A diversidade da comunidade endofítica de fungos associada à cana-de-açúcar transgênica tolerante a imazapyr e suas linhas de cultivo não transgênicas foi avaliada por isolamento e ARDRA (Amplified rDNA Restriction Analysis). Cultivares transgênicos e não-transgênicos, e seu manejo (aplicação do herbicida ou remoção manual de daninhas), foram considerados para verificar o possível efeito indireto da cana-deaçúcar geneticamente modificada (GM) sobre a comunidade de fungos endofíticos. O total de quatorze haplótipos de ARDRA foram observados na comunidade endofítica de cana-de-açúcar. O seqüenciamento da região ITS1-5.8S-ITS2 revelou uma comunidade rica representada por doze famílias diferentes do filo Ascomicota. Alguns dos isolados demonstraram alta similaridade com gêneros que ocorrem comumente como endófitos em plantas de clima tropical, como Cladosporium, Eppicoccum, Fusarium, Guignardia, Pestalotiopsis e Xylaria. A análise de variância molecular (AMOVA) indicou que as flutuações observadas na composição dos haplótipos estão relacionadas tanto ao cultivar transgênico quanto a aplicação do herbicida. Enquanto a aplicação do herbicida induziu mudanças rápidas e transientes na comunidade de fungos, as plantas transgênicas induziram mudanças mais lentas que foram mantidas ao longo do tempo. Uma abordagem independente de cultivo baseada em bibliotecas de 18S ambiental revelou a presença de clones com seqüências similares a gêneros das famílias Ustilaginaceae e Filobasidiaceae, e das ordens Sporidiobolales e Tremellales, todos do filo Basidiomicota. Os resultados aqui demonstrados representam o primeiro relato sobre a composição de fungos endofíticos associados a plantas de cana-de-açúcar e também representam um passo importante para o entendimento dos efeitos que plantas transgênicas e seu manejo podem induzir sobre a comunidade de fungos endofíticos. / The diversity of fungal endophytic community associated with transgenic imazapyr-tolerant sugarcane plants and its non-transgenic lines was evaluated by isolation and ARDRA (Amplified rDNA Restriction Analysis). Transgenic and nontransgenic cultivars and their crop management (herbicide application or manual weed control) were considered in order to assess the possible non-target effects of genetically modified (GM) sugarcane on the fungal endophytic community. A total of fourteen ARDRA haplotypes were observed in the endophytic community of sugarcane. ITS1- 5.8S-ITS2 sequencing revealed a rich community represented by twelve different families from the Ascomycota phylum. Some of the isolates showed a high sequence similarity with genera that commonly occur as endophytes in plants from tropical climates, such as Cladosporium, Eppicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of Molecular Variance (AMOVA) indicated that fluctuations observed in haplotypes composition were related to both transgenic cultivar and herbicide application. While herbicide applications induced quickly transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. A cultivation-independent approach based on libraries of environmental 18S revealed the presence of clones with high sequence similarity with genera from Ustilaginaceae and Filobasidiaceae families and Sporidiobolales and Tremellales orders, all from Basidiomycota phylum. The results demonstrated here represent the first draft on the composition of fungal endophytes associated with sugarcane plants and also represent an important step to understand the effects that transgenic plants and their crop management may induce on fungal endophytic community.

