<p> Meiosis is a highly conserved specialized cell division that occurs in many organisms, including budding yeast and mammals. Meiosis divides the chromosome number of the cell in half to create gametes for sexual reproduction. A single round of chromosome duplication is followed by two rounds of chromosome segregation, Meiosis I (homologs segregate) and Meiosis II (sister chromatids segregate). Proper segregation at Meiosis I requires that homologs are connected by both crossovers and sister chromatid cohesion. Crossovers are formed by the repair of double strand breaks (DSBs) preferentially by the homolog. The choice of repair template is determined at the time of strand invasion, which is mediated by two recombinases, Rad51 and the meiosis-specific Dmc1. Rad51 is necessary for Dmc1 to function properly but its strand exchange activity is inhibited both by Dmc1 and Mek1, a meiosis-specific kinase, which is activated by DSBs. Mek1 suppresses interaction between Rad51 and its accessory factor Rad54 in two ways. First, phosphorylation of Rad54 lowers its affinity for Rad51. Second, phosphorylation stabilizes Hed1, a meiosis-specific protein that binds to Rad51 and excludes Rad54. Although <i>RAD54</i> is not required for wild-type levels of interhomolog recombination, <i> rad54</i>&Delta; diploids exhibit decreased sporulation and spore viability, indicating the presence of unrepaired DSBs. My thesis tested the idea that Mek1 kinase activity is down-regulated after interhomolog recombination to allow Rad51-mediated repair of any remaining DSBs. </p><p> Meiotic recombination occurs in the context of a proteinaecous structure called the synaptonemal complex (SC). The SC is formed when sister chromatids condense along protein cores called axial elements (AEs) comprised of the meiosis-specific proteins, Hop1, Red1 and Rec8. AEs are brought together by interhomolog recombination, which creates stable connections and the gluing together of the AEs by the insertion of the transverse filament protein, Zip1, in a process called synapsis. Pachynema is the stage of meiotic prophase in which chromosomes are fully synapsed and where interhomolog recombination has proceeded to the double Holliday junction (dHJ) stage. </p><p> Meiotic progression requires transcription factor <i>NDT80</i>, a middle meiosis transcription factor required to express >200 genes, including the polo-like kinase, CDC5 (required for Holliday junction resolution and SC disassembly) and <i>CLB1</i> (required for meiotic progression). Diploids deleted for <i>NDT80</i> arrest in pachynema with unresolved dHJs. I used an inducible version of <i>NDT80</i> (<i>NDT80-IN </i>) to separate prophase into two phases: pre-<i>NDT80</i>, when interhomolog recombination occurs and post-<i>NDT80</i>, when it is proposed that inactivation of Mek1 allows intersister recombination to repair residual DSBs. <i>RAD54</i> is sufficient to function after interhomolog recombination, as inducing both <i>RAD54</i> and <i>NDT80</i> simultaneously rescues the spore inviability defects observed in <i>NDT80-IN rad54&Delta;</i> diploids. Using an antibody specific for phosphorylated Hed1 as an indicator of Mek1 kinase activity, I showed that Mek1 is constitutively active in <i>ndt80</i>-arrested cells and that induction of <i>NDT80</i> is sufficient to abolish Mek1 activity. Furthermore, inactivation of Mek1 by Ndt80 can occur in the absence of interhomolog strand invasion and synapsis. Mek1 inactivation correlates with the appearance of <i>CDC5</i> and the degradation of Red1. My work demonstrates that the sole target of <i>NDT80</i> responsible for inactivating Mek1 is <i>CDC5</i>. </p><p> Unrepaired DSBs trigger the meiotic recombination checkpoint resulting in prophase arrest, which requires Mek1 and works by sequestering Ndt80 in the cytoplasm. Mek1 also delays meiotic progression in wild-type cells, likely through inactivation of Ndt80. My work shows that Ndt80 in turn negatively regulates Mek1. Based on my observations, as well as published work showing that synapsis results in the removal of Mek1 from chromosomes, I propose that recombination and meiotic progression are coordinated by regulation of Mek1. </p>

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Kristi A. Wharton. Includes bibliographical references.

