Accurate and complete DNA replication is essential for maintaining the integrity of the genome. In eukaryotes, this process requires the coordinated action of numerous molecular machines. Based on yeast and animal model systems, we defined a set of fifty-one ?core DNA replication proteins? that are integral to the initiation, DNA synthesis, and Okazaki fragment maturation functions of DNA replication. We used computational analyses to identify putative homologs in the genomes of two plants, Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), providing the first comprehensive view of the core DNA replication machinery in plants. Our results indicated that the overall composition of this apparatus is conserved, but plants are unique in that multiple DNA replication genes exist as small gene families. Fourteen of the genes we annotated in this study have not been previously reported in the literature, and we have provided revised gene models for seventeen plant proteins. To better understand how the DNA replication machinery functions in plants, we cloned multiple subunits of the pre-replication complex (pre-RC) from Arabidopsis and generated antibodies against four key components of this complex ? AtORC1, AtORC2, AtMCM5, and AtMCM7. We demonstrated that the pre-RC is developmentally regulated in Arabidopsis and, consistent with a role in DNA replication, is abundant in proliferating tissues. We used immunocytochemical and biochemical methods to characterize MCM7 in plants. We observed two distinct localization patterns for plant MCM7 proteins. In most cells, MCM7 was nuclear and colocalized with DNA. In a small fraction of cells, MCM7 was dispersed throughout the cytoplasmic compartment. Biochemical analysis confirmed that MCM7 binds to chromatin and that it is present in the nucleus at least during the G1, S and G2 cell cycle stages. Together, these analyses support a model where the MCM complex is loaded onto DNA in late M and early G1, released into the nucleoplasm during S phase followed by a brief dispersion into the cytoplasmic compartment concurrent with nuclear envelope breakdown in mitosis.

Bacteria are remarkable in their ability to adapt to environmental conditions that are continually in flux between growth-promoting and growth-limiting. Responses to a host of environmental situations are equally varied, ranging from the secretion of antimicrobial compounds and polymer degrading enzymes, to the up-regulation of alternative cellular developmental pathways leading to complete physiological transformation. In endospore forming bacteria this results in a metabolically inert, yet highly resistant endospore. The first study presented here deals with the NMR structural and dynamic characterization of a class of proteins in Bacillus subtilis known as transition-state regulators, responsible for global gene regulation during the transition from the vegetative mode of growth to the semi-quiescent stationary phase. The utilization of protein-DNA docking protocols further allows for the first description of a structural model for the interaction between these DNA-binding proteins and a cognate DNA promoter sequence. The later portions of this dissertation deal with the characterization of proteins involved in the ubiquitous bacterial signal transduction system known as the two-component signal transduction pathway. In the basic two-component signal transduction paradigm, an environmental signal is detected by a multi-domain sensor kinase that, via phosphorylation, activates a response regulator protein for its cellular role (be it DNA-binding, RNA-binding, enzymatic, etc). In the second study, a comparative modeling analysis of the predicted receiver domains the response regulators from Vibrio vulnificus YJ016 was performed, using the hydrophobic characteristics of the response regulator surface known to interact with the four-helix bundle of the cognate sensor kinase as the basis for sub-classification. In the final study, a new mass spectrometric technique to detail the structural changes in proteins resulting from oxidative damage was applied to the single domain response regulator Spo0F from B. subtilis.

Protein disulfide isomerases (PDIs) contain thioredoxin domains and aid in the formation of proper disulfide bonds during protein folding. Iterative BLAST searches of sequence databases were used to identify 22 PDI-like (PDIL) genes in Arabidopsis thaliana and maize (Zea mays) and 19 in rice (Oryza sativa). The PDIL genes were resolved into 10 phylogenetic groups. Genes in groups I-V had two active thioredoxin domains while members of groups VI-X had one active thioredoxin domain. One single domain PDIL, maize PDIL5-1, showed increased accumulation in the endosperm mutants that produced defective storage proteins, but PDIL5-1 was not localized to endomembrane fractions. Expression analysis was done in eighteen PDIL genes in maize endosperm, a storage tissue, and two vegetative organs, embryo and leaves. Eight PDIL genes were expressed mainly in endosperm, and two of these genes (PDIL1-2 and 2-3) had increased expression levels only in the endosperm that produced defective storage proteins. There were three PDIL genes (1-3, 1-4, 1-5) that showed the highest expression levels in the embryo, while two other genes, adenosine 5?-phosphosulfate reductase-like (APRL) 2 and APRL8, had elevated expression levels in leaves. To further characterize members of the gene family, isomerase and reductase assays were conducted to test for recombinant maize PDIL1-1, 1-3, 2-3 and 5-1 enzymatic activity in vitro. Recombinant and endogenous maize PDIL1-1 showed both isomerase and reductase activity while recombinant PDIL1-3 showed anti-chaperone activity. Recombinant PDIL2-3 and PDIL5-1 showed no activity under the assay conditions tested.