Casos de malária importada, contraídos em região endêmica, mas diagnosticados em um país não-endêmico, são um evento raro mas com desfecho potencialmente fatal. Nosso objetivo foi investigar se a análise de genomas mitocondriais permite inferir a origem geográfica de casos importados de malária vivax diagnosticados nos EUA, comparando os resultados com aqueles obtidos por análise de DNA microssatélite. Foi sequenciado o genoma mitocondrial completo de 63 amostras de P. vivax provenientes de infecções importadas dos EUA, além de 7 amostras do Brasil e 6 do Panamá. A rede de haplótipos com DNA mitocondrial foi construída com 412 sequências e foi possível classificar com precisão a origem geográfica presumida dos isolados da América do Sul, Coréia, Sudeste Asiático e Melanésia, porém os isolados do Sul da Ásia, América Central e África não puderam ser classificados geograficamente. A análise bayesiana realizada com a tipagem de marcadores de microssatélites não apresentou sucesso quanto à classificação geográfica dos isolados de P. vivax. / Cases of imported malaria, infection acquired in an endemic region, but diagnosed in a non-endemic country, are rare but can lead to a fatal outcome. Our objectives were investigate whether the analysis of mitochondrial genomes allows inferring the geographic origin of isolates of P. vivax derived from cases of imported malaria diagnosed in the USA, and compare the performance of mitochondrial genome and DNA microsatellite analysis. We sequenced full mitochondrial genomes from 63 P. vivax isolates collected at the USA from imported infections, and 7 samples from Brazil and 6 Panama. A network of mitochondrial DNA haplotypes was built with 412 genomic sequences and were able to classify accurately isolates from South America, Korea, Southeast Asia and Melanesia according to their presumed geographic origin, but failed to do so with samples from South Asia, Central America and Africa. The Bayesian analysis performed by typing microsatellite markers showed no success on the classification of geographical isolates of P. vivax.

Enzymes, as nature’s catalysts, show remarkable abilities that can revolutionize the chemical, biotechnological, bioremediation, agricultural and pharmaceutical industries. However, the narrow range of stability of the majority of described biocatalysts limits their use for many applications. To overcome these restrictions, extremozymes derived from microorganisms thriving under harsh conditions can be used. Extremophiles living in high salinity are especially interesting as they operate at low water activity, which is similar to conditions used in standard chemical applications. Because only about 0.1 % of all microorganisms can be cultured, the traditional way of culture-based enzyme function determination needs to be overcome. The rise of high-throughput next-generation-sequencing technologies allows for deep insight into nature’s variety. Single amplified genomes (SAGs) specifically allow for whole genome assemblies from small sample volumes with low cell yields, as are typical for extreme environments. Although these technologies have been available for years, the expected boost in biotechnology has held off. One of the main reasons is the lack of reliable functional annotation of the genomic data, which is caused by the low amount (0.15 %) of experimentally described genes. Here, we present a novel annotation algorithm, designed to annotate the enzymatic function of genomes from microorganisms with low homologies to described microorganisms. The algorithm was established on SAGs from the extreme environment of selected hypersaline Red Sea brine pools with 4.3 M salinity and temperatures up to 68°C. Additionally, a novel consensus pattern for the identification of γ-carbonic anhydrases was created and applied in the algorithm. To verify the annotation, selected genes were expressed in the hypersaline expression system Halobacterium salinarum. This expression system was established and optimized in a continuously stirred tank reactor, leading to substantially increased cell amounts and protein yields. The resulting gene expression products were assessed for function in vivo and/or in vitro. Our functional evaluation of the tested genes confirmed our annotation algorithm. Our developed strategy offers a general guide for using SAGs as a source of scientific and industrial investigations into “microbial dark matter” and may help to develop new catalysts, applicable for novel reactions in green chemistry.

