Methodological advances in directed evolution have already made it possible to discover useful biomolecules within months to years. A further acceleration of this process might make it possible to address outstanding challenges, or needs for which the current timescale is a fundamental barrier. To realize these goals would require transformative methodological advances in directed evolution. In Chapter One, current methods in directed evolution are briefly reviewed. In Chapter Two, a general platform for continuous directed evolution is presented. The method is used to evolve T7 RNA polymerase enzymes with novel promoter activity on the days timescale. In Chapter Three, a method is developed for tuning selection stringency during continuous evolution, thus obviating the requirement for a minimal starting library activity. In Chapter Four, a method is developed for simultaneous positive and negative selection, thus allowing explicit selection for substrate specific enzymes. In Chapter Five, the advances in stringency modulation and negative selection are combined to evolve highly substrate specific enzymes starting from an inactive starting library. In a continuous evolutionary arc of less than three days, we discover T7 RNA polymerase enzymes with a degree of specificity for the T3 promoter exceeding that of the wild type enzyme for its native substrate.

<p>DNA replication is the process of synthesizing an exact copy of a genome during S-phase. DNA replication must only occur once and only once in the cell cycle. Therefore, the DNA replication program is a highly regulated process that is established prior to S-phase. In G1, the protein complexes that define an origin of replication are assembled stepwise onto chromatin. Potential origins of replication are first bound by ORC, origin recognition complex. ORC then recruits other factors that load the Mcm2-7 helicase onto the chromatin to assemble a pre-RC, pre-replication complex. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 are broadly distributed on chromatin, exhibit little co-localization with ORC or replication foci, and can function as dormant origins. I used biochemical and genomic approaches to dissect the mechanisms regulating the assembly and distribution of the Mcm2-7 complex in Drosophila tissue culture cells. I found that Mcm2-7 loading occurs in two distinct phases during G1. In the first phase, limiting amounts of Mcm2-7 are loaded at ORC binding sites in a cyclin E/Cdk2 independent manner. Subsequently, there is a cyclin E/Cdk2 kinase activity dependent phase of Mcm2-7 loading that results in a 15-fold increase in chromatin associated Mcm2-7 and a dramatic genome-wide reorganization of the distribution of Mcm2-7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 kinase activity over the course of G1 is not only critical for Mcm2-7 loading, but also the distribution of the Mcm2-7 helicase prior to S-phase entry. </p><p>The assembly of the pre-RC is not only required for DNA replication, but it has been implicated in being required for cohesin loading. The cohesin complex imparts cohesion between sister chromatids as they are replicated and remains in place until the sister chromatids are separated in mitosis. I assessed if pre-RC assembly is required for cohesin loading using genomic and biochemical approaches in Drosophila tissue culture cells. I found that pre-RC components co-localize with cohesin subunits throughout the Drosophila genome. I was unable to detect any cohesin loading onto chromatin mediated by pre-RC assembly or components in vivo. However, this result does not mean that they are not coordinated. </p><p>Any errors during DNA replication can cause genomic instability through rereplication, fragile sites, or stalled forks. In addition, other processes like sister chromatid cohesion that are coordinated with DNA replication can also introduce genomic instability. Aneuploidy is a potential consequence of sister chromatid cohesion defects resulting in unequal multiples of a genome within a cell. Aneuploidy can be detrimental to a cell or organism due to copy number variation (CNV) causing differences in expression of genes. However, cells are able to compensate for CNV between the sexes due to the differences in the number of sex chromosomes. I used genomic approaches to characterize three aneuploid Drosophila cell lines for the modENCODE project. I further characterized the S2 Drosophila cell line using immunofluorescence microcopy approaches to identify the chromosomal rearrangements that were mapped by de novo assembly of the genome. Both approaches showed that the S2 cell line has highly rearranged chromosomes. The S2 cell line was also analyzed to address if cells can compensate for CNV on autosomes using genomic approaches. In collaboration with the Oliver group (NIH) we found that S2 cells are able to compensate for CNV of autosomal genes by buffering gene expression.</p><p>In summary, my research explored mechanisms that a cell can employ to maintain genomic stability: assembly of dormant origins, chromosome segregation, and CNV compensation.</p> / Dissertation

