Current life on earth is believed to have descended from self-replicating RNA molecules, and evidence of its deep history is apparent in its diversity of function. Discerning the structure of RNAs will help inform our understanding of RNA function as well as develop future tools in medicine. The most basic structural information lies in its secondary structure, the first level of structural organization within an RNA molecule. Techniques such as crystallography, comparative analysis, and computational algorithms have been developed to predict secondary structure of RNA, though performance becomes hindered when analyzing longer RNAs. In order to find a more efficient method of predicting the structure of long RNAs, we combine data gathered from DMS probing experiments (Structure-Seq) and input subsections of the RNA into the RNAStructure prediction algorithm. By dividing the structure into smaller sections, we find that predictive capabilities can be vastly improved, though inclusion of DMS probing data has varying effects in improving prediction accuracy. We test this subdividing of RNA of prediction in both a user-directed and naive manner in the 18S RNA in Arabidopsis thaliana. Overall, these improvements in computation and experimentation suggest a more efficient and accurate strategy to predict RNA secondary structure in long RNAs.
Plants and animals have evolved and developed different methods of immunity to defend themselves against various attacking pathogens. One technique is by recognizing such pathogens through recognition receptor proteins located on the extracellular membrane and then signaling a response pathway. In this lab, the MAPK6 enzyme of the MAP/ERK pathway in Arabidopsis thaliana was studied and biochemically characterized. The gene for the protein was cloned, expressed in bacteria and the protein then purified through extraction. By running an analytical gel exclusion chromatography, MAPK6 was then determined to be monomeric structurally. Also, through an in vitro reaction with ATP, the protein was determined to be an active kinase that utilizes phosphorylation to transmit immune responses within the cascading pathway.
To accommodate a rapidly drier climate, Californians need to cut water from landscaping. The goal of this study is to evaluate the effectiveness of adding extra water to young frontier elms (Ulmus “Frontier), using hydration bags on young valley oaks, and irrigating young valley oaks with MP rotators and hydration bags. The plant-based measurement of water stress, midday stem water potential (MSWP), was collected in the early afternoon through the process of pressure bombing. This study found that adding extra water did not reduce the water stress of frontier elms and had no prolonged effect, that using hydration bags was three times as water efficient as using MP Rotators at watering the young valley oaks, and that removing hydration bags increased water stress in young valley oaks.
Arabidopsis Thaliana Cell Plate Formation: an Interdisciplinary Approach to Characterizing ES7 Resistant Mutants
There is currently a lack of knowledge surrounding the signals and factors that lead to cell plate formation during cytokinesis. Although scientists in the field know the multistep process of the cell plate formation, there is still much to be learned about the molecular events occurring during formation. The Drakakaki lab at UC Davis is studying endomembrane trafficking and polysaccharide deposition at the cell plate. By using the chemical Endosidin 7(ES7), which inhibits cell plate maturation by specifically impeding callose deposition, the researchers have developed mutant strains of the Arabidopsis thaliana that are resistant (Park et. al 2013). Characterization of two mutants es7r-1 (ems 13) and es7r-3 (ems 70) by root length and subcellular effect in response to ES7 will yield the target gene and therefore a novel protein active during cell plate formation. In addition, a cell plate vesicle proteomics approach will lead to a collection of cell plate-destined proteins which to study further
Cloning and Preliminary Characterization of STY46 from Arabidopsis thaliana, a Candidate Protein Kinase Involved in the Plant Sugar Starvation Response
To study how plants respond to conditions under which the carbohydrates needed for growth and development are exhausted, this project focuses on cloning and functional characterization of a cytosolic protein kinase, STY46 that may potentially be involved in the sugar starvation response (SSR). Generating A. thaliana STY46 knockout (-KO) and overexpression (-OE) lines is the main method in this project to determine the function of the protein. A SALK line (SALK_116340) was confirmed to contain a T-DNA insert in the STY46 gene and the aim was to prove that this reduced the expression of this gene. Highly intact RNA was isolated and reverse transcription PCR performed to determine the level of transcript of STY46 in STY46-KO. However the results were inconclusive. This may be due to not enough amount of RNA use for the cDNA reverse transcription. To develop an overexpression line, STY46 was cloned from wild type Arabidopsis genotype. Primers were designed to flank the open read frame of the STY46. They also contained restriction enzyme recognition sites and a myc-tag engineered at their 5’ ends to facilitate subsequent synthesis of a plant transformation construct. A cDNA fragment of the expected size was successfully amplified by PCR.
