T7 RNA polymerase is a single protein capable of driving transcription from a simple promoter in virtually any context. This has made it a powerful tool in a range of biotechnology applications. In this work, previous efforts to evolve or engineer T7 RNA polymerase are reviewed. This work is then expanded upon, first with the development of a method for the cell-free evolution of T7 RNA polymerase based on the functioning of an autogene. The autogene is a transcriptional feedback circuit in which active T7 RNA polymerase proteins transcribe their own gene, resulting in exponential amplification of their genetic information. While this system is doomed by an error catastrophe, this can be delayed by the use of in vitro compartmentalization. In response to the limits of the autogene, a novel directed evolution approach termed compartmentalized partnered replication (CPR) is presented. CPR couples the in vivo functionality of a gene to its subsequent in vitro amplification by emulsion PCR. The use of CPR to generate a panel of six versions of T7 RNA polymerase, each specific to one of six promoters, is described. Separately, a rational engineering approach, taken to facilitate the high-yield transcription of fully 2′-modified RNA, is detailed. Two sets of mutations to T7 RNA polymerase, previously known to confer thermal stability and enhance promoter clearance respectively, can be used to enhance the activity of existing T7 RNA polymerase mutants that utilize non-standard nucleotides as their substrates. Next, CPR and random mutagenesis is used to populate the functional fitness landscape of T7 RNA polymerase. This neutral drift library is then challenged to increase the processivity of T7 RNA polymerase, enabling long-range transcription. Finally, the lessons that can be learned about T7 RNA polymerase specifically and molecular evolution and protein engineering generally are discussed. / text

<p> <i>Myo-inositol</i> is a six-carbon sugar alcohol. It is essential as a precursor of the phospholipid membrane component phosphatidylinositol (PI) and the phosphoinositide signaling pathway in all eukaryotes. It aids in cellular metabolism, osmoregulation, and plays an important role in fertilization and diseases such as diabetes, bipolar disorder, and Alzheimer&rsquo;s disease. <i> Myo</i>-inositol metabolism is comprised of synthesis, transport, catabolism, and recycling. <i>Myo</i>-inositol synthesis is catalyzed by myo-inositol-3-phosphate synthase (MIPS). Surprisingly, synthesis of <i>myo</i>-inositol and its role in fertilization has not yet been studied in the model organism <span style="text-decoration:overline"> Drosophila melanogaster</span> (fruit fly). We hypothesize that MIPS expression is essential for growth and development of <i>D. melanogaster.</i> In this study, a precise deletion of the entire MIPS gene was generated and confirmed through PCR amplification and sequencing of the resultant DNA fragments. The lack of the MIPS transcript in homozygous MIPS deletion flies was confirmed by RT-PCR. During that experiment, two additional isoforms of MIPS were identified in wild-type flies (CS). Supplementation of chemically defined food with 0.5mM inositol was required to sustain all homozygous MIPS deletion fly strains. Fully-grown homozygous deletion flies could live without additional inositol in the food, but newly emerged larvae only survived to the first instar larval stage. However, even while on rich media supplemented with 170mM inositol, a homozygous MIPS deletion stock was unable to produce viable offspring. Homozygous MIPS deletion strains were identified as male-sterile, incapable of producing offspring when mated to any strain of females (including wild-type). Homozygous female MIPS deletion flies were fertile and maintained a high fecundity rate when mated to any strain (with an exception of homozygous male MIPS deletion flies). The male-sterility was complemented with the addition of a wild-type MIPS gene to chromosome 3. Testes dissections of homozygous male MIPS deletion flies revealed improper progression of spermatogenesis, specifically during sperm individualization. These studies contribute to the understanding of the role of inositol synthesis in growth, development, and fertilization.</p>

