The purpose of this study was two-fold. The first objective was to determine if Saccharomyces cerevisiae is a useful system for investigating the expression of T-DNA (it takes several months to obtain sufficient bacteria-free transformed plant tissue to investigate T-DNA transcription). A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid in an attempt to investigate the expression of gene 7 in yeast. The second objective was to determine the significance of a heat shock related sequence identified in the 5¹ region of T-DNA gene 7. Primer extension analysis, SI nuclease mapping, and Northern hybridizations indicate that transcription of T-DNA gene 7 in yeast is different from that of transcription of gene 7 in crown gall tumors. Transcription is different because the distance between the TATA box and the transcription initiation sites must be at least 40 nucleotides in yeast. Therefore, Saccharomyces cerevisiae does not appear to be a useful system for investigating the expression of T-DNA. Crown gall tumors were subjected to a number of stress agents, including heat shock, to determine the significance of the heat shock related sequence identified in gene 7. Primer extension analyses indicate that only cadmium and mercury have a significant effect on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase in transcription does not appear to be heat shock or metallothionein related, indicating that another mechanism is involved in the enhanced transcription of T-DNA gene 7 in crown gall tumors. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

DNA profiling of exhibits that originate from forensic stock theft cases is routinely used as a tool to link suspects to the crime or scene. DNA derived from aged or degraded samples is often highly fragmented which compromises the efficiency for obtaining a complete genotypic profile using PCR. Conventional polymerases such as Taq, lack certain repair mechanisms for use on degraded DNA templates. New generation polymerases are known to have high fidelity characteristics. The aim of this study was to determine the efficiency of Restorase®, a novel DNA polymerase blend that is known to repair damaged DNA and the FastStart High Fidelity PCR System enzymes, on degraded forensic bovine samples using PCR-based methodology. Bovine meat samples were subjected to different degrees of degradation in the sun and in the shade during summer and winter seasons. DNA was extracted, subjected to PCR amplification using 16 bovine microsatellites and genotypes were generated for analyses. Rapid degradation of samples was observed during winter while during summer samples tend to dry out. Restorase® exhibited high enzyme activity on degraded samples as compared with FastStart and Taq DNA polymerase. Some of the markers that failed to be successfully amplified by Taq polymerase, such as ETH10 and SPS115 were recovered using Restorase®. Markers such as BM1818, BM2113, ETH3, INRA23 and TGLA227 remained active throughout the experiment using all the enzymes, and therefore can form a basis of the bovine marker panel. Restorase® was found to be an alternative enzyme for use in bovine forensic analysis. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