Role of Mitochondrial Inner Membrane Complex Organizing System in Fibroblasts from FMR1 Premutation Carriers
FXTAS is an adult onset neurodegenerative disorder known to cause symptoms such as intention tremor and gait ataxia, which grow in severity with age. It is also possible to have the FMR1 premutation that causes FXTAS without experiencing symptoms of the disorder. In this study, fibroblast samples isolated from FMR1 premutation carriers with and without FXTAS and age and sex matched controls were tested through PCR in order to determine the gene expression of MICOS protein encoding genes and the FMR1 gene. Samples from male premutation carriers displayed a downward trend in expression of MICOS protein encoding genes and the FMR1 gene as age increased, whereas samples from female premutation carriers exhibited the opposite trend. Although this pilot study does not provide enough conclusive data to accurately and precisely justify any conclusions, it can be inferred from these data that males overexpress the genes tested while young in order to compensate for deficits associated with the premutation and resist pathogenesis until they grow too old to do so effectively, whereas FMR1 premutation heterozygous females underexpress the genes tested while young due to a compensatory response in the remaining unaffected allele.
Chemotaxis allows some organisms like Pseudomonas bacteria to move in response to chemical stimuli. Methyl-accepting chemotaxis proteins (MCPs) are membrane-bound chemoreceptors that facilitate this process. Understanding MCPs could lead to potential application of chemotaxis in fields like bioremediation, where it could improve efficiency since bacteria could not only break down pollutants but seek them out as well. In order to characterize the MCPs of species like Pseudomonas putida F1, hybrid sensor proteins comprised of the sensory domain of bacterial MCPs and the signaling domain of a 2-component regulator were used in the optimization of a screening method. By using a filter paper disk to diffuse potential attractant/repellants in a plate, bacteria with these hybrid sensor proteins will show an inhibition of growth if they sense the compound. This screening method was optimized by testing various concentrations and volumes of compounds as well as plate media content. This method will allow for quick screening of MCP functions in P. putida F1 and other bacteria.
The buffering capacity of foods plays a pivotal role in food breakdown during gastric digestion. The ability of foods to resist changes in pH alters the acidity of the stomach, influencing acid secretion, enzyme function, and chyme viscosity[SO1] . All of these properties directly influence the stomach’s ability to break down foods. Therefore, a comprehensive and replicable method to determine the buffering capacity of foods would prove beneficial for helping to predict and compare digestive properties [SO2] of different foods. We have developed a preliminary methodology for determining the buffering capacities of certain categories and subcategories of undigested foods, and used these to determine buffering properties of certain semisolid foods before digestion.
Research and application of fullerenes have been limited by the work and cost of separation and purification. This paper studies sulfur as a separation agent with C60 and C70 solutions. Crystal tubes were made of sulfur and 1:1 molar ratio mix of C60 and C70. Resulting crystals were tested with HPLC and single crystal X-Ray diffraction. It was found that sulfur cocrystallizes with both C60 and C70, but the two fullerenes crystallize separately. The results of this experiment can be used to develop new separation methods for fullerenes and adds to existing knowledge of sulfur as a cocrystallizing agent with fullerenes.
The outer membrane protein 85 (Omp85) family proteins found in the outer envelope of chloroplasts are remnants of ancestral cyanobacterium protein since their endosymbiosis by eukaryotes. Flowering plants in particular have an additional beta barrel protein in the outer membrane of their chloroplasts, OEP80tr, whose exact function is unknown. This study attempts to determine the function of OEP80tr by investigating the effects of null expression mutations of the gene. Through analysis of germination and plant growth (in roots, hypochotyl, and leaves), this study compares the phenotypes of wild type plants with the functioning gene to plants without the gene. The resulting observations could then be used to determine the role in development of the OEP80tr protein.
Restoration of native grasslands in California is expensive, mostly due to the high cost of seeds. Although restoration can increase biodiversity and ecosystem services, ecosystem services do not generate revenue. In order to reconcile economics and the need for biodiversity in California annual grasslands, this study focuses on discovering how a grass species’ status as an annual or perennial plant affects biodiversity and relates to cost. We studied several restoration methods based on strip seeding, where strips of various widths were alternatively seeded or left unseeded. We hypothesized that over time seeds from the seeded strips would establish in the unseeded strips, a cost-efficient means of restoration. Requiring $825 and $1250 respectively, the 33% seeded and 50% seeded (wide) treatment demonstrated potential as cost efficient means of habitat restoration. The 33% treatment resulted in approximately 12.5 species at the plot level for species richness, while the 50% wide treatment yielded 12.4 species. Botanical composition of seeded and unseeded strips indicated that strip seeding is a successful low-cost method for Elymus glaucus, but other native perennial grasses failed to establish in unseeded strips.
