Recent advances in genome sequencing have allowed for the study of metagenomes, genetic material recovered directly from environmental samples. This has produced an enormous number of enzyme sequences without efficient methods for characterization. Since it is unknown whether a characterized enzyme is the best catalyst for a specific reaction, uncharacterized, natural enzymes in the metagenome may be better candidates for catalyzing the specific reaction. Thus, I am developing methods to search for and computationally characterize metagenomic enzymes with the same function as enzymes of therapeutic and commercial interest. By utilizing the Rosetta Software Suite to produce highly accurate models of enzyme structures, this method will evaluate an enzyme sequence’s potential for catalysis directly from a gene sequence. In order to validate my computational techniques, I will experimentally determine catalytic rates of enzyme candidates. As the method for an efficient metagenome search is still unknown, I am working to determine proper parameters for a homology search by using visual and statistical analysis of enzyme structure models in order to produce a diverse set of genes for experimental characterization.

Transcription, the first step in gene regulation in which RNA is created from DNA, is a process that occurs in the nucleus. When messenger RNA is transcribed, its function is to code for a protein product. This message is transported from the nucleus to the cytoplasm, where it can serve its purpose, and over time gets degraded. Transcription and degradation were always thought to be uncoupled, but recent evidence suggests otherwise.  Coordination of synthesis and decay rates of an mRNA transcript could be beneficial to the cell. It has been shown that copies of the exact same message encoded by different promoters can have different rates of degradation in the cytoplasm depending on the promoter used to activate transcription. I hypothesize that an RNA-binding motif on a protein could affect the lifespan of a specific message, and plan to test this hypothesis using the awesome power of yeast genetics and molecular biology.  By culturing wild type and deletion strains of yeast, and de-repressing the activity of gluconeogenic enzymes by starving the cells of glucose, I can test my prediction. SNF1 is a protein kinase that is active only during gluconeogenesis, and I have shown that when it is inhibited, the rate of mRNA degradation is more rapid than under normal conditions. I will test my prediction by deleting genes encoding putative RNA binding proteins that are phosphorylated by SNF1 and measuring mRNA levels by Reverse Transcriptase-quantitative polymerase chain reaction.  The idea of mRNA degradation being linked to the way that mRNA was transcribed is new, and I am interested to see what I will find.  I am hoping to identify both positive and negative effectors of mRNA stability using this approach.

Gluten intolerance disorders such as Celiac sprue elicit a painful immunological response in the intestines of patients who consume gluten. Gluten is a polypeptide found in wheat, barley, and rye, and contains repeating Pro-Gln-Leu-Pro (PQLP) motifs. Currently the only treatment for gluten intolerance is elimination of gluten from the diet. Our goal is to design an enzyme therapeutic that can be taken as a drug therapeutic in pill form. The engineered enzyme will break down the immunogenic PQLP motifs before gluten reaches the small intestine and can elicit an inflammatory response. Kumamolisin-As, a protein isolated from Alicyclobacillus sendaiensis, demonstrates promising proteolysis on PQ residues, and works in the low pH of the stomach. Using the protein-engineering program “FoldIt,” we designed novel mutations to Kumamolisin to improve its activity on PQLP. Our best version of the enzyme, dubbed KumaMax, degrades gluten over 700 times faster at pH 4 than SC-PEP, an enzyme currently in clinical trials. After further protein characterization, we hope to start clinical trials on KumaMax with the future goal of creating an effective treatment for gluten intolerance.

Duchenne Muscular Dystrophy (DMD) is a degenerative disease leading to fatal respiratory and cardiac complications. It is caused by loss of functional dystrophin through mutation of the dystrophin gene. Possible treatments could use Adeno-associated virus (AAV) to introduce genes into muscle cells. This strategy requires muscle specific regulatory cassettes containing sequences specific for transcribing the therapeutic gene in muscle cells. My studies seek to increase the activity of one such cassette, MHCK7. Since AAV vectors can package only ~5000 basepairs of DNA, the dystrophin cDNA and regulatory cassette must be miniaturized. I am modifying MHCK7 by adding a 95 bp intronic enhancer (SIE) from the muscle creatine kinase (MCK) gene that is important for MCK expression in slow muscle fibers and cardiac muscle. The SIE has been placed ~1kb 3’ of the MHCK7 luciferase reporter cDNA to mimic its endogenous genomic location. SIE addition was performed by introducing a HindIII site 3’ of the luciferase polyA sequence by site directed mutagenesis, and the subsequent opening of DNA at that site. The SIE was transferred from a previously made plasmid and ligated into the opened MHCK7. Restriction digests and gel electrophoresis were used to assess successful introduction of HindIII and to confirm digests for opening the DNA and isolating the SIE. New recombinant MHCK7-3’-SIE will be transfected into skeletal and cardiac muscle cultures, and its activity will be assessed through luciferase activity. The SIE’s contribution to expression will be compared to that of the original MHCK7 plasmid as well as to MHCK7 with the SIE located immediately 5’ of the cassette. If the new regulatory cassette is more active than MHCK7 and 5’-SIE-MHCK7, further studies will test the cassette in conjunction with a microdystrophin cDNA. If activity is not increased, I will test alternative locations of the SIE.