O DNA repetitivo constitui uma fração considerável do genoma de muitos organismos eucarióticos. Composto tanto por sequências funcionais, como os genes ribossômicos, quanto não codificantes, como é o caso dos elementos transponíveis, mini/microssatélites e o DNA satélite, essa porção do genoma tem sido amplamente utilizada como objeto de estudo, uma vez que sequências repetitivas podem estar associadas, por exemplo, a processos de diferenciação sexual. Esses estudos têm auxiliado tanto na melhor compreensão da dinâmica dessas regiões cromossômicas, como salientado a importância, a conservação e a evolução da porção repetitiva no genoma. O gênero Eigenmannia (Gymnotiformes, Sternopygidae) compreende espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW nos quais os elementos do par sexual diferem pela presença de blocos heterocromáticos maiores do que os encontrados em cromossomos autossomos, ou sistemas múltiplos envolvendo translocação Y-autossomo. O presente trabalho tem por objetivos o estudo sobre do gene Citocromo Oxidase I (COI), de forma a verificar a capacidade discriminatória desse gene mitocondrial e sugerir possíveis espécies dos então cariomorfos do gênero Eigenmannia no estado de São Paulo, continuidade do estudo do DNA repetitivo no gênero Eigenmannia, tanto de regiões funcionais do genoma, no caso o gene ribossômico 5S, bem como de elementos transponíveis, permitindo assim uma melhor compreensão sobre a distribuição, conservação nos cariomorfos e verificar sua eventual participação no processo de diferenciação não só de cromossomos sexuais, mas também na evolução cariotípica do grupo. Os resultados obtidos com o gene COI, assim como aqueles obtidos pelo gene ribossômico 5S evidenciam distâncias genéticas consistentes com a hipótese de que os cinco cariomorfos possam ser considerados como espécies distintas. Além disso, a hibridação in situ do gene ribossômico 5S forneceu uma nova evidência para a fusão cromossômica que deu origem ao cromossomo sexual Y, já descrita na literatura, enquanto que a hibridação de sequências teloméricas não forneceu evidências de processos de fusão recentes envolvendo os cariomorfos. Com relação aos elementos transponíveis foi possível verificar padrões distintos nos elementos TC1 e Rex1 no que diz respeito às sequências, uma vez que o elemento TC1 delimitou dois grandes grupos o que pode indicar uma invasão simultânea nos grupos e no retrotransposon Rex1 a invasão tenha ocorrido em um ancestral comum a todos os cariomorfos / The repetitive DNA constitutes a considerable fraction of the genome of many eukaryotic organisms. Compound by both functional sequences, such as ribosomal genes, and non-coding, such as transposable elements, mini / microsatellite DNA and the satellite, this portion of the genome has been widely used as a study object, since the repetitive sequences may be associated with, for example, the processes of sexual differentiation. These studies helped to understand the dynamics of these chromosomal regions, pointing the importance, conservation and evolution of the repetitive portion of the genome. The genus Eigenmannia (Gymnotiformes, Sternopygidae) comprises a morphological cryptic species that exhibit variation in chromosome number and may have sexual XY or ZW systems in which the elements of sexual pair differ by the presence of heterochromatic blocks larger than those found in chromosomes autosomes, or systems involving multiple Y-autosome translocation. The present work aims to study the gene Cytochrome Oxidase I (COI) to verify the discriminatory capacity of this mitochondrial gene and suggest possible species of the so called karyomorphs of the genus Eigenmannia in the state of São Paulo. The study of repetitive DNA in Eigenmannia genus, includes 5S ribosomal gene and transposable elements, thus allowing a better understanding of the distribution, conservation in karyomorphs and verify their possible participation in the process of differentiation not only of sex chromosomes, karyotypic evolution but also in the group. The results obtained with the COI gene, as well as those obtained by the 5S ribosomal gene demonstrate genetic distances consistent with the hypothesis that the five karyomorphs can be regarded as separate species. In addition, in situ hybridization of ribosomal 5S gene provided new evidence for chromosomal fusion which led to the Y sex chromosome, as described in the literature, whereas hybridization of telomeric sequences did not provide evidence of recent fusion events involving the karyomorphs. Regarding transposable elements, it could be verified distinct sequence patterns between TC1 and Rex1 elements, since the TC1 element delimited two groups which may indicate a simultaneously invasion in those groups and retrotransposon Rex1 invasion has occurred in a common ancestor to all karyomorphs

This study provides information about the effects of polyamines on the restriction enzymatic cleavage of bacteriophage lambda DNA. The polyamines studied were spermine, spermidine, Nl-acetylspermidine and N8- acetylspermidine. The restriction enzymes studied were Xhoi, BamHI, EcoRI, and Hindiii. The electrophoretic pattern of lambda DNA digests by these enzymes were recorded photographically. These results were further analyzed by spectrographic digitization and replotting. Polyamines affect the electrophoretic pattern of restriction fragments in two ways: by causing DNA streaking and by decreasing ethidium bromide binding to DNA, which in turn affects DNA staining properties. The polyamines studied have effects which are increasingly dependent on the charge of the polyamine. The concentration necssary to alter the electrophoretic pattern decreases with increased positive charge of the polyamines. Spermine, the most highly charged polyamine studied, resulted in alterations at a lower concentration than any other polyamines studied. Following spermine was spermidine, and then the two acetylated polyamines, Nl-acetylspermidine, and N8- acetylspermidine.

Fingernail evidence is often collected in criminal cases of violent and/or sexual assault. In acts of aggression and self-defense, foreign deoxyribonucleic acid (DNA) can be transferred from the perpetrator to beneath the surface of the fingernail of the victim. It is possible to recover this foreign, exogenous DNA from the victim’s fingernails and potentially identify the perpetrator via DNA analysis. When attempting to recover this DNA from fingernail clippings, a couple of problems can occur. Often times, not enough exogenous DNA gets trapped underneath fingernails, so there is not usually much DNA to work with for recovery. The other major problem is the presence of endogenous DNA from the fingernail donor. Not only is there donor DNA in the fingernail itself, but the donor’s own DNA can build up underneath their nails simply by rubbing their face or combing their fingers through their hair. This means that there can be more donor DNA present that can mask the presence of the foreign DNA and cloud the results. In an attempt to improve the recovery of foreign DNA and produce a reportable, informative profile, a time course study was developed. Typically, when using forensicGEM as an extraction method, the samples would incubate for 15 minutes before going through protease inactivation. For this study, the extraction period was broken up into four 5-minute periods of incubating, for a total of 20 minutes, before inactivating the protease. This was done to pinpoint the time period at which more foreign DNA is being extracted from the surface of the nail before endogenous DNA is extracted in excess and clouds or even hides the presence of foreign DNA altogether. Female fingernail clippings were spiked with neat male saliva to observe the ratio of male to female DNA during quantitation and on the electropherograms. The quantitation results depicted a strong presence of male DNA through the entirety of the time course, and female DNA did not appear to be extracted in greater levels until the 15 minutes of incubation. The resulting profiles exhibited the male saliva profile as the major contributor for most of the samples, especially at the 5- and 10-minute markers. In 50% of the profiles, a minor female contributor could be identified as the nail donor. One sample produced a single, male profile for each time point with no indication of a female donor present in the extract; another sample produced a profile with a male major contributor with only 3 to 6 loci having additional detectable alleles of a minor contributor at each time point. These alleles could not be conclusively attributed to the female nail donor, but she could not be excluded. These preliminary results indicate that a shortening of the forensicGEM extraction period could be beneficial for improving the recovery ratio of exogenous to endogenous DNA from fingernail evidence.