<p> There are two important features that characterize the canonical genetic code. First, the genetic code is redundant. There are 64 three-letter words that can be assembled from four letters (4³), but only 20 types of amino acids are translated. Combinations of only 20 chemistries have been more than sufficient to generate the amazing diversity of life. However, if we could minimize this redundancy to encode additional nonstandard amino acids (NSAAs), this could permit the evolution of entirely new biological functions and possibly, new organisms.</p><p> Second, the genetic code is conserved. All species utilize the same biochemical foundation to sustain life. This conservation permits organisms to share beneficial traits via horizontal gene transfer (<i>4</i>) and enables accurate expression of heterologous genes in non-native organisms (<i>10</i>). However, the conservation of the genetic code also allows viruses to hijack host translation machinery (<i>15</i>) and compromise cell viability. It further allows unwanted sharing of information between genetically modified organisms (GMOs) and their environment. Virus resistance and intrinsic biocontainment &ndash; biological barriers limiting the spread and survival of microorganisms in natural environments &ndash; are among today's major unsolved problems in biotechnology.</p><p> Changing the genetic code to create genomically recoded organisms (GROs, whose codons have been reassigned to create an alternate genetic code) could solve these challenges. The focus of my graduate work was to construct and characterize GROs that: 1) provide dedicated codons to enable sustained incorporation of more than 20 amino acids as part of an expanded genetic code, and 2) depend on synthetic biochemical building blocks for viability, to advance orthogonal barriers between organisms and their environment.</p><p> <b>Chapter 2.</b> Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of an orthogonal translation system (OTS) consisting of an engineered elongation factor and archaeal aminoacyl-tRNA synthetase (aaRS)/tRNA pair to the normal <i>E. coli</i> translation machinery (<i>16</i>). However, protein yield suffered horn expression of the OTS and competition with release factor 1 (RF1). In a strain lacking RF1, phosphoserine phosphatase, and where only seven essential TAG codons were converted to TAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability. </p><p> <b>Chapter 3.</b> To create a GRO we replaced all known TAG stop codons in <i>E. coli</i> with synonymous TAA codons, which permitted the deletion of RF1 and reassignment of UAG translation function. This GRO exhibited improved cellular fitness and improved properties for incorporation of NSAAs that expand the chemical diversity of proteins <i>in vivo.</i> The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.</p><p> <b>Chapter 4.</b> GMOs are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals (<i> 17</i>). Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems (<i>18, 19</i>), which include bioremediation (<i>20</i>) and probiotics (<i>21</i>). Here, we describe the construction of a series of GROs (<i>22</i>) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced in-frame TAG codons into 22 essential genes of a GRO containing an OTS, linking their expression to the incorporation of sAAs. Of the 60 variants isolated, notable strains were not rescued by environmental cross-feeding, maintained robust growth, and exhibited undetectable escape frequencies upon culturing &sim;10¹¹ cells in liquid media for 20 days, a significant improvement over existing biocontainment approaches (<i>18, 19, 23&ndash;27</i>). </p>

<p>Dover sole (<i>Microstomus pacificus</i>) is a commercially important, slope dwelling flatfish of the northeast Pacific coast. Its genetic diversity at the mitochondrial DNA control region appears substantially lower than another commercially important flatfish, Pacific sanddab (<i>Citharichthys sordidus</i>). I designed a comparative study along depth and latitudinal gradients using five flatfishes and one brotula. In the control region's left domain, genetic diversity of six species trended lower with increasing habitat depth at Palos Verdes: shallow species had high genetic diversity and deep dwelling species (ex. Dover sole) had low genetic diversity. This diversity gradient may follow decreases in mass specific metabolic rates as Dover sole grow, invade the oxygen minimum zone and assume higher tissue water content. The left domain from 64 Dover sole specimens was compared across 4 latitudinal locations. Genetic diversity trended higher with increasing latitude, possibly due to cold water emergence as biomass shift shallower with increasing latitude. </p>

