X-Ray crystallographic structure and deletion mutagenesis of LacI have demonstrated that three-leucine-heptad repeats (LHR3), located at the C-terminus, are critical for dimer-dimer interaction to form tetramer. We are interested in exploring the potential for dimeric proteins, such as purine repressor, which share significant homology to lac repressor, to form higher oligomerization states when this assembly element is present. This information will expand our understanding of the general mechanism of protein oligomer formation. To assess whether introduction of a LHR motif will elicit tetramer formation, two mutants of PurR, PL3 and PL3+, with different lengths of the LHR3 sequences of lac repressor, have been constructed. A monomer-dimer-tetramer equilibrium is observed for PL3, along with wild-type corepressor binding affinity and decreased operator binding affinity. PL3+ is a trimer, and has no detectable corepressor binding affinity and decreased operator binding affinity. These data indicate that construction of higher order oligomers may be complex.

In the biosynthesis of cholesterol in mammals and ergosterol in fungi, oxidosqualene is cyclized to lanosterol by lanosterol synthase. Similarly, the biosynthesis of plant sterols includes the conversion of the same substrate, oxidosqualene, to cycloartenol by cycloartenol synthase. Cloned lanosterol synthases and cycloartenol synthases are about 30% similar, have over 100 conserved residues and have structurally similar cyclization products. I explored the dissimilarities between these two cyclases by inducing the evolution of a yeast lanosterol synthase mutant expressing cycloartenol synthase. Cycloartenol synthase mutants with lanosterol synthase activity were identified by selecting transformants that were no longer sterol auxotrophs. Once I identified the site of the mutation, I partially characterized the cyclization products of two cycloartenol synthase mutants with lanosterol synthase activity. I also cloned a large fragment of the Oryza sativa cycloartenol synthase gene, as concluded by the deduced amino acid sequence.

<p> A new class of promising drug carriers based on collagen mimetic peptides assembled into triple helical peptide (THP) is described. The peptides fold into a triple helical conformation forming high aspect ratio, rigid rod nanoparticles. The fluorescein tag is used to visualize cellular uptake. Incorporation of the non-viral cell-penetrating vector (RRG)₂ and R₆ into the sequence does not alter its helical conformation, allowing efficient delivery of peptide carrier to the nucleus. The delivery is not effective for non-helical peptides. THP is stable against enzymatic degradation, as studied in human serum. Because of its size and shape, the THP nanoparticle carrier, a rigid rod with 1.5 nm diameters and adjustable length between 7.5 and 10 nm, has the potential to be efficiently delivered to tumor cells via the Enhanced Permeation and Retention (EPR) effect. The ease of replacement of hydroxyproline with lysine during the synthesis of the peptide allows for simple drug conjugation to the carrier.</p>

<p> Many molecules in living systems are present in charged forms, and these molecules are often highly regulated. The work presented in the following chapters addresses two main topics involving charged molecules using the model plant <i>Arabidopsis thaliana</i>: elemental homeostasis and lipid flippases. The study of elemental homeostasis is referred to as ionomics and is the topic of Chapter II. P₄-ATPases are thought to be the principle class of proteins with lipid flippase activity and are the topics of Chapter III and Chapter IV. </p><p> Plants, especially seed crops, are an important source of mineral nutrition in the human diet and are thus important targets for biofortification and toxic element exclusion. Here, we report the results of a pilot ionomic screen in which we quantified the concentrations of 14 elements in Arabidopsis seeds. To identify conditional ionomic phenotypes, plants were grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. To help identify the genetic networks regulating the seed ionome, elemental concentrations were evaluated in mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes observed in the mutant screen supports an estimate that up to 11% of the Arabidopsis genome encodes proteins of functional relevance to the seed ionome. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: <i>SOS2, ABH1, CCC, At3g14280,</i> and <i>CNGC2.</i> Reproducible ionomic differences were also observed between the Col-0 reference accession and eight of the other nine accessions screened. Significantly, all 15 mutants with reproducible ionomic phenotypes showed at least one change under standard soil conditions. This suggests that the sole use of a standard growth environment might be the most effective strategy for continued reverse-genetic efforts to identify genes that impact the Arabidopsis seed ionome. Nonetheless, each soil modification had a unique impact on the Col-0 seed ionome and elicited several conditional phenotypes in both the mutant and accession screens, indicating that seed elemental homeostasis is sensitive to soil conditions. Together, the results of this study establish that elemental analysis is a sensitive approach to identify genes and environmental conditions that impact elemental accumulation in Arabidopsis seed. </p><p> By flipping lipids between membrane leaflets, P₄-ATPases are thought to help create and maintain asymmetry in biological membranes. Lipid asymmetry between membrane leaflets has been implicated in a wide range of biological processes including: vesicular trafficking, cell signaling, modulation of membrane permeability, protein recruitment, and regulation of protein activity. Additionally, one P₄-ATPase, Neo1p, is essential in yeast. In <i>Arabidopsis thaliana,</i> 12 P₄-ATPases have been identified: <u>A</u>minophospho<u>l</u>ipid <u> A</u>TPase 1 (ALA1) to ALA12. However, very little is known about P₄-ATPases in the context of plant systems. </p><p> Of the 12 ALA isoforms, only ALA3 has been extensively studied. Previous studies have shown that loss of ALA3 results in pleiotropic phenotypes affecting root, shoot, and reproductive development. Here, we expand on the previous studies by showing that multiple phenotypes for <i>ala3</i> mutants are strongly sensitive to growth conditions. We also expand on the <i> ala3</i> pollen phenotype by identifying three points of defect in <i> ala3</i> pollen tubes: delayed germination, slow growth, and reduced overall length. Furthermore, we show that <i>ala3</i> pistils have reduced ovule production, thus providing the first evidence of a female reproductive defect in <i>ala3</i> mutants. Together, these results support a model in which ALA3 functions in multiple cell types and is critical to plants for development and adaptation to varied growth conditions. </p><p> Two other ALA isoforms, ALA6 and ALA7, were also examined in this study. We provide <i>in-vitro</i> and <i>in-vivo</i> evidence that ALA6 and ALA7 are important for rapid, sustained pollen tube growth. Expression of fluorescently labeled ALA6 fusion proteins indicates that the subcellular localization of ALA6 includes the plasma membrane and highly mobile endomembrane structures. We also show that staining by lipophilic FM dyes is reduced by &sim;10-fold in <i>ala6-1/7-2</i> pollen tubes relative to wild-type, suggesting differences in plasma membrane composition. Furthermore, tandem mass spectroscopy analysis revealed significant differences between the lipid compositions of <i>ala6-1/7-2</i> and wild-type pollen grains, both in the concentrations of different headgroups and in the average number of double bonds present within acyl side chains. Together, these results support a model in which ALA6 and ALA7 function to directly or indirectly regulate the distribution and concentration of lipids in pollen and are thus critical for pollen fitness.</p>

