Retrotransposons In Miniature (TRIM) elements were previously unknown types. The identification of Related to Empty Sites (RESites) provide evidence for past transposition events. Examples of elements contributing to coding regions, acquiring cellular sequences, along with in-depth analysis of the insertions, their target sites and their distribution illustrate the impact of transposons on gene and genome structures. Computer-based searches of genomic sequences has also improved our understanding of previously identified transposon families, such as the origin, classification and mobilization of Tourist elements. In addition, information on transposons gathered from in silico analysis of genomic sequences has served to design in vivo experiments. In a whole genome strategy, Transposon Display was used to investigate transposition and regulation of mobility of Tourist-like elements in A. thaliana and in the nematode Caenorhabditis elegans.

Transposons are DNA segments that can move or transpose themselves to new positions within the genome of an organism. Biologists need to predict preferred insertion sites of transposons to devise strategies in functional genomics and gene therapy studies. It has been found that the deformability property of the local DNA structure of the integration sites, called Vstep, is of significant importance in the target-site selection process. We considered the Vstep profiles of insertion sites and developed predictors based on Artificial Neural Networks (ANN) and Support Vector Machines (SVM). We trained our ANN and SVM predictors with the Sleeping Beauty transposonal data, and used them for identifying preferred individual insertion sites (each 12bp in length) and regions (each 100bp in length). Running a five-fold cross-validation showed that (1) Both ANN and SVM predictors are more successful in recognizing preferred regions than preferred individual sites; (2) Both ANN and SVM predictors have excellent performance in finding the most preferred regions (more than 90% sensitivity and specificity); and (3) The SVM predictor outperforms the ANN predictor in recognizing preferred individual sites and regions. The SVM has 83% sensitivity and 72% specificity in identifying preferred individual insertion sites, and 85% sensitivity and 90% specificity in recognizing preferred insertion regions.

Transposons, also known as transposable elements (TEs), are genetic elements capable of changing their location in the genome and amplifying in number. Because of their ability to cause mutations in the host genome, often with detrimental consequences to the host, yet avoid being eliminated by natural selection, transposons have been labeled selfish elements or genomic parasites. However, the advent of genomics has allowed the identification of numerous instances where transposons have played a crucial role in host genome evolution. In this thesis, I evaluate the extent to which transposons have influenced the genomes of their hosts, with an emphasis on plant genomes. I review the present knowledge of different mechanisms by which this is achieved and provide examples to illustrate them. Next, I tackle the problem of annotating transposons in the completed genomic sequence of domestic rice by comparing RepeatMasker, the standard approach used in transposon annotation, with an alternative approach employing hidden Markov models. In addition, I perform a genome-wide analysis of gene fragment capture by rice Mutator-like transposons. I conclude that, while this is a widespread phenomenon in rice, it is unlikely to represent a major force in generating novel protein-coding genes. Nevertheless, the duplicated gene fragments that are transcribed may playa role in the regulation of host genes they arose from via an RNAi-like mechanism. Finally, I conduct an in silico analysis of a gene family derived from a domesticated Mutator-like transposase, called MUSTANG (MUG), in conjunction with an experimental characterization of the MUG family in Arabidopsis. The results of the study indicate that the MUG family arose in a common ancestor of flowering plants and that the Arabidopsis genes AtMUG1 and/or AtMUG2 may act as global regulators of mitochondrial function. I conclude that our appreciation of the role of transposons in host function and evolution will undoubtedly continue to grow as our understanding of these processes deepens.

Since their discovery, transposable elements (TEs) have been regarded either as useful building blocks of genomes, or as "selfish DNA": genetic parasites that exploit the sexual cycle to spread in copy number within populations to the detriment of their hosts. If the "selfish DNA" hypothesis is correct, TEs are expected to deteriorate and be lost from asexual populations. This thesis tests the predictions of the "selfish DNA" hypothesis in four asexual plant species, focusing on patterns of nucleotide diversity and nucleotide substitution. Sequences bearing strong resemblance to known TE families including Ty1/copia, Ty3/gypsy, and LINE-like elements were successfully isolated from all four plant species, and showed patterns of nucleotide substitution consistent with a long history of purifying selection. Stochastic simulations were also conducted, and suggested that this result is expected if the host species has been asexual for less than tens of thousands of generations.