Understanding the Structural and Molecular Basis of Postharvest Chilling Injury in Tomato Fruit (Solanum lycopersicum L.)
Postharvest Chilling Injury (PCI) is a devastating disorder that leads to loss of produce. In this study two approaches were taken to better understand the development of PCI in tomato (Solanum lycopersicum L.) a species susceptible to PCI. First, the spatio-temporal development of PCI symptoms in developing fruit was assessed using Magnetic Resonance Imaging (MRI). Second, the CBF1 gene from Arabidopsis thaliana was cloned so that the gene may be engineered into tomato fruit to improve the tomato fruit response to chilling. MRI data indicates that chilling injury affects each tomato tissue type (the pericarp, the locular tissue and the columella) differently with greater effects seen in green fruit due to their continuation of ripening and various metabolic processes. A 780 bp fragment corresponding to the coding sequence (CDS) of CBF1 was amplified from Arabidopsis genomic DNA and attempts to sub-clone into pGEM-T Easy Vector® are underway.
The primary function of proteasomes is to degrade mis-folded and unwanted proteins. Tripeptidyl peptidase II (TPPII) is a newly discovered protease that works downstream of the ubiquitin-proteasome system. It is required in eukaryotic cells for cleaving of larger protein degradation products. However, little is known about its function in other cellular processes. TPPII’s responses to stress and DNA damage could be areas of research for cancer therapy, if TPPII can be studied better. Homogenization is a process used to break down cells for subsequent analysis or purification of proteins. Accurate analysis of protein expression and protease activity in the cell can be studied by good homogenization procedures and sample preparation. This study investigated the effect of various homogenization and assay buffers to determine the optimal buffer for homogenization and activity assays of cardiac TPPII. The results demonstrated that a homogenization buffer containing polyvinylpyrrolidone and glycerol produced more desirable results compared to buffers currently used for TPPII analysis
Structural and kinetic analysis of hydrolase mutants and subsequent impact on predicting functionality with Michaelis-Menten mechanics
Understanding about enzymes has grown due to the use of computational algorithms in predicting the correlation between structure and function. These algorithms have had critical roles in the redesign and reengineering of native enzymes to enhance catalytic behavior. In this paper, front-end programs were used to model, interact with, and redesign a glycoside hydrolase enzyme. The structural model was coupled with Michaelis-Menten kinetics to yield the Kcat and Km kinetic constants for three mutants. The compiled dataset will be essential to tracking patterns and trends across enzyme families. It may also be useful in future efforts to redesign enzymes using predictive methods.
Heliotropism, also known as solar-tracking, is the diurnal motion of plant leaves and flowers in response to the direction of the sun. Many studies of solar-tracking plants indicate that heliotropism is correlated with higher light absorption, photosynthetic efficiency, and crop productivity. Sunflowers undergo this process by orienting their leaves, as well as their stem toward the sun throughout the day. The flowers continue to move at night in order to readjust their direction in preparation of the sun’s rise in the east the next day. This paper reports two experimental tests of the hypothesis that disrupting the normal solar cycle of the sunflower will slow the rate at which the plant develops and decrease yield. In the first experiment, 83 HA412 inbred line sunflowers were planted in a field. Half of the plants were treated by being tethered to bamboo stick in order to restrain heliotropic stem movement. In the second experiment, 28 HA412 inbred line sunflowers were planted in 3 gallon pots placed in the field. Half of the plants were rotated 180 degrees at sundown to face the other direction. Leaf area, leaf count, and plant height was measured for all plants in both experiments. The angle of head movement was also measured for the plants in the second experiment. The results of both experiments show that plant growth and development was less in treated plants then the control plants, indicating that heliotropism plays a part in plant growth. However, more research and trials are needed to confirm this. The objective of this study is to determine the effect of stem heliotropism on sunflower growth and productivity in order to arbitrate whether genetically modifying other crop-bearing plants to elicit stem heliotropism would be beneficial to crop yield.