<p> Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death in the United States. Twin cohort studies indicate that inherited susceptibility accounts for approximately 35% of all CRC cases, but only 5-6% of CRC cases can be attributed to known functional mutations. We were the first to identify a germline mutation in Transforming Growth Factor Beta Receptor 1 (<i>TGFBR1</i>) that is also somatically acquired in tumors, a 9 bp in frame deletion within exon 1 (rs11466445), which results in a receptor with decreased TGF-&beta; signaling properties. The observed association between this hypomorphic variant and cancer risk led us to hypothesize that constitutively decreased TGF-&beta; signaling may contribute to the development of CRC. </p><p> In this dissertation, we developed a novel mouse model of <i>Tgfbr1 </i> haploinsufficiency (<i>Tgfbr1</i>^+/&minus;) and found that <i>Tgfbr1</i>^+/&minus; mice were twice as likely as <i>Tgfbr1</i>^+/+ mice to develop CRC. We subsequently identified two human haplotypes associated with constitutively decreased <i>TGFBR1</i> expression and CRC risk and found that decreased <i> TGFBR1</i> expression is strongly associated with three SNPs: rs7034462, rs11466445 and rs11568785. Further examination of <i>TGFBR1</i> haplotype tagging SNPs suggests that the <i>TGFBR1</i> rs7034462-TT is a novel moderate penetrance risk genotype, which has high penetrance among African Americans, the ethnic group with the highest risk for CRC. Our results provide strong support for the novel notion that rs7034462-TT is a potentially clinically relevant CRC susceptibility genotype that may identify individuals at high risk of dying from CRC.</p>

<p> Leaf senescence is the final stage of leaf development where older leaves undergo an active degenerative process. This highly coordinated event is characterized by a cascade of differential gene expression resulting in senescence upregulated and senescence downregulated genes. Cytosine methylation, a mechanism of epigenetic control, has been shown to play a role in regulating gene expression. Gene body cytosine methylation is correlated with transcriptional activation while promoter cytosine methylation is correlated with transcriptional repression. Evidence from previous work suggests CG methylation (^mCG) in promoter regions plays a role in repressing gene expression and that a correlation between demethylation and mRNA induction is most likely within 500 bp up- and downstream of TSSs. The purpose of this study is to investigate the correlation between promoter cytosine methylation and transcriptional repression by identifying potential cytosine methylation-regulated senescence upregulated genes (CMR-SURGs). Four candidate CMR-SURGs were identified from previously generated RNA-seq data and an online cytosine methylome. We hypothesized that the four CMR-SURGs would display a correlation between mRNA induction and loss of promoter ^mCG. mRNA expression was measured by real-time qPCR, and cytosine methylation was quantified by bisulfite treatment of genomic DNA followed by PCR, cloning, and sequencing of PCR products. These data however, showed that only <i>UGT78D1</i> displayed a negative correlation between promoter cytosine methylation and age-related mRNA induction.</p><p>

<p> The integrity of the mechanisms that control gene transcription during development and in post-natal life is essential to maintain tissue homeostasis and impede the development of genetic diseases such as cancer. Inheritance of a defective hyperpatahyroidism 2 (<i>HRPT2</i>) allele, an essential regulator of gene transcription, predisposes individuals to a constellation of symptoms ranging from endocrine abnormalities to parathyroid adenomas and jaw bone tumors (HPT-JT). In order to elucidate the function of the <i> HRPT2</i> gene and the pathogenesis that results upon spontaneous inactivation in familial cases of parathyroid cancer and HPT-JT, mouse models were generated that allow for deletion of <i>Hrpt2</i> within different stages and tissues during development. We have used the <i>Hrpt2</i> flox mouse model to delete <i>Hrpt2</i> in mesenchymal progenitor cells as well as committed, terminally differentiated osteoblasts and osteocytes. Whereas loss of <i>Hrpt2</i> in mesenchymal progenitors was embryonic lethal, genetic deletion of <i>Hrpt2</i> in mature bone forming cells led to increased bone mass and bone strength. However, <i>Hrpt2 </i> conditional knockout bones had increased cortical porosity and osteocyte apoptosis associated with increased osteoblast specific gene expression. This work suggests that <i>Hrpt2</i> is required for cell proliferation and differentiation and acts as a transcriptional repressor in terminally differentiated cell types.</p><p> Control of gene transcription defines cell identity and fate. Ascorbic acid (AA, also known as vitamin C) is an essential vitamin for humans and is well known for its role in collagen synthesis. AA acts as a cofactor for TET enzymes, which hydroxylate methylated cytosines. Here, we characterize how 7 days of AA treatment causes changes in gene transcription, 5-hydroxymethylcytosine deposition, and the active chromatin marks H3K4me3 and H3k27ac in MC3T3-E1 murine pre-osteoblasts cells, initiating cell differentiation and expression of the osteoblast phenotype. Though 5hmC deposition was not specific for only highly expressed genes, it was highly enriched at transcriptional start sites and CpG islands. While H3K4me3 was mostly unchanged, H3K27ac was predictive of driving gene expression. This work suggests that AA causes dramatic changes to the epigenome through epigenetic modifiers to impact cell differentiation. </p><p>

Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53-/- mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere-forming assays and the DsRed-let7c-sensor. The frequency of long-term label-retaining epithelial cells (LRECs) was decreased in Trp53-/- mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ-secretase (DAPT) reduced the number of Trp53-/- mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells. Notch is a target of γ-secretase suggesting that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. The expansion of p53-deficient mammary stem/progenitor cells can also be reversed after the expression of C-terminal p53, suggesting that the C-terminal domains of p53 may be responsible for the regulation of mammary stem/progenitor cells self-renewal. In parous mammary gland, increased p53 responsiveness sensitized mammary stem/progenitor cells to ionizing radiation without affecting the self-renewal of these cells, which may be responsible for the parity-induced protection against breast cancer.

The Drosophila morgue gene was identified as a regulator of programmed cell death and protein ubiquitination. It has been shown to enhance programmed cell death via promoting the turnover of DIAP1, a conserved anti-apoptotic protein. Morgue protein contains a zinc finger motif, an F box domain and a ubiquitin E2 conjugase variant domain with a Cysteine to Glycine substitution at the catalytic site. This unique domain/motif architecture suggests that Morgue may have very distinctive activities. However, how and what each domain/motif contributes to Morgue function remains unexplored. My dissertation project focused on a study of Morgue protein evolution and function using a combination of bioinformatics, genetics and biochemical methods. The results suggest that Morgue exhibits widespread but restricted phylogenetic distribution among invertebrate metazoans; the study of Morgue's origin provides an example of how multi-domain proteins may evolve. Results of functional studies revealed that over-expression of Morgue can induce a homozygous lethal phenotype that is independent of either F-box or the Glycine in the UEV domain. In addition, co-immunoprecipitation experiments have shown that Morgue associates with SkpA and Lys48 linked polyubiquitin chains, indicating that Morgue might be a multi-functional protein in PCD and ubiquitination.

Brachypodium distachyon (brachypodium) is a temperate grass that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In maize, Yellow Stripe1 (ZmYS1) is the transporter responsible for uptake of Fe(III)-PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. One aim of this study was to establish brachypodium as a model system to study Fe homeostasis in grasses. HDMA was detected as the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated in parallel to that of related species such as barley and wheat. Nineteen YSL family members were identified in brachypodium, and their expression profiles in response to Fe deficiency were analyzed. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. The Fe status of the plant can regulate expression of several BdYSL genes in both shoots and roots suggesting roles in Fe homeostasis. Brachypodium YS1 knockdown lines were also generated to test Fe(III)-PS transport abilities of certain YSL proteins. In the context of this dissertation, an EMS mutagenized population of brachypodium was produced. One mutant with the iron deficiency symptom interveinal chlorosis was characterized. Metal content analyses revealed that mutant plants are low in Fe, Zn and Cu. Mutant plants are capable of synthesizing PS in normal amounts, but they do not release the PS from roots into the soil solution, suggesting that gene underlying this mutation functions to allow secretion of PS. Performing bulk segregant array mapping and fine mapping using SNP markers, the location of this new mutation was mapped to a 1.7Mb interval on chromosome 5. Cloning of this gene is likely to fill in the missing part of the iron uptake mechanism in grasses.