<p> In order to elucidate the function of Myc in the maintenance of pluripotency and self-renewal in mouse embryonic stem cells (mESCs), we screened for novel ESC-specific interactors of Myc by mass spectrometry. Undifferentiated Embryonic Cell Transcription Factor 1 (Utf1) was identified in the screen as a putative Myc binding protein in mESCs. Utf1 is a chromatin-associated factor required for maintaining pluripotency and self-renewal in mESCs. It can also replace <i>c-myc</i> during induced pluripotent stem cell (iPSC) generation with relatively high efficiency, and shares target genes with Myc in mESCs highlighting a potentially redundant functional role between Myc and Utf1. </p><p> We validated that Myc and Utf1 directly interact in an in vitro context by GST-pull down. From in vitro truncation mutant studies of both proteins, a large region of Utf1 was found to be necessary for direct interaction with N-Myc, while the basic helix-loop-helix leucine zipper domain of N-Myc is required for direct interaction with Utf1. Utf1 was not found to directly interact with Myc's heterodimerizing partner, Max. </p><p> Utf1 and Myc complex formation could not be recapitulated in vivo, in both an ESC context and overexpressed in 293FT cells. Also, analysis of transcriptional function of Myc and Utf1 in 293FT cells using an E-box reporter construct did not result in changes to reporter expression levels, further supporting lack of complex formation in this cellular context. </p><p> Using pre-existing data sets two putative direct common target genes of Myc and Utf1, <i>Slc27a2</i> and <i>Ttyh1</i> were identified in mESCs. Due to the low level of data set overlap and technical variables that arise from overlapping data sets from independent studies a more optimal technical approach for identifying common genomic binding sites and target genes of both proteins was identified. Utf1, c-Myc, N-Myc, H3K27me3, and H3K4me3 ChIP-Seq, as well as RNA-Seq after diminished levels of Myc and/or Utf1 would lead to a better understanding of the combinatorial role of Myc and Utf1 in mESCs.</p>

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Since the first isolation of allyl isothiocyanate from the seeds of black mustard over a century ago, the occurrence of isothiocyanates in nature has frequently been reported. Organic isothiocyanates are commonly called mustard oils because of their long recognized distribution in plants of the mustard family, Cruciferae, which derived the name originally from the French condiment moustarde. Various of the mustards have been important items of commerce since ancient times. Mustard oils may be conveniently grouped as volatile and non-volatile. Volatile oils, such as allyl mustard oil, have a characteristically sharp taste and odor, whereas the non-volatile, such as p-hydroxybenzyl isothiocyanate (the active principle of table mustard), are odorless but also possess a sharp taste. Volatile mustard oils may be easily separated from the seed mixture by steam distillation, and consequently have been more extensively investigated than non-volatile mustard oils. The purpose of this investigation is to elucidate the chemical nature of the precursors of mustard oils in plants, the mustard oil glucosides.

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