Positive natural selection leaves detectable, distinctive patterns in the genome in the form of a selective sweep. Identifying areas of the genome that have undergone selective sweeps is an area of high interest as it enables understanding of species and population evolution. Previous work has accomplished this by evaluating patterns within summary statistics computed across the genome and through application of machine learning techniques to raw population genomic data. When using raw population genomic data, convolutional neural networks have most recently been employed as they can handle large input arrays and maintain correlations among elements. Yet, such models often require massive amounts of training data and can be computationally expensive to train for a given problem. Instead, transfer learning has recently been used in the image analysis literature to improve machine learning models by learning the important features of images from large unrelated datasets beforehand, and then refining these models through subsequent application on smaller and more relevant datasets. We combine transfer learning with convolutional neural networks to improve classification of selective sweeps from raw population genomic data. We show that the combination of transfer learning with convolutional neural networks allows for accurate classification of selective sweeps. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for gross chromosomal rearrangements (GCRs) that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Numerous studies have provided insights into the types of spontaneous GCRs that can occur in various Saccharomyces cerevisiae mutants. However, understanding the mechanism and frequency of formation of these GCR without knowledge of the initiating lesions is limited. Here, we focus on DSB-induced repair pathways that lead to GCRs. Inverted duplications occur at a surprisingly high frequency when a DSB is formed near short inverted repeats in cells deficient for the nuclease activity of Mre11. Similar to previously proposed models, the inverted duplications occur through intra-strand foldback annealing at resected inverted repeats to form a hairpin-capped chromosome that is a precursor to dicentric chromosomes. Surprisingly, we found that DNA polymerase δ proof-reading activity but not the Rad1-Rad10 nuclease is required for inverted duplication formation, suggesting a role for Pol δ in the removal of the heterologous tails formed during foldback annealing. Contrary to previous work on spontaneous inverted duplications, we find that DSB-induced inverted duplications require the Pol δ processivity subunit Pol32 and that RPA plays little role in their inhibition, suggesting that spontaneous inverted duplications arise differently than those induced by DSBs. We show that stabilization of dicentric chromosomes after breakage involves telomere capture through a strand-invasion step mediated by repeat sequences and requires Rad51. Previous work on spontaneous inverted duplications suggested that Tel1, but not Mre11-Sae2, inhibits inverted duplications that initiate from inverted repeats separated by long spacers (> 12 bp). However, we do not find evidence for this requirement. Cells with Tel1 deletion can still resolve hairpins containing loops up to 30 nt long. Furthermore, deletion of Sae2, but not Tel1, increases the frequency of inverted duplications when a DSB is induced near an inverted repeat separated by a 20 bp-long spacer. This highlights another difference between spontaneous and DSB-induced GCRs. Finally, we find that the sequence context of a DSB affects the type of GCR outcome. Inverted repeats are required for the formation of inverted duplications, as the deletion of a DSB-proximal inverted repeat significantly reduces the incidence of this type of rearrangement. Furthermore, introduction of a DSB near short telomere-like sequence is required for chromosome truncations stabilized by de novo telomere addition. The effect of the sequence context can partly explain how repair pathways can be channeled to different mutagenic outcomes. Our results highlight the importance of considering how the initiating lesion can affect the type of resulting GCRs and the mechanisms by which they occur.

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.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

No description available.

No description available.

Mitochondria are essential cellular organelles and the location of the electron transport chain, the site of the majority of energy production in the cell. Mitochondria contain their own circular genome approximately 16,000 base pairs in length. The mitochondrial genome (mtDNA) encodes 11 protein-coding genes essential for the electron transport chain, 22 tRNA genes, and two rRNA genes. Mitochondrial malfunction occurs in many diseases, and changes in the mitochondrial genome lead to numerous disorders. Multiple mitochondrial haplotypes and sequence features are associated with Alzheimer's disease. In this dissertation we utilized TreeScanning, an evolutionary-based haplotype approach to identify haplotypes and sequence variation associated with specific phenotypes: Alzheimer's disease case-control status, mitochondrial copy number, and 16 neuroimaging phenotypes related to Alzheimer's disease neurodegeneration. In the first two studies we utilized 1007 complete mitochondrial genomes from participants in the Cache County Study on Memory Health and Aging. First, individuals with mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD. Our study is the largest to date and the only study with complete mtDNA genome sequence data. Next, each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. Each of these three variants was associated with higher mitochondrial copy number and we suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that lead to changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes. Lastly, we used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This was the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Together, these projects provide evidence of mtDNA involvement in the risk and physiological changes of Alzheimer's disease.