<p> Ticks are obligate blood sucking parasites which transmit a wide range of pathogens worldwide including protozoa, bacteria and viruses. Additionally, tick feeding alone may result in anemia, dermatosis and toxin-induced paralysis. <i> Dermacentor andersoni</i> is a species of tick found in the western United States that transmits pathogens of public health importance including <i> Rickettsia rickettsii, Francisella tularensis,</i> and Colorado Tick Fever Virus, as well as <i>Anaplasma marginale</i>, a rickettsial pathogen that causes economic losses in both the dairy and beef industries worldwide. <i>D. andersoni</i> ticks are obligate blood sucking parasites that require a blood meal through all stages of their lifecycle. During feeding, ticks secrete factors that modulate both innate and acquired immune responses in the host which enables them to feed for several days without detection. The pathogens transmitted by ticks exploit these immunomodulatory properties to facilitate invasion of and replication in the host. Molecular characterization of these immunomodulatory proteins secreted in tick saliva offers an opportunity to develop novel anti-tick vaccines as well as anti-inflammatory drug targets. To this end we performed deep sequence analysis on unfed ticks and ticks fed for 2 or 5 days. The pooled data generated a database of 21,797 consensus sequences. Salivary gland gene expression levels of unfed ticks were compared to 2- and 5-day fed ticks to identify genes upregulated early during tick feeding. Next we performed mass spectrometry on saliva from 2- and 5-day fed ticks and used the database to identify 677 proteins. We cross referenced the protein data with the transcriptome data to identify 157 proteins of interest for immunomodulation and blood feeding. Both proteins of unknown function and known immunomodulators were identified. We expressed four of these proteins and tested them for inhibition of macrophage activation and/or cytokine expression in vitro. The results showed diverse effects of the various test proteins on the inflammatory response of mouse macrophage cell lines. The proteins upregulated some cytokines while downregulating others. However, all the proteins upregulated the regulatory cytokine IL-10.</p>

In conditions of phosphate deprivation, the budding yeast, Saccharomyces cerevisiae activates the phosphate starvation response pathway (PHO pathway). Induction of the PHO pathway causes the transcription of genes involved with phosphate metabolism. Phosphate transport genes are activated during starvation, giving rise to the presence of Pho84, the high affinity transporter. In high phosphate conditions low affinity transporters reside at the plasma membrane. Here we show that Spl2, a suppressor of phospholipase-C, is involved in the down-regulation of the low affinity transport system. This phenomenon gives rise to complex population dynamics and bistability. Furthermore, we demonstrate how the phenotype of strains lacking Pho84 can be explained in context of unconstrained positive feedback through Spl2. We then turn our attention towards comparative studies of the PHO pathway, comparing the transcriptional response of S. cerevisiae to C. glabrata. Using expression microarrays and deep sequencing, we find that the transcriptional circuit in C. glabrata has been altered such that transcriptional cooperativity is lost, nucleosome positioning is altered, and transcriptional competition between the transcription factor Pho4 and centromere binding factor, Cbf1, is relatively conserved.

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. The previously uncharacterized pre-NH2-terminal domain (residues 1-140) within HSV-1 Pol is unique to the herpesvirus Pol family. We sought to investigate the importance of this domain for viral replication in cell culture and an animal model of infection. We evaluated the enzymatic activity of purified pre-NH2-terminal Pol mutant proteins in which conserved residues had been deleted or substituted. Subsequently, the corresponding pol mutant viruses were engineered for viral genetic analyses. We found that the extreme N-terminal 51 residues were not required for wild type 5’-3’ polymerase activity in vitro. Interestingly, the extreme N-terminal 42 residues were dispensable for viral replication in cell culture while a conserved motif at residues 44-49 was necessary for efficient viral DNA synthesis and production of infectious virus. Viral replication proteins have proven to be particularly important in the context of acute and latent infections in animals. Characterization of pol mutant virus replication in a mouse ocular model of infection revealed that the extreme N-terminal 42 residues were not required for viral replication and reactivation from latency. The conserved motif, however, was shown to be required for robust acute ganglionic replication and efficient latency establishment. We hypothesized that the conserved motif at residues 44-49 mediates a protein- protein interaction that positively impacts viral DNA synthesis during infection. Specific protein candidates were evaluated using purified proteins in vitro, and proteins that coprecipitated with wild type and mutant polymerases from infected cell lysates were analyzed. To date, we have yet to identify a protein whose binding was disrupted as a result of the mutation. Ultimately, we have established a role for the pre-NH2-terminal domain of HSV-1 Pol during viral replication that is distinct from 5’-3’ polymerase activity. The conserved motif mediates a function that is required for efficient viral DNA synthesis in cell culture and is of even greater importance for acute ganglionic replication in mice. The mechanism of action more than likely reflects a conserved mechanism for herpesvirus replication.