The effects of amphetamine on learning and memory are important areas of research due to the widespread use of amphetamine among people, ranging from prescription to recreational. It has been shown in animals that repeated treatment of amphetamine will result in sensitization to the drug. Sensitization is a process through which treated subjects develop an elevated response to a drug. Long term potentiation (LTP) is a physical change strengthening the connection between synapses that is thought to underlie learning and memory. Enhancement of LTP in the striatum has been shown to occur as a result of amphetamine use. The striatum is a brain region important in mediating a large number of behaviors including motor control and learning. For this reason it is possible that sensitization to amphetamine may affect learning in mice. To investigate this possibility, wild-type mice sensitized to amphetamine were tested in behavioral paradigms designed to measure learning ability. It was found that they do not show enhanced performance in motor learning, appetitive learning, or spatial learning, but they may show an improvement over controls when they are required to alter their strategy to escape from a water-maze. In order to explore the neurological pathway through which sensitization to amphetamine may affect learning, we also tested knockout mice that lack N-methyl-D–aspartate-type glutamate receptors (NMDARs) in medium spiny neurons, the primary neuron type in the striatum. In many brain regions, NMDARs are important for various forms of learning and memory, and it has been shown that these knockout mice display impaired motor and appetitive learning and yet are still able to sensitize to amphetamine. Using the same learning paradigms as with our wild-type mice, we found that knockout mice did not show any enhancement in motor or appetitive learning as a result of amphetamine sensitization.

Ubiquitin is a small post-translational signaling molecule. Its roles in repairing damaged DNA, apoptosis and protein degradation make it critical for tumor suppression. Genetic mutations affecting proteins in the ubiquitination pathway have been implicated in multiple inherited human cancers. The ubiquitination pathway attaches the protein ubiquitin (Ub) onto a substrate molecule with the help of three enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). Transfer of Ub onto the substrate requires the formation of a thioester bond between Ub and E2, denoted as E2~Ub. A catalytically active E3 ligase which can bind E2~Ub, but never directly binds Ub, is also necessary. E3’s catalytic mechanism is not fully understood. One function of E3 is to bring E2~Ub and substrate in close enough proximity to facilitate a reaction. However, E3 enhances the release of Ub from E2~Ub in the absence of substrate molecules. This suggests E3 plays a role in priming E2~Ub for transfer of Ub. Using site directed mutagenesis and protein-protein interaction assays visualized through western blotting, I have investigated what electrostatic properties of Ub are important for E3’s catalytic activity. Informative Ub mutations don’t stop formation of E2~Ub, but disrupt the catalytic activity of E3. I propose that polar/charged Ub residues in the contact region between Ub and E2 affect E2~Ub binding modes, promoting inhabitance of E2~Ub conformations favorable to Ub transfer upon binding of E3.

Embryonic stem cells are isolated from the early developing embryo and are capable of forming the different cell types found in the body. Understanding how these cells develop and maintain their specialized state is critical to understanding how they function. We hypothesize that embryonic stem cells acquire a unique metabolic state that aids them in maintaining their specialized state. Using two distinct mouse pluripotent states, embryonic stem cells (ESC’s) and epiblast stem cells (EpiSC’s) representing cells isolated from the inner cell mass of the blastocyst and the epiblast, a later stage in embryonic development, we found that EpiSC’s are much more glycolytic in terms of their energy usage compared to ESC’s as measured by oxygen consumption and extracellular acidification rates. Furthermore, using quantitative polymerase chain reaction experiments, we found that genes key to regulating and maintaining high rates of glycolysis are significantly upregulated in EpiSC’s compared to ESC’s. We showed that the overexpression of HIF1α, a known key regulator of these glycolytic genes, in ESC’s is able to shift the ESC’s towards a more glycolytic metabolic state and cellular morphology reminiscent of EpiSC’s. In addition, transition of ESC’s to EpiSC’s using the known chemical inducers Activin and Fibroblast Growth Factor 2 revealed that changes in the expression of metabolic genes occur prior to changes in expression of cell fate markers, which suggest that a metabolic transition in the ESC’s may be key in its transition to an EpiSC-like state.