As electronic devices became smaller, interest in quantum-confined semiconductor nanostructures increased. Self-assembled mesoscale semiconductor structures of II-VI nanocrystals are an especially exciting subject because of their controllable band gap and unique photophysical properties. Several preparative methods to synthesize and control the sizes of the individual nanocrystallites and the electronic and optical properties have been intensively studied. Fabrication of patterned nanostructures composed of quantum-confined nanoparticles is the next step toward practical applications. We have developed an innovative method to fabricate diverse nanostructures which relies on the size and a shape of a chosen deoxyribonucleic acid (DNA) template.

Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...

Chromosomal translocation is a characteristic feature of human lymphoid malignancies and a driver of the initiation and progression of the disease. They arise from the mis-repair of physiological DNA double-strand breaks (DSBs) generated during the assembly and subsequent modifications of the antigen receptor gene loci, namely V(D)J recombination and class switch recombination (CSR). Mammalian cells have three DSB repair pathways –classical non-homologous end-joining (cNHEJ), alternative end-joining (A-EJ), and homologous recombination. DNA end-resection that generates a single-strand 3’ overhang is a critical regulator for the repair pathway choice. Specifically, localized end-resection prevents cNHEJ and exposes flanking microhomology (MH) to promote error-prone A-EJ. In addition to DNA repair, DNA end-resection generates extended single-strand DNA, which activates the ATR-mediated cell cycle checkpoint and indirectly contributes to genomic integrity. The central goal of my thesis research is to investigate the physiological role of DNA end-resection initiation in lymphocyte development and lymphomagenesis. DNA end-resection in mammalian cells is mostly initiated by the endonuclease activity of MRE11-RAD50-NBS1 (MRN) complex aided by CtIP. In addition, MRN protein also recruits EXO1 and DNA2 nucleases in combination with Top3 helicase complex for more extensive resection. The CtIP protein is essential for the endonuclease activity of the MRN complex that initiates DNA end-resection. CtIP is essential for embryonic development. Here I utilized B cell-specific conditional deletion models and loss-of-function mutations to investigate the role and regulation of CtIP and CtIP-mediated DNA end-resection in lymphocyte development and tumorigenesis. The level and extent of CtIP-mediated resection are tightly regulated. For the first aim, we applied the ATAC-Seq and EndSeq methods to test whether chromatin accessibility determines the level of DNA end-resection. Specially, we found that chromatin-bound DNA damage response factors – H2AX and 53BP1- reduced the accessibility of the DNA around the DSBs and antagonized end-resection. Our data also suggest that during DNA damage response, the nucleosome-free or accessible regions are more prone to secondary DNA breakages. Mechanistically, the preferential vulnerability is correlated with the availability of chromatin-bound DNA damage response factor 53BP1, which protects the nucleosome covered region at the price of the nucleosome-free regions. The work provides one explanation for tissue and cell type-specific translocations in transcriptionally active regions and super-enhancers. For the second and third aims, I investigated the role of CtIP and CtIP-mediated end-resection in lymphocyte development and lymphomagenesis in vivo using the conditional deletional CtIP allele and a phosphorylation-deficient CtIP-T855A mutant. T855 phosphorylation promotes end-resection but is not essential for cellular viability. I identified a sequence-context-dependent role of CtIP and end-resection in A-EJ mediated repair. We found that the reduced level of end-resection did not alter the frequency of the A-EJ mediated joining during B cell CSR, nor the levels of micro-homology at the junction, a defining feature of A-EJ mediated repair. These findings, for the first time, showed that DNA end-resection is not essential for A-EJ-mediated chromosomal DSBs repair nor for the generation of MH at the junction in vivo. This unexpected observation also highlights a tissue- and cell type-specific regulation of A-EJ and the importance of sequence context for A-EJ. Moreover, we found that ATM kinase suppresses A-EJ mediated translocation and reported the very first cell cycle-dependent analyses of CSR junctions. In cNHEJ-deficient B cells (e.g., Xrcc4- or DNA-PKcs- deficient), the A-EJ pathway is responsible for both the residual CSR events and the generation of the oncogenic IgH-Myc chromosomal translocations. In the last chapter, I determined how CtIP contributes to oncogenesis using the CtIP-T855A phospho-deficient mouse model. The result showed that CtIP T855 phosphorylation is critical for the neonatal development of Xrcc4-/-p53-/- mice and IgH-Myc translocation driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, phospho-deficient CtIP compromises the extent of end-resection without affecting the initiation. Reduced end-resection in CtIP-T855A mice and cells attenuated G2/M checkpoints and reduced the tolerance to the oncogene-induced replication stress, thereby limit lymphomagenesis. Collectively, our data provided the first in vivo characterization for the role of CtIP and its related end-resection pathway in lymphocyte development and lymphomagenesis. The results highlight the importance of end-resection for checkpoint maintenance (§ 4) and the context-dependent regulation of A-EJ and DNA repair pathway choice in vivo (§ 3), explaining why A-EJ is more robust at the repetitive switch regions. Finally, the application of HTGTS, EndSeq, and ATAC-Seq technologies in lymphocyte-specific gene rearrangements markedly improved the analysis depth and sensitivity while reducing the cost of repair-junction sequencing, allowing the quantitative detection of subtle changes and additional mechanistic insights. Specifically, we showed that end-resection could be regulated at the level of chromatin accessibility, which is determined by both baseline chromatin occupancy and DNA damage-induced changes (§ 2). These findings provide one explanation for the tissue and cell type-specificity of translocation targeting. These techniques can be used to analyze the impact of other DNA repair factors during lymphocyte development and lymphomagenesis and in translocation in general.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

