Bacterial species and communities play foundational roles in human health and therapeutics, in vital ecological and environmental processes, and in industrial applications for the biosynthesis of valuable compounds and materials. However, existing genetic engineering methods and technologies available for bacterial functional genetics or large-scale genomic integration are inefficient, unable to translate between different target species, or lacking precise targeting or reprogramming capabilities. In this work, we describe a novel class of CRISPR- associated transposons (CRISPR-Tn) that facilitate programmable RNA-guided DNA insertions. In particular, the Tn6677 CRISPR-Tn system from Vibrio cholerae comprises a Tn7-like transposase machinery that has co-opted a nuclease-deficient Type I-F3 CRISPR-Cas system to guide its target selection. We show that, similar to canonical CRISPR-Cas systems, this CRISPR- Tn system can be easily programmed using the CRISPR RNA (crRNA) spacer sequence, and directs highly target-specific DNA integration into the Escherichia coli genome. After defining their core biological and mechanistic principles, we developed these CRISPR-Tn systems into a genome engineering platform, which we named INTEGRATE (Insertions of Transposable Elements by Guide RNA-Assisted Targeting). Particularly, optimization of V. cholerae Tn6677 (Vch INTEGRATE, or VchINT) produced a system capable of programmable, broad-bacterial- host, and multiplexed integration of DNA payloads up to 10 kilobases in length, with genomic editing efficiencies reaching 100%. Our single-plasmid expression of system components enabled, for the first time, genome engineering of specific target strains within a complex fecal bacterial community. In addition, we performed extensive deep sequencing within transposition experiments to characterize and examine non-conventional transposition products, including cointegrates formed through replicative transposition, and long-range integration events resulting from on-target DNA binding. Finally, by individually inserting transposon ends into the E. coli genome, we demonstrated successful transposition-mediated mobilization of a genomic fragment 100 kilobases (kb) in length, demonstrating engineering at the genome-scale using VchINT. Altogether, this work highlights the potential of VchINT and other CRISPR-Tn systems as next- generation genome engineering technologies in bacteria and beyond.

<p>RNA-directed DNA methylation (RdDM) is a process by which epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on <i>Mediator of Paramutation 1 (Mop1</i>), a putative RNA dependent RNA polymerase. Here I show that although RdDM is essential for the maintenance of DNA methylation of a silenced <i>MuDR</i> transposon in maize, a loss of that methylation does not result in a restoration of activity of that element. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of the element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. I find that a brief exposure of high temperature in a <i>mop1</i> mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in <i>mop1</i> wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once it has been reversed. To leverage the specificity of our observations made at bench, I also performed a transcriptome analysis in <i>mop1</i> mutants under heat. I found that a substantial number of genes as well as a subset of TEs are reactivated in <i>mop1</i> mutants under heat, which is consistent with the effects I observed on <i>MuDR</i>. Interestingly, I found that <i>mop1</i>-specific reactivation of TEs is closely correlated with changes in expression of nearby genes, most of which are involved in metabolic transportation and sensing. This suggests that one function of <i>MOP1</i> is to prevent inappropriate expression of genes in this pathway when they are close to TEs. Taken together, my work will provide an opportunity to better understand the causes and consequences of TE silencing and reactivation, as well as the effects of TEs on gene regulation under stress conditions.</p>

No description available.

No description available.

