Thesis (Ph. D.)--University of Idaho, 2006. / Abstract. "May 2006." Includes bibliographical references (leaves 107-127). Also available online in PDF format.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 26, 2010) Thesis advisor: Dr. Timothy P. Holtsford. Includes bibliographical references.

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

The biodiversity of Durban (eThekwini municipality) in KwaZulu Natal is primarily threatened by urbanization although other factors such as climate change and the spread of invasive species also pose a significant threat. Knowledge of what species exist within the city is important for biodiversity surveillance, detecting invasive taxa and uncovering cryptic species. Conducting a comprehensive biodiversity inventory is a daunting task, especially for hyperdiverse groups such as terrestrial arthropods, where closely related species can often only be separated by subtle morphological characters. This study investigated whether the barcoding marker, Cytochrome Oxidase C Subunit 1 (COI) can be used to efficiently and accurately delineate species of ants (family Formicidae) in comparison to traditional taxonomic approaches. The feasibility of DNA barcoding for assembling biodiversity inventories for urban areas which could be useful in conservation planning was also evaluated. A total of 619 individuals were sequenced from 23 geographic localities within the eThekwini region and surrounding regions. DNA barcoding revealed 80 provisional species/ “barcode clusters” or monophyletic lineages which could represent distinct species, while morphology revealed 51 different morphospecies. Extrapolation measures of species richness indicated that as many as 153 species of ants could occur in the city. Phylogenetic and phylogeographic analyses were performed on co-distributed species belonging to the genera Lepisiota, Camponotus, Pheidole and Pachycondyla to better understand the spatial distribution of genetic variability in the eThekwini region. Nuclear markers 18S rDNA and 28S rDNA were also sequenced and compared for a subsample of individuals from Camponotus and Pachycondyla. There was genetic variation at COI and the nuclear markers for each of the species examined. In order to fully elucidate the population genetic patterns which could be expected in eThekwini and surrounding regions, further sampling across more localities is essential. The use of more nuclear markers could also assist in uncovering these unique patterns of genetic variation in an urban setting. In this study, the utility of COI as a species diagnostic tool in ants was confirmed. The barcoding library constructed showed promise in highlighting reserves that should be preserved and possible cryptic speciation for further investigation. / M. Sc. University of KwaZulu-Natal, Durban 2014.

A genome sequence can be conceptualized as a 'book' written with four nucleotide 'letters' in oligonucleotide (oligo) 'words'. These words can be used in genomics, bioinformatics and the development of molecular markers. The whole-genome sequence for rice (Oryza sativa L.) is almost finished and has been assembled into pseudomolecules. For barley ( Hordeum vulgare L.) expressed sequence tags (ESTs) have been assembled into 21,981 tentative consensus sequences (TCs). The availability of such sequence information provides opportunities to investigate oligo usage within and between genomes. For the first of three studies reported in this thesis, a C++ program was written to automatically design oligos that are conserved between two sets of sequence information. In silico mapping between rice coding sequences (CDS) and barley TCs indicated that oligos between 18 and 24 bp provide good specificity and sensitivity (83% and 86%, respectively, for 20mers). Conserved oligos used as PCR primers had a high (91%) success rate on barley lines. Sequencing of PCR products revealed conservation in exon sequence, size and order between barley and rice. Introns were not conserved in sequence but were relatively stable in size. Map locations of eight new markers in barley revealed both genome colinearity and rearrangements between barley and rice. The second study reported in this thesis examined word frequency within the rice genome. A non-random landscape composed of high-frequency and low-frequency zones was observed. Interestingly, high-frequency words seemed to be rice specific while single-copy words were gene specific and conserved across species. As in the first study, oligos of 12 bp or less were not specific, and 18 bp seemed to be a critical length for the specificity of oligos. The third study reported in this thesis involved the development of molecular markers for known genes using public sequence information. Six new polymorphic markers were d