Understanding stem cell maintenance is crucial for delineating conditions such as tissue loss, aging, and cancer. Using the Drosophila female germ-line stem cell (GSC) model to observe stem cells in vivo, several extrinsic and intrinsic factors have been demonstrated to regulate maintenance of Drosophila GSCs; they have also been found to be conserved in mammalian systems. The search is on for novel regulatory factors of stem cell maintenance with the hope of extending our understanding to stem cells in mammalian systems. My project involves the characterization of the molecular function of Pineapple eye gene (Pie), a novel regulator of GSC maintenance. Through a loss-of-function screen in GSCs, it has been demonstrated that mutations of pie result in GSC maintenance failure. The amino acid sequence of pie revealed that it contains ring-domains suggesting possible E3 ubiquitin ligase function. Such proteins are key components of the ubiquitination pathway, which are involved in protein tagging and degradation. To test pie’s function as an E3 ligase, I am using a novel assay that employs the fact that bacteria do not have endogenous ubiquitination pathway and therefore utilizes Escheria coli as an in vivo system to express the components of the ubiquitination pathway and detect the formation of ubiquitin chains, a key indicator of E3 Ligase activity. I have demonstrated that the expression of pie’s ring-domains without a substrate is not sufficient for the detection of ubiquitin chains. I will now test whether the full length pie is necessary for proper ubiquitination. Further, I will also test whether pie’s functional domain needs a substrate in order to function effectively. Successful characterization of pie can reveal new pathways to target for conditions involving stem cell maintenance.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. MiRNA biogenesis involves two key ribonucleases, Drosha and Dicer. I am investigating the Drosha enzyme. Previous research from the Ruohola-Baker lab reveals that reducing levels of Drosha in stem cells significantly reduces levels of some miRNAs, while only slightly reducing levels of another group of miRNAs. This surprising result led us to conduct a bioinformatics analysis to identify characteristics that differentiate these two groups of miRNAs. We discovered structural differences, specifically that bulges in the central region of miRNAs were more frequent in heavily reduced miRNAs than in miRNAs that were not affected. My mentor, Henrik Sperber and I propose a model explaining why miRNAs with mismatches (bulges) in the central region of the sequence are more sensitive to reduced levels of Drosha than rigid miRNAs with no mismatches in that region. To test whether a bulge causes differential miRNA processing when Drosha is the limiting factor, I am adding and removing bulges to primary miRNA transcripts, overexpressing them in cells with low Drosha levels, and comparing their mature levels using qPCR. In addition, I am testing whether in nature cells vary Drosha levels as a mode of regulation. A previous study shows that T cells at specific stages of differentiation reduce Drosha expression by as much as 10 fold. My own bioinformatics analysis reveals that certain tissues in mice have an 8-fold reduction of Drosha. To see whether reduction of Drosha levels correlates with differential microRNA processing, I am using microarray data to find subsets of microRNAs that have dramatically reduced levels when Drosha expression is significantly low. With my research, I hope to discover a potentially novel mechanism of regulation in which cells alter Drosha levels to selectively process a subgroup of miRNAs.

Differentiated human embryonic stem cells (hESCs) demonstrate the potential for regenerative medicine and drug discovery utilizing rationally assigned cell fate. But hESC are limited by the technical difficulty of recapitulating an ever growing set of cellular phenotypes using a finite set of pluripotent cell lines. The resulting molecular deficiencies limit the ability of differentiated cells to integrate into host tissue, and diminish the translational potential of findings based on differentiated hESC. Induced pluripotent stem cells (iPSCs) can be derived from patients with or without genetic disorders, and have much greater use for drug discovery and basic research, and patient derived iPSCs are less prone to rejection by host tissue. However, iPSC generated by insertion of genes into the host genome, increasing risk of rejection and limiting maturation of iPSC-derived cells. RNA-induced pluripotent stem cells (RiPSC) offer a solution to problems of genomic modification by inducing pluripotency exclusively through the transfection of mRNA. MicroRNA (miRNA) exhibit widespread regulation of mRNA translation and degradation, and do not directly alter the genome. A bioinformatics study curating publicly available microarray data for components of the miRNA biogenesis pathway, pluripotency factors, and miRNA expression will generate hypotheses about the functions of miRNA and relevant proteins, with a focus on differentiation and neural cell fate. Quantification of miRNA and mRNA expression in hESC and RiPSC during retinal differentiation will support or disprove hypothesized relationships between protein and miRNA expression. Transfection of miRNA will be used to determine the ability of these miRNA to enhance differentiation and maturation of retinal cells. Additionally, the resulting retinal progenitors will be implanted into mouse retina, directly testing the potential of miRNA to improve clinical outcomes of stem cell based therapies for the nervous system.