We analyzed gene expression during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (N=185) were allotted to a 2x2 factorial experiment consisting of a non-obese (ICR-control) and an obese (M16-selected) line of mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) gain by day 14 was 12% lower in CLA compared to LA fed mice (P<0.0001). By day 14, t10c12-CLA reduced weights of epididymal, mesenteric and brown adipose tissues as a percentage of BW in both lines by 30, 27 and 58%, respectively, and increased liver weight/BW by 34% (P<0.0001). Total RNA was isolated and pooled (4-5 mice per composite) from epididymal adipose (day 5 & 14) and liver (day 14) of the obese mice to analyze gene expression profiles using Agilent mouse oligo microarray slides (4 per tissue?day) representing >20,000 genes. Numbers of genes differentially expressed by ≥ two fold in epididymal adipose (day 5 & 14) and liver (day 14) were 29, 125, and 80, respectively. Of particular interest in adipose, CLA putatively increased expression of the uncoupling proteins (1 and 2), carnitine palmitoyltransferase (L and M), and carnitine translocase, but decreased expression of PPAR-gamma, GLUT-4, perilipin, caveolin-1, adiponectin and resistin (P<0.01). In conclusion, this experiment has revealed candidate genes that will be useful in elucidating mechanisms underlying the potent anti-adipogenic effects of t10c12-CLA.

Here I used microarrays to identify genes that are activated or repressed by nicotine and caffeine in Drosophila melanogaster. I compared genotypes with differential resistance to each drug in order to select genes that may be involved in resistance to the drugs. Comparison of the genes differentially expressed by both drugs leads me to propose that there are common mechanisms of metabolic resistance to caffeine and nicotine, in particular cytochrome P-450-mediated mechanisms. Caffeine seems to have a more dramatic influence on gene expression than nicotine, in particular on expression of genes involved in energy metabolism. Next, I extended the studies on nicotine resistance to ask whether there are differences in response between two populations of Drosophila. The gene expression patterns of both populations were evaluated separately and in a combined analysis. Most of the differentially expressed genes were up-regulated by nicotine in both populations and in the combined analysis. The induced transcripts were mainly related to protein, nucleic acid, amino acid and energy metabolism, and response to stimulus and stress. Those findings suggest that amino acid and energy metabolism may be important biological processes affected by nicotine and be interesting targets for further investigation related to the nicotine response in Drosophila. The two populations displayed considerable differences in gene expression profiling that may be the result of the observed phenotypic variation for nicotine response between the two populations. Most of the differential expression induced by nicotine seems to be specific to the more resistant population. Finally, I focused on genes whose expression showed significant correlation with survival time on nicotine food. Using a regression approach, it was possible to map quantitative trait transcripts associated to nicotine response. Control expression of alkaline phosphatase and ornithine aminotransferase displayed significant correlation to survival time in drug food. They seem to be linked to regulation of GABA/glutamate neurotransmission and detoxification mechanisms, which ultimately counteracts the stimulatory effects of nicotine.

The genomes of six Streptococcus thermophilus bacteriophages were compared to identify genes that could be targeted by engineered phage defense systems with potentially widespread efficacy. The genes associated with the S. thermophilus phage Sfi21-prototype genome replication module, including a putative primase and a putative helicase, were found to be among the best candidates due to their frequency of distribution in industrial phage isolates, striking sequence conservation between independent isolates, and intrinsic strategic importance in early phage development. Fourteen antisense RNA cassettes targeting the phage k3-derived helicase (hel3) or primase (pri3) genes were expressed in S. thermophilus NCK1125. These constructs consistently reduced the efficiency of plaquing (EOP) of phage k3 to between 5 x 10-1 and 2.0 x 10-3 depending on the (i) gene targeted and (ii) region of the gene that was targeted. The largest antisense RNAs were generally found to confer the largest reductions in EOP, however shorter antisense RNAs designed to the 5' region of the gene retained much of the inhibitory function, especially if they contained sequences complementary to the ribosome binding site. Expression of antisense RNAs correlated with decreased levels of phage encoded primase transcripts, likely due to increased degradation of the dsRNA complex. This, in turn, correlated with diminished phage genome replication and aborted phage development. In a separate study, invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutation within the S. thermophilus phage k3-encoded putative primase. PCR products containing the desired mutation(s) were cloned and expressed in S. thermophilus NCK1125. The majority of the examined constructs remained sensitive to phage k3, however four constructs conferred strong phage resistance to the bacterial host. The mutated residues resided within a putative ATPase/helicase domain suspected to be critical for primase function in vivo. The co-expression in trans of the K238(A/T) or RR340-341AA mutant proteins suppressed the function of the native, phage-encoded primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. According to this model, the plasmid-encoded mutant primase subunits are structurally intact and form stable interactions with the native, phage-encoded primase subunits, thus inhibiting their activity. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing more that nine log cycles. Given the magnitude of the resistance conferred, it was concluded that the putative primase is essential for genome replication in S. thermophilus Sfi21-type phages. Further, it was also clear that host-encoded factors were unable to complement the resultant deficiency. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and phage sensitivity of the host, indicating that translation was required to confer phage resistance. Residues within a critical oligomerization domain were also identified through genetic analysis. Introduction of an E437A mutation downstream of the transdominant K238T mutation completely suppressed phage resistance, indicating that the E437A mutation precluded the association of the mutant and native subunits. To our knowledge, this is the first use of subunit poisoning to inhibit phage replication in the lactic acid bacteria.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