Although transposable elements (TEs) have been subjected to detailed study in various organisms such as humans, maize, and drosophila, this is not the case for all organisms. Despite numerous studies on the effects of TEs in the field of evolution and functional genomics, there has not been many studies yet on how much variation these elements show in populations. To address these questions, we identified TEs in Leptidea sinapis based on a newly produced high-quality genome assembly and identified novel TEs in this project. In the first step of the project, we manually curated consensus sequences of the 150 most abundant TE subfamilies. We could identify 145 of these subfamilies: two of which were non-curatable because of bad consensus sequences, three that were uncertain where they start and end, and one of the subfamilies were divided into two different subfamilies. Hence, we ended up with 146 different TE subfamilies, and the remaining part of the project was carried out using these. In the second step, we examined how the manually curated 146 subfamilies were distributed in 83 different L. sinapis individuals in the Swedish population. Before performing manual curation for our selected TEs, we looked at the TE landscape of the long-read sequenced L. sinapis genome and showed that 58.2% of the L. sinapis genome consists of TEs. In a recent study, it has been shown that 40% of L. sinapis consists of TEs. So, when compared to previous studies, our result showed that the L. sinapis genome contained more TEs than previously reported. When we made the same analysis after manual curation, we showed that this amount increased to 62.4%. The distribution of classified TEs by groups is as follows: LINE 22.6%, DNA 7.43%, SINE 4.76%, LTR 3.10%.  After creating the final TE landscape for our reference genome, we analyzed 83 different individuals collected from different regions of Sweden such as Uppland, Östergötland, Västmanland, Närke, Värmland, Dalarna, Hälsingland, Småland, Medelpad, and Västerbotten for the individual number of non-reference insertions using RelocaTE2. We observed that these 146 subfamilies showed different distributions among individuals based on their sequence coverage. We couldn’t find any correlation between the number of insertions and the latitude of locations where individuals had been collected. When we look at the total number of insertions, we realized type I transposable elements were more abundant compared to type II transposable elements. Also, we checked the percentage of covered bases per individual in our dataset and observed that individuals with greater coverage had more TE insertions. After realizing this, when we analyzed individuals from different locations with very similar coverage, we could not see a significant correlation between the number of TE insertions and the latitude of locations of butterflies from different locations. For this reason, we can say that for the most abundant 146 TE subfamilies in the reference genome, there is not a significant difference between regions of Sweden. This study contributes to a better analysis of TE content in L. sinapis, and the know-how and possible problems with technical bias for individual TE insertion studies in general.

The Spok genes are a group of selfish genetic elements in the fungus Podespora anserina which kills spores lacking them. These Spoks function through a toxin and resistance domain. Homologues of these have been found across other species such as in the Fusarium genus. Nechadraft_82228 is a Spok homologue present in the species Fusarium vanettenii, with uncertain killer action. In this study the killer action is examined through site directed mutagenesis (SDM) of the resistance domain in Nechadraft_82228. The site directed mutagenesis was performed successfully and showed negative results regarding whether the Nechadraft_82228 had functioning killer action and resistance. Growth pattern for spot assays hinted at the existence of a different underlying reason for the lack of growth, opposed to a functional toxin/killer action. Namely respiratory dysfunction in the transformed Saccharomyces. Cerevisiae, potentially due to unfit heat shock temperature.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 134-144). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 134-144). Also available on the Internet.

This thesis provides theoretical resources for the design of a new bioinformatics tool for transposon annotation with focus on their additional structural elements. There is a biological description of transposons, the mobile elements in DNA, their classification and structure. It further deals with the overview and classification of available transposon identification and annotation bioinformatics tools, description of function and implementation of a select few. Next we state the scheme of a new bioinformatics tool for LTR retrotransposon identification and annotation with a focus on extra ORFs and tandem repeats. The functionality of this new tool was tested on the A. thaliana genome. We identified 95 groups of conserved extra ORFs and 10 groups of conserved tandem repeats.

Main motive of this master thesis was the need of good bioinformatics tools for genome comparison and improvement of one of the existing tools - RepeatExplorer. This work offers an overview of transposable elements in DNA, existing tools for identification and analysis of repetitions in sequenced genomes, summary of currently used genome sequencing methods. This work describes shortcomings of RepeatExplorer tool with focus on comparative analysis of genomes. Two solutions to remove these problems were designed and implemented. The first solution is designed for comparing pairs of genomes. The principle of this solution is based on comparison of similarity of distribution of contigs coverages using Kolmogorov-Smirnov test, thanks to which we are able to determine different parts in the genomes.The second solution, which is used to compare multiple genomes, is based on the method of mapping reads from compared genomes to the reference genome contigs and provides contigs coverage graphs, by which we are able to determine the variability of the repeats.Their functionality was verified on real NGS data of organism Silene latifolia.