Malaria has plagued mankind for millennia. Antimalarial drug use over the last century has generated highly drug-resistant parasites, which amplify the burden of this disease and pose a serious obstacle to control efforts. This dissertation is motivated by the simple fact that malaria parasites have become resistant to nearly every antimalarial drug that has ever been used, yet the precise genetic mechanisms of parasite drug resistance remain largely unknown. Our work pairs genomics-age technologies with molecular biology, genetics and molecular epidemiology in order to identify and characterize novel genes that contribute to drug resistance in P. falciparum. In the Introduction, we highlight relevant opportunities and challenges in trying to identify and understand the genetic basis of malaria drug resistance as it emerges to currently used therapies. In Chapter One, we demonstrate how genome-wide association studies (GWAS) can be applied to P. falciparum in order to identify novel drug resistance loci. Functional follow-up revealed that overexpression of the novel candidate gene PF10_0355 made parasites more resistant to the drugs halofantrine, lumefantrine and mefloquine. These findings show that PF10_0355 plays a role in parasite drug response, as well as provides validation of our GWAS approach. In Chapter Two, we further characterize PF10_0355 and show that modulation of the gene by either knockout or allelic replacement changes parasite drug sensitivity. Furthermore, we show that moderate changes in drug response measured in the short-term can have dramatic effects when parasites are competed with one another under drug pressure. In Chapter Three, we use an overexpression approach to functionally follow up other novel drug resistance genes generated by GWAS in P. falciparum. We find that overexpression is a useful way to begin to screen candidate drug resistance loci in the malaria parasite. In Chapter Four, we use a DAPI-based ex vivo drug assay to monitor drug resistance among parasites circulating in Thiés, Senegal. In the future, we will look for genetic markers of parasite drug resistance in this population by GWAS. Finally, in the Discussion we present an essay about malaria evolution and eradication written for non-specialists. Our hope is that the work presented in this dissertation furthers understanding of drug resistance in the malaria parasite, both within and beyond the malaria research community.

The human brain is one of the most exquisite structures in nature, featuring extreme functional complexity and capacities that allow for advanced cognitive abilities. During the development of the human brain, neural progenitors undergo massive proliferation, which is known to inevitably result in spontaneous mutations; yet the degree of somatic mosaicism within the human brain is unexplored. Several hypotheses have been proposed that various types of somatic mosaicism may serve as an adaptive mechanism to diversify neurons and thereby promote the functional complexity of human brains. Previously proposed mechanisms to increase somatic mosaicism within the brain include elevated somatic LINE-1 element retrotransposition, and the creation of somatic aneuploidy during neurogenesis. On the other hand, genomic diversity needs to be balanced by genomic stability, in order to protect against deleterious mutations that reduce the fitness of the cells, or oncogenic mutations that might promote cancers. In fact, brain-specific somatic mutations have also been proposed to contribute to the unexplained burden of neurological diseases. To directly study genomic variability from cell-to-cell within the human brain, we developed a method to isolate and amplify single neuronal genomes from postmortem and surgically resected human brain tissues. We quantified the frequency of somatic LINE-1 retrotransposition events and aneuploidy in human cortical neurons, and found that the frequencies of both are low, with no sign of brain-specific elevation, arguing against the hypotheses that these two mutational sources are obligate generators of neuronal diversity. Additionally, aneuploidy analysis was performed on bulk and single cortical cells from a hemimegalencephaly brain. Hemimegalencephaly is an asymmetrical brain overgrowth syndrome caused by somatic mutations in brain. Single-cell analysis identified an unexpected mosaic tetrasomy of chromosome 1q, affecting both neuronal and glial populations, as a genetic cause of hemimegalencephaly. These results demonstrate that single-neuron sequencing allows systematic assessment of genomic diversity in the human brain and the identification and characterization of pathogenic somatic mutations underlying neurological disorders.