The ability to confidently obtain deoxyribonucleic acid (DNA) profiles from a variety of sample types in a crime laboratory is very important. The first step in the analysis of DNA in a forensic crime laboratory is the extraction of the nucleic acid material from the rest of the cellular material contained in the sample. There are many different extraction methods available for use in the field of forensics but one that is reliable, cost effective, and easy to use is necessary. One of the methods that meet these requirements is a solid-phase extraction utilizing a silica membrane that binds the DNA in the presence of chaotropic salts. This solid-phase extraction using a silica membrane is ideal for automation and use with a bio-robot. One commonly used instrument is the BioRobot EZ1® system (Hilden, Germany) from Qiagen®. Automation using these robots was the first step in decreasing the time it takes to perform extraction as well as reduce the potential for contamination, but there are still opportunities to improve in both of these areas. In this study the Qiagen® Lyse&Spin Basket Kit was evaluated due to its potential to further decrease the time needed for extraction and the eliminate a step of the extraction process where contamination could be introduced. Laboratories around the country have reported problems with using Buffer G2 as an extraction buffer when used with samples such as blood on fabric. The reason for this is currently unknown, but in order to continue to confidently extract samples of this type, a change to diluted ATL buffer was suggested. The current extraction buffer, G2, used in the extraction protocol at the Kansas City Police Crime Laboratory was not yielding results in some situations such as blood on fabric. Switching to diluted ATL yielded the same quantity of DNA and the same quality of DNA profiles with mock casework samples. When diluted ATL was used with Quality Control (QC stain) samples it yielded significantly more DNA during extraction. When the Qiagen® Lyse&Spin Basket Kit was used during the extraction of whole blood on fabric (QC stain samples) they showed a significantly lower quantitation value than the tubes currently used in the Kansas City Police Department Crime Laboratory. The same results were obtained when the Qiagen® Lyse&Spin Basket Kit was used to extract 1:2 diluted saliva samples. When samples with lower quantities of DNA, such as 1:100 blood dilutions, 1:50 saliva dilutions, and mock casework samples, were examined the Qiagen® Lyse&Spin Basket Kit there were no significant differences seen when compared to the currently used baskets. When the quantitation data was analyzed for the QC stain samples extracted with the Qiagen® Lyse&Spin Basket Kit, abnormally high degradation index (DI) values were observed. It was determined that these high values did not affect the integrity of the sample. In order to determine the possible cause behind the poor performance of the Qiagen® Lyse&Spin Basket Kit when used to extract samples with higher starting amounts of DNA an experiment was designed to determine if genetic material was left on the cotton swatch in the spin basket. It was seen that DNA was left behind on the cotton swatches from both the Qiagen® Lyse&Spin Baskets and the currently used baskets. It appeared that more DNA was contained on the fabric used with the Qiagen® Lyse&Spin Basket, but further research is needed to determine if this difference is significant.