<p>The t(10;11) chromosomal translocation gives rise to the <italic>CALM-AF10</italic> fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. <italic>CALM-AF10</italic>-driven leukemias are characterized by a perturbed epigenetic and transcriptional state. Specifically, the <italic>HOXA</italic> cluster genes are hypermethylated on Histone H3 lysine 79 (H3K79), which corresponds with their transcriptional upregulation. Conversely, <italic>CALM-AF10</italic> cells display global H3K79 hypomethylation. DOT1L, the H3K79 histone methyltransferase, interacts with the OM-LZ domain of AF10, and the AF10 OM-LZ domain has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. These data have suggested a critical role for the AF10-DOT1L interaction in <italic>CALM-AF10</italic> leukemias. However, the mechanism(s) by which DOT1L-mediated epigenetics are perturbed and the precise role of CALM in leukemogenesis have remained unclear. </p><p>In this dissertation, we examine the contribution of CALM to CALM-AF10-mediated leukemogenesis. We determine that CALM contains a functional nuclear export signal (NES) that mediates steady-state cytoplasmic localization of CALM-AF10. An NES is a highly conserved leucine-rich amino acid sequence that is recognized by the nuclear export receptor, CRM1. Classically, CRM1 binds to NES-containing proteins and mediates their export from the nucleus to the cytoplasm through the nuclear pore complex. Through structure-function analyses, we determine that the CALM-derived NES is necessary and sufficient for CALM-AF10-dependent leukemogenesis. In addition, fusions of NES motifs from heterologous proteins (ABL1, Rev, PKIA, and APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors <italic>in vitro</italic>. From these data, we conclude that a CRM1-dependent NES represents the functional contribution of CALM for CALM-AF10-mediated leukemogenesis. </p><p>In the second part of this dissertation, we examine the mechanism(s) by which the CALM NES imparts transformation potential to AF10. We determine that the CALM NES is essential for CALM-AF10-dependent <italic>Hoxa</italic> gene upregulation and aberrant H3K79 methylation. Using co-immunofluorescence microscopy, we observe increased cytoplasmic localization of DOT1L in the presence of CALM-AF10, suggesting that mislocalization of DOT1L may lead to a global loss of H3K79 methylation. In addition to mediating nuclear export, we find that the CALM-CRM1 interaction is critical for targeting CALM-AF10 to the <italic>Hoxa</italic> locus. Inhibition of CRM1 with Leptomycin B prevents transcription of <italic>Hoxa</italic> genes in <italic>CALM-AF10</italic> leukemia cells. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of CRM1 inhibitors as therapeutic agents for patients with <italic>CALM-AF10</italic> leukemias.</p> / Dissertation

<p> Transforming Growth Factor Beta Induced Gene Human Clone 3, BIGH3, is an extra cellular matrix protein expressed by different cell types. BIGH3 promotes cell adhesion and has been recognized as a tumor suppressor protein in many studies, a function consistent with the finding that the expression of BIGH3 is reduced in various tumors and transformed cells when compared to healthy counterparts. </p><p> In the present study, we found that BIGH3 induces MG63 multi tumor spheroid (MTS) cells apoptosis and antagonized the development of tumor cells into large aggregate, supporting BIGH3 tumor suppressor role. MG63 spheroids were cultured in recombinant BIGH3 and vascular smooth muscle cells (VSMCs) conditioned medium . We have shown BIGH3 to be abundantly expressed by VSMCs. In addition, stimulation of BIGH3 gene by TGF-&beta;1 in MG63 cells resulted in overexpression of BIGH3 and subsequent increase in apoptosis by almost 3 fold. TUNEL assay was performed to detect apoptotic cells. Smaller and scattered tumor spheroids were observed in TGF-&beta;1 treated cells. Importantly, in-house developed anti- BIGH3 antibody reduced apoptosis percentage by almost one-half and antagonized the development of osteosarcoma cells into large aggregate spheroids. Within the formed spheroids, BIGH3 was immunologically detected in in the stroma and at cell bodies, suggesting a possible binding of BIGH3 to the cell surface. Collectively, these data suggest that BIGH3 plays a suppressive role in development of osteosarcoma tumor spheroids. </p><p> MG63 were cultured in agar-coated wells, where they developed into 3D aggregates. Unlike classical monolayer-based models (2D), multicellular tumor spheroid (MTS) cell culture system mimics the in vivo 3D structure of a solid tumor.</p>