Stachybotrys elegans (Pidopl.) W. Gams is a destructive mycoparasite of the soilborne plant pathogen Rhizoctonia solani. It colonizes effectively all types of cells of R. solani, and is considered as an effective biological control agent (BCA). Monitoring the presence of this mycoparasite in the field trials requires the development of a reliable and sensitive diagnostic assay that is able to detect and differentiate the BCA from their target host. To achieve this, designed SCAR (sequenced characterized amplified regions) primers designated as SE-13F and SE-13R were generated from informative RAPD markers. They were tested in conventional PCR assays alone or in conjunction with the recently developed SCAR primers (SBU-177/336) designed for Rhizoctonia solani (Kuhn) on several types of DNA. These included DNA extracted from pure cultures, co-cultures of the BCA and the pathogen, plant tissue and several types of soils inoculated with both the BCA and the pathogen. Irrespective of the type of the biological samples from which the DNA was extracted, the primers SE-13F/SE-13R successfully amplified only S. elegans. No cross-reaction was observed when the primers were used to amplify DNA of other fungi, bacteria and plant tissues. Likewise, the primer pair SBU-177/336 detected only its target organism, i.e., R. solani. The detection limit using these primers on amplified DNA was as little as 1 pg DNA extracted from pure cultures of S. elegans, 100 pg DNA extracted from greenhouse soil and 33 pg DNA extracted from natural soil. This work is the first report on the development of SCAR markers for the BCA, S. elegans. These molecular markers offer not only an alternative diagnostic assay to conventional detection methods, but also the possibility of being used in ecological studies.

Two studies were conducted on gene mapping analysis. For the first study, genetic simulation experiments were conducted to address the effects of marker density, method of mapping analysis, and gaps in a marker map on the efficiency of QTL detection and the accuracy of QTL parameter estimation. The simulated genome consisted of seven chromosomes with seven or eight segregating QTL affecting the simulated quantitative trait. A set of six randomly segregating QTL outside the test region was consistently used to represent 40% of phenotypic variation. An individual QTL or a linkage block of two QTL on a target chromosome contributed 10% of phenotypic variation. The marker map was either dense (with markers every 4 cM) or sparse (with markers every 20 cM). The gap in the marker map was either 32 cM or 56 cM. Interval mapping and composite interval mapping were used to map QTL on the target chromosome. A dense map provided more power of QTL detection, better accuracy of QTL parameter estimation, and higher false-positive error rates for the target chromosome than a sparse map. Composite interval mapping provided more power of QTL detection, better accuracy of QTL parameter estimation, and lower false-positive error rates than interval mapping. Presence of a large gap in a marker map affected QTL detection and QTL parameter estimation for a QTL inside or near the gap. The use of a dense map with composite interval mapping was the most efficient combination tested in this study. For the second study, a mixed factorial regression model for interval mapping was developed for conducting QTL-by-environment interaction analysis and for providing inferences about QTL that are applicable beyond the environments used in the experiments. Genetic simulation was used to test the model for the power of detecting QTL-by-environment interaction and identifying the types of such interaction as crossover or non-crossover, and for the accuracy of estimating QTL parameters. The model prov