Autophagy, or “self-eating”, is a process cells use during times of stress or starvation to break down their own proteins or organelles to provide the required nutrients. I focused on the autophagy-related protein 13 (Atg13), a regulatory protein that controls the initial stages of autophagy. Specifically, I was interested in defining the distinct roles of the N and C-termini of the Atg13 protein. Using Saccharomyces cerevisiae expressing the full length, N-terminal, and C-terminal half of Atg13, I tested how these protein domains affected autophagy processing. To monitor this I looked at the autophagy dependent processing of aminopeptidase 1 (Ape1) and alkaline phosphotase 8 (pho8). Western blots were used to monitor the changes in Ape1 protein length after processing, and a colormetric phosphatase assay measured autophagy-dependent increases in pho8activity. Using these techniques I found that the two domains of Atg13 play distinct roles in the process of autophagy.

Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. Last summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. We constructed a strain of Escherichia coli that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. Using this method, we were able to produce on average 160 mg of alkanes per liter. Also, we identified a protease with gluten-degradation potential, and then reengineered it to have 100 times increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. Finally, to enable next-generation cloning of standard biological parts, BioBrick vectors optimized for Gibson assembly, an emerging DNA cloning technique, were constructed and used to construct the Magnetosome Toolkit: genes for biofabrication of magnetic particles.

The mitotic spindle orchestrates proper alignment of chromosomes during cell division. The spindle is composed of microtubules that attach to kinetochores, multi-protein complexes that assemble at the centromeres of chromosomes. Understanding kinetochore function is essential to understanding the process of cell division. Through mass spectrometry analysis, the Davis Lab has found an abundance of the urmylation component Uba4 at the kinetochore. The urmylation pathway is highly similar to ubiquitination, and involves conjugation of a ubiquitin related modifier (Urm1) to proteins and RNAs. The role of urmylation at the kinetochore is not known. We hypothesize that post-translational modification of kinetochore proteins through urmylation is a regulator of chromosome segregation. To examine the effects of urmylation on the kinetochore I will be using an Ncs2 mutant, a protein involved in the urmylation pathway, By fluorescently labeling the kinetochore protein Spc105 and spindle pole body protein Spc110, I am using fluorescence microscopy and computational analysis to look for abnormalities in spindle appearance. These results will help elucidate the role of the urmylation pathway in the regulation of cell division.

Blindness is a condition that impacts people across the globe. Some of these people are born blind due to a genetic defect while others become blind through an accident or as the result of disease. Thus, dissecting the underlying molecular causes of blindness is a critical component in improving the lives of many people. To study causes of blindness, we are analyzing disease causing mutations in the model organism Danio Rerio, or zebrafish. The recessive ZVM10 mutation causes early degeneration of the retinal photoreceptors, causing complete blindness. To study this mutation we are determining the identity of the mutated gene. The first step is to determine the position of the mutation within the zebrafish genome (i.e. map the mutation). This is done by mating the ZVM10 AB fish with a polymorphic fish strain. The crossovers between the different DNAs are traced, and used to identify the area homozygous for the AB allele. By continually reducing this region we specify a small group of genes that potentially have mutated. Using this method, I have established a general region on chromosome 20 between Z markers G40149 and G47173 containing the ZVM10 mutation. Identification and study of this mutation will enable scientists to better understand the role of this gene in vision. It will also permit the identification of people who are blind due to this genetic mutation, through genetic screening of the patient. Finally, if this gene is also involved in human blindness, it will become a candidate for rescuing blindness through gene therapy.

The spindle assembly checkpoint is important for detecting errors in spindle assembly during mitosis. When the checkpoint is activated, it arrests the cell cycle so that errors such as unattached kinetochores can be detected and corrected before progressing through the cell cycle. Problems with kinetochore attachment can be detrimental and lead to cells with an abnormal number of chromosomes which can then cause birth defects. Misregulation of the cell cycle can lead to abnormal cell division and cancer. Spindle pole bodies in yeast are functionally equivalent to human centrosomes, which organize microtubules in the cell cycle. Spindle pole bodies increase in size when the cell cycle is arrested at the checkpoint. Mad1 is an essential component of the checkpoint and interacts with the Nup60 protein in the nuclear pore complex, a channel through which information traffics between the nucleus and cytoplasm. The N-terminus of