Adipose Fatty Acid Binding Protein 4 (FABP4) or aP2, plays an important role in several immunometabolic pathologies such as type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Long considered to be a cytosolic protein, aP2 has recently been detected in conditioned media of adipocytes. Interestingly, there is a growing body of literature showing association of increased circulating levels of aP2 with cardiovascular disease and metabolic syndrome. Our lab has discovered a role for aP2 secreted from adipocytes in regulating liver glucose output and blood glucose levels in diabetes. The emerging biology of this novel adipokine makes it critical to understand the route and mechanisms that lead to its secretion.

<p> Pierce&rsquo;s disease (PD), caused by bacterium Xylella fastidiosa, seriously hampers the cultivation of <i>Vitis vinifera</i> also known as bunch grapes, in different parts of the world. The bacterium clogs xylem vessels and forms a biofilm, resulting in the wilting of the plant. Bunch grape cultivars exhibit certain degree of tolerance to PD, however most commercial cultivars suffer heavy loss due to this devastating disease. Therefore, studies on genetic variation for disease tolerance will assist in identification of key molecular components that confer tolerance to PD. <i>Vitis</i> species, such as, Florida hybrid bunch (FH) and muscadine grape (<i> Vitis rotundifolia</i>) are widely cultivated in southeastern United States, and are known for their tolerance to PD. A detailed proteomic profile study of contrasting grape species is vital to understand the biological molecules associated with the PD tolerance. However information on total protein composition of <i>Vitis xylem</i> and sap is limited. The overall goals of this study are to determine the signal sequences associated with xylem and sap for the delivery of therapeutic proteins to control <i>Xylella fastidiosa. </i> The specific objectives of this research project are: 1) to compare the proteome profiles of xylem tissue and xylem sap from PD tolerant and -susceptible grapevine cultivars, and 2) to determine the role of proteins in the tissue and sap associated with PD tolerance mechanism. In this study, we used Bunch, FH, and Muscadine grape cultivars to characterize differentially expressed and unique proteins. Differentially expressed proteins were identified using LC MS/MS spectrometry searched against <i>Vitis</i> database. A total of 2519 and 402 proteins were identified in xylem and sap respectively, of which 151 proteins were common to both tissues. Bunch, FH, and muscadine sap showed 52, 53, and 30 unique proteins respectively. The cluster dendrogram analysis of the sap proteome showed that all of the <i>Vitis</i> species are bifolious. Based on the aforementioned, Florida hybrid bunch and muscadines are more closely related to each other than to bunch grape. Functional analysis and gene ontology revealed that proteins involved in carbohydrate metabolic process are more abundant in bunch grape, while FH and muscadine grape have more defense related proteins. Therefore, it is plausible to conclude that major functions of sap proteins in Bunch, FH, and Muscadine grapes are carbohydrate metabolic process and proteolysis (23%), protein phosphorylation (38%), and oxidation and reduction process (16%), respectively. Proteins involved in the defense and peroxidase activity are abundantly present in xylem and sap of FH and muscadine, and these proteins are relatively in reduced levels in bunch xylem and sap. Together, our findings highlight the possible roles of the identified unique proteins towards PD tolerance to Florida hybrid bunch and muscadine cultivars.</p>

Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.