<p> Many fungal species are studied to understand pheromone and receptor interaction associated with mating behavior. An established genetic model for mating in higher fungi is heterothallic <i>Schizophyllum commune </i>. This fungus uses its pheromones to communicate compatibility among mates, typically leading to the production of haploid meiotic basidiospores. <i> S. commune</i> has thousands of mating types, as part of its tetrapolar mating system in which mating is determined by two unlinked complex genetic loci, one of which codes for both mating pheromones and receptors (<i> matB</i>). <i>Schizophyllum umbrinum</i> is a fungal species which was described by Raper (1959) as a homothallic species but has not been studied in depth. <i>S. umbrinum</i> produces viable basidiospores, but has the means to bypass outcrossing. The studies described in this thesis were designed to examine components of a putative <i>S. umbrinum</i> <i> matB</i> locus and possible pheromone communication between the two <i> Schizophyllum</i> species. Two ways we explored <i>S. umbrinum</i>&rsquo;s reproductive behavior was to look for evidence of functional pheromone genes and pheromones, and to search for variation among progeny and strains collected from different locations that might be indicative of more than one <i> matB</i> type and outcrossing. <i>S. umbrinum</i> <i> matB</i> noncoding regions were examined with PCR for variation among progeny of a wild-collected mushroom. There were no DNA length differences detected among 11 siblings for PCR covering 1.9 kb of amplified DNA and spaced over 11kb in the genome. Using Southern blot analysis, the whole <i> matB</i> coding and noncoding regions between putative pheromone genes <i> suph1</i> and <i>suph9</i> were examined. These results verified and expanded beyond the PCR assay. The results suggest that the mushroom that produced the 11 sibling strains was not produced by outcrossing that involved two different <i>matB</i> idiomorphs but was likely produced from a homothallic mating where no <i>matB</i> mating type idiomorphs exist. The conservation and similarities in amino acid sequences between the predicted mature pheromones of <i>S. commune</i> and <i>S. umbrinum</i> suggested possible function of the <i>S. umbrinum</i> pheromone genes products. Heterologous expression and activity were tested in baker&rsquo;s yeast, <i>S. cerevisiae</i>, and <i>S. umbrinum </i> genes were also introduced into mushroom <i>S. commune</i>. In the yeast mating assay, successful communication between <i>S. umbrinum </i> pheromone SuPh3 and suph9 was demonstrated. In the <i>S. commune </i> mating assay, successful communication of <i>S. umbrinum</i> pheromones SuPh6, SuPh7, and suph9 with <i>S. commune</i> receptors <i> in vivo</i> was demonstrated. The ability to elicit signal transduction in both heterologous assay was observed by successful mating indication pheromone response. In conclusion, <i>S. commune</i> can express some <i> S. umbrinum</i> pheromone genes and process some of those gene products that resemble <i>S. commune</i> <i>matB</i> pheromones from their precursor to facilitate a response from <i>S. commune</i> receptors. Factors other than molecular compatibility may keep these signaling molecules in nature from interacting in these two sympatric species. To date, no evidence of self-activation of pheromone signaling within <i>S. umbrinum </i> has been discovered to explain the role of the pheromones in this homothallic species.</p>

<p>Eukaryotic translation initiation is a rate-limiting step of protein synthesis and is controlled by signal transduction in response to various extracellular cues and stresses. This thesis is focused on the eukaryotic initiation factor 4G (eIF4G), which participates in multiple steps of initiation: (i) eIF4G interaction with polyA-binding protein (PABP) links the 5' pre-initiation complex to the 3' poly(A) tail of mRNAs; (ii) eIF4G recruits 40S ribosomal subunit to mRNAs through interactions with the m7-G cap binding protein eIF4E; (iii) eIF4G binds to the eIF4E kinase, mitogen activated protein kinase (MAPK) interacting kinase 1 (Mnk1), modulating eIF4E phosphorylation. I studied how eIF4G function is affected by viral infection, mitogenic stimulation and during mitosis.</p><p>First, I reported that herpes simplex virus 1 (HSV-1) infection leads to re-localization of PABP to the nucleus, dissociating it from translation initiation machinery. Next, I showed that MAPK-mediated phosphorylation of Mnk1 leads to Mnk1 conformational changes, enhancing its binding to eIF4G and, hence, increasing phosphorylation of its substrate, eIF4E. Finally, I demonstrated that Cdk1/cyclin B1 directly phosphorylates eIF4G in mitosis and speculated on the role of this phosphorylation event in mitotic translation. In summary, my work demonstrated that eIF4G plays key roles in the regulation of the translational response to viral infection, growth signaling and cell cycle progression.</p> / Dissertation

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 10 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.