Colorectal cancer (CRC) is the fourth most common malignancy in the developed Western countries and represents the second leading cause of death from cancer after lung cancer. Despite better diagnostic tools and improvement of surgical standards, the prognosis of this disease is still unsatisfactory with a mean 5-year cancer-specific survival of approximately 50%, mainly due to our inability to predict liver failure secondary to hepatic involvement and still poorly effective palliative treatments. The metastatic cascade is a complex, multistep process driven by progressive accumulation of genetic alterations which result in proto-oncogene activation and inactivation of tumour suppressor genes. Among the additional pathways involved in this process, secretion of angiogenesis factors, proteolysis of the extra-cellular matrix (ECM) molecules, and probably inhibition of apoptosis are also known to facilitate tumour progression. We undertook a retrospective study in a series of paraffin-embedded human colonic tissues to investigate the prognostic significance of new tumour markers as predictors of liver metastasis and survival in sporadic CRC. This research was conducted in three parts: first, immunohistochemical studies of protein markers and development of a new quantitative method of measurement in a subset of the immunostained sections using a colour video image analysis (VIA) procedure; second, concomitant determination of the expression of the bcl-2 gene at the messenger RNA (mRNA) level, using a more specific methodology of in situ hybridisation (ISH) and investigation of the relationship between bcl-2 mRNA and its encoded protein expression; and third, investigation of microsatellite alterations at four loci using a fluorescent microsatelllite polymerase chain reaction (PCR) assay coupled with an automated DNA sequencer. In our initial immunohistochemical experiment, we found a good correlation between colour VIA and semiquantitative evaluation of nm23 immunoreactivity (IR) confirming the validity of such quantitative analysis (Pearson's correlation coefficient r=0.88; P<0.001). Furthermore, overexpression of nm23 was associated with an increased risk of developing liver metastases (logrank test for trend, P<0.001) and cancer-related death (P=0.002). We used the same quantitative method to determine the expression of urokinase-type plasminogen activator (u-PA) and c-erbB-2 (HER2/neu) proteins and found that neither predicted patient outcome. However, CRC showing overexpression of u-PA (above 85 pixels) had an increased risk of liver metastasis (P=0.013). Since this was a post hoc analysis we can not be confident that this represents a real effect. There were significant positive correlations among expression of all three markers, u-PA, c-erbB-2 and nm23 proteins (u-PA vs c-erbB-2, Spearman rank correlation coefficient, P=0.003; u-PA vs nm23, P<0.001; c-erbB-2 vs nm23, P=0.001) suggesting that, in vivo, all proteins interact or are similarly regulated. Semiquantitative analysis of the vascular endothelial growth factor (VEGF) protein showed that expression of VEGF was significantly reduced in the metastatic liver tumours compared to their matched primary ones (Wilcoxon test, P=0.002), suggesting VEGF activity to be secondarily down-regulated once the tumour cells reach the hepatic parenchyma. There was no strong evidence from our data that the level of VEGF in the primary tumour could predict risk of liver metastasis or survival duration. Finally, when semiquantitatively assessing five protein markers (nm23, p53, c-erbB- 2, u-PA, and VEGF) individually or in combination, we found that only nm23 protein expression was positively related to the risk of liver metastasis (logrank test, P<0.001); p53 protein expression was only marginally associated (P=0.091). Furthermore, patients with Dukes' stage B tumours showing positive expression of nm23 protein also demonstrated an increased risk of liver metastasis (P=0.001). Although the risk of developing liver secondaries was statistically correlated with the number of positive markers (NPM) and the cumulative intensity score (CIS) (logrank test for trend, P=0.004 and P=0.001 respectively), these two parameters did not improve the predictive value over and above that of nm23 protein alone. These results suggest that the only marker of the five we studied that provides prognostic information about the risk of developing liver metastasis in CRC is nm23. Its evaluation may be particularly useful in selecting high risk Dukes' stage B patients who should be considered for adjuvant chemotherapy. In the second part of this study, immunohistochemical analysis using a monoclonal mouse antibody to the bcl-2 protein and in situ hybridisation using digoxigenin-labelled bcl-2 cRNA probes were carried out on the same paraffin-embedded specimens and semiquantitatively assessed. These specimens also included adenomas with various degrees of dysplasia. The expression of bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary CRC [moderate/severe dysplasia: Mann-Whitney U-test, P=0.0001; severe dysplasia/primary CRC: P=0.027], whereas the cellular expression of bcl-2 mRNA was progressively increased during the dysplasia/adenoma-carcinoma neoplastic sequence. Our observation suggests that in a proportion of CRCs the bcl-2 proto-oncogene expression may be down-regulated at a post-transcriptional level. In the final section of this study, we investigated the possible relationship between loss of heterozygosity (LOH) and microsatellite instability (MIN) at four microsatellite loci spanning the 17q21-23 region that includes the nm23-H1 and nm23- H2 genes, to the risk of liver metastasis and nm23 protein expression. Genomic DNA was extracted from the same series of paraffin-embedded colorectal specimens and a fluorescent PCR coupled with DNA fragment analysis in an automated DNA sequencer was applied. In 45% and 48% of the primary and secondary lesions, LOH was present in at least one locus. We found a positive correlation when looking for a trend comparing the fraction of sites with LOH at these loci to risk of liver recurrence (logrank test for trend, P=0.005). This remained an independent predictor after adjusting to T-stage (Multivariate Cox regression, P=0.022), N-stage (P=0.007), or Dukes' stage (P=0.012). MIN was present in 2 loci in 41% and 30% of the primary and secondary tumours respectively. Considering only the Dukes' B tumours, we found that an increasing number of sites showing MIN was associated with a reduced risk of liver recurrence (logrank test for trend, P=0.032). When comparing LOH and MIN status of the primary and matched liver secondary tumours with their corresponding normal tissue samples, we found concordant genomic alterations in 72% (NME1 locus) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity ( ?? 2 test for trend, P=0.024). These findings suggest that, genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via a stillmechanism.

Thesis(M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.