Embryonic stem cells (ESCs) are a special type of cell marked by two key properties: The capacity to create an unlimited number of identical copies of themselves (self-renewal) and the ability to give rise to differentiated progeny that can contribute to all tissues of the adult body (pluripotency). Decades of past research have identified many of the genetic determinants of the state of these cells, such as the transcription factors Pou5f1, Sox2 and Nanog. Many other transcription factors and, more recently, epigenetic determinants like histone modifications, have been implicated in the establishment, maintenance and loss of pluripotent stem cell identity. The study of these regulators has been boosted by technological advances in the field of high-throughput sequencing (HTS) that have made it possible to investigate the binding and modification of many proteins on a genome-wide level, resulting in an explosion of the amount of genomic data available to researchers. The challenge is now to effectively use these data and to integrate the manifold measurements into coherent and intelligible models that will actually help to better understand the way in which gene expression in stem cells is regulated to maintain their precarious identity. In this thesis, I first explore the potential of HTS by describing two pilot studies using the technology to investigate global differences in the transcriptional profiles of different cell populations. In both cases, I was able to identify a number of promising candidates that mark and, possibly, explain the phenotypic and functional differences between the cells studied. The pilot studies highlighted a strong requirement for specialised software to deal with the analysis of HTS data. I have developed GeneProf, a powerful computational framework for the integrated analysis of functional genomics experiments. This software platform solves many recurring data analysis challenges and streamlines, simplifies and standardises data analysis work flows promoting transparent and reproducible methodologies. The software offers a graphical, user-friendly interface and integrates expert knowledge to guide researchers through the analysis process. All primary analysis results are supplemented with a range of informative plots and summaries that ease the interpretation of the results. Behind the scenes, computationally demanding tasks are handled remotely on a distributed network of high-performance computers, removing rate-limiting requirements on local hardware set-up. A flexible and modular software design lays the foundations for a scalable and extensible framework that will be expanded to address an even wider range of data analysis tasks in future. Using GeneProf, billions of data points from over a hundred published studies have been re-analysed. The results of these analyses are stored in an web-accessible database as part of the GeneProf system, building up an accessible resource for all life scientists. All results, together with details about the analysis procedures used, can be browsed and examined in detail and all final and intermediate results are available and can instantly be reused and compared with new findings. In an attempt to elucidate the regulatory mechanisms of ESCs, I use this knowledge base to identify high-confidence candidate genes relevant to stem cell characteristics by comparing the transcriptional profiles of ESCs with those of other cell types. Doing so, I describe 229 genes with highly ESC-specific transcription. I then integrate the expression data for these ES-specific genes with genome-wide transcription factor binding and histone modification data. After investigating the global characteristics of these "regulatory inputs", I employ machine learning methods to first cluster subgroups of genes with ESC-specific expression patterns and then to define a "regulatory code" that marks one of the subgroups based on their regulatory signatures. The tightly co-regulated core cluster of genes identified in this analysis contains many known members of the transcriptional circuitry of ESCs and a number of novel candidates that I deem worthy of further investigations thanks to their similarity to their better known counterparts. Integrating these candidates and the regulatory code that drives them into our models of the workings of ESCs might eventually help to refine the ways in which we derive, culture and manipulate these cells - with all its prospective benefits to research and medicine.

Functional genomics uses high throughput genome-wide technologies to investigate the functional consequences of genetic variants on gene expression and protein products. In the context of disease, using integrative functional genomic approaches to understand the genetic variation which underlies disease susceptibility and aetiology may contribute to better diagnosis, treatment and prevention. This thesis investigated genetic determinants of variation in response to infection by applying a functional genomics approach to investigate three clinical cohorts: patients with severe sepsis, an influenza challenge study and patients with common variable immune deficiency disorders. The transcriptomic response to severe sepsis is reported here for the largest known adult severe sepsis community acquired pneumonia cohort. Two clusters within the cohort, based on gene expression signatures, which have different survival rates and identify individuals with distinct immune responses to sepsis, highlight the value of functional genomics for identifying heterogeneity within patient cohorts. This was further complemented by the resolution of gene expression signatures in healthy individuals following influenza challenge which identified individuals with moderate to severe disease. Shared gene expression signatures between the cohorts highlighted components of the immune response to viral infection important across multiple clinical settings and may assist with the identification of viral infections in the sepsis patients. Gene expression was mapped as a quantitative trait (eQTL). Comparison to data sets for healthy individuals and from innate immune stimulated cells identified regulatory variants specific to the context of sepsis. Whole genome sequencing for a cohort of patients with common variable immune deficiency disorders was performed. This identified novel variants and pathways which may play a role in the underlying immunopathogenesis of disease. Integration with RNA-seq for a small number of patients allowed prioritisation of non-coding variants based on evidence of altered gene expression. Comparison to the sepsis cohort analysis identified key immune related genes involved in rare and common responses to bacterial infection. This thesis has demonstrated the value of integrating multiple functional genomic techniques to further our understanding of the mechanisms underlying variation in the response to infection.