Thesis (Ph. D.)--University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 191-192)

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Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 43 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 37-43).

Efficient expression of recombinant protein was achieved in E. coli through the use of vectors capable of copy number amplification. Expression was regulated by the tryptophan promoter and plasmid copy number by the temperature sensitive Lambda Pg promoter. Reproducible expression of a variety of recombinant proteins was achieved in defined medium under fermentation conditions. In all cases, cell growth was strongly inhibited after amplification of both plasmid DNA and product expression. Plasmid copy number control was studied in continuous culture. Below 36°G, plasmid DNA was maintained at a low and constant level. At 37°G and 38°C however, plasmid DNA amplification was activated. In order to study the effect of physiological parameters on expression from these plasmids, the CAT protein was chosen as a model. The effect of specific growth rate on copy number amplification was studied in batch and continuous culture. Prior to induction, higher growth rates increased CAT expression. After induction however, clear trends were difficult to determine although growth rate clearly influenced the lag period before DNA amplification occurred. In continuous culture, a combination of reduced growth rate and overgrowth by the plasmid free cells caused transient product formation. Therefore to conduct extended experiments under inducing conditions, two stage continuous culture was evaluated. Cells grown at 34°C were fed continuously to a second fermenter held at 38°C. Stable production was achieved over a 160 hour period - sufficient time to perform reproducible experiments. In these experiments, specific growth rate in the second stage was determined to be a significant factor influencing high level GAT expression. In addition, the efficiency by which it was both transcibed and translated from the plasmid DNA could be altered. The residence time in the second stage played a secondary role - mainly influencing the point at which plasmid-free cells could overgrow the culture. Trends in plasmid stability were also studied. The development of more sensitive methods to detect plasmid instability revealed the gradual accumulation of plasmid-free cells in these cultures. This was due to overgrowth of these plasmid-free cells rather than plasmid loss due to segregational deficiencies. Higher rates of overgrowth were observed at greater product expression levels.

A cluster of QUT genes within A. nidulans, encoding the enzymes required for the catabolism of quinate to protocatechuic acid, has previously been isolated within the recombinant phage Q1 by hybridization to certain genes in the equivalent N. crassa qa cluster. The location and functional integrity of the QUTE, QUTD and QUTA genes within the QUT gene cluster has been confirmed by the transformation of appropriate A. nidulans qut mutant strains. A.nidulans DNA homologous to the N. crassa qa-2 gene, encoding catabolic dehydroquinase, is able to transform a qutE mutant strain. Biochemical analysis of QUTE transformants containing multiple copies of the QUTE gene has shown that upon induction by quinate there is no increase in the level of catabolic dehydroquinase over that observed in a wild-type strain and that the transformants are subject to normal regulatory control. A. nidulans DNA homologous to the N. crassa qa-y gene is able to transform a qutD mutant strain. Biochemical studies of a number of qutD mutants suggests that in A.nidulans the QUTD gene encodes an essential component of a permease system required for the uptake of quinate. A.nidulans DNA homologous to the N. crassa qa-1F gene is able to transform a qutA mutant strain showing that the QUTA gene is equivalent to the N. crassa qa-1F gene and encodes a positively-acting regulatory protein. A small number of QUTA transformants exhibited constitutive expression of the QUT genes but these strains were subsequently found to be phenotypically unstable and therefore unsuitable for further analysis. DNA sequence analysis of the genes described above by the research group has confirmed their location within Q1 and their physical organisation in chromosome VIII of Aspergillus nidulans.

The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for β-galactosidase showed that cells harbouring the 19S-lacZ gene expressed β-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. β-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for β-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in β-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.

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The process by which information encoded m an organism's DNA is used in the synthesis of functional cell products is known as gene expression. In recent years, sequencing of RNA (RNA-seq) has emerged as the preferred technology for the simultaneous measurement of transcript sequences and their abundance. The analysis of RNA-seq data presents novel challenges and many methods have been developed for the purpose of mapping reads to genomic features and expression quantification. In the first part of my thesis I developed an R based pipeline for pre-processing, expression estimation and data quality assessment of RNA-seq datasets, which formed the basis for my subsequent work on the evolution of gene expression regulation in mammals. Since changes in gene expression levels are thought to underlie many of the phenotypic differences between species, identifying and characterising the regulatory mechanisms responsible for these changes is an important goal of molecular biology. For this, I studied the regulatory divergence of liver gene expression and of isoform usage between mouse strains. I demonstrate that gene expression diverges extensively between the strains and propose that the regulatory mechanism underlying divergent expression between two closely related mammalian species is a combination of variants that arise in cis and in trans. Isoform usage diverges to a lesser extent and appears to display a larger contribution of trans acting regulatory elements to its regulation, suggesting that isoform usage may be under different evolutionary constraints. These observations have important implications for understanding mammalian gene expression divergence and for understanding how speciation occurs.

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Plant-parasitic nematodes are the cause of devastating yield loss in vital agricultural crops around the world. Heterodera glycines, also referred to as soybean cyst nematode, is the main pathogen of Glycine max (soybean) causing more loss than all other pathogens of G. max combined. The resultant economic impact due to H. glycines in United States soybean production alone is estimated to account for an annual one-billion-dollar loss. Natural resistant genotypes have been found in trials to combat this pathogen. Of the resistant varieties identified, G. max[Peking/PI 548402] and G. max[PI 88788] are the major sources of resistance. Identification of genes expressed in the cells of which the nematode parasitizes, the syncytia, exclusively undergoing the resistant/incompatible reaction from the two major sources of resistance mentioned previously have identified a number of candidate genes presumed to function in defense to H. glycines parasitism. Prior to this work, success has been obtained by selection of a number of these candidate genes in functional analysis to show involvement in defense. This work is aimed at functionally identifying signaling components involved in the defense reaction. Reverse genetic studies of NON-RACE SPECIFIC DISEASE RESISTANCE 1 Glycine max homolog, Gm-NDR1-1, has confirmed a functional role in the defense to H. glycines to G. max. Gene expression studies revealed both effector-triggered immunity (ETI) and pattern-triggered immunity (PTI) components to be regulated by Gm-NDR1-1. Furthermore, induction in the heterologous expression of Gm-NDR1-1 in Gossypium hirsutum (cotton) suppressed Meloidogyne incognita parasitism. Harpin treatment has been evaluated due to the knowledge of NDR1’s capability of being harpin-induced (HIN1). Expression studies of the harpin treatment did in fact induce Gm-NDR1-1. The analysis further provides evidence of NDR1 role in defense by displaying the harpin-induced response of NDR1 in resistance to infection of Rotylenchulus reniformis. Receptors are known to function through signaling components in plant defense. Therefore, the conserved downstream signaling component of multiple diverse stimuli, mitogen-activated protein kinases (MAPKs) were functionally characterized in G. max for their role in resistance to H. glycines via the reverse genetic parasitism assays and evaluated to observe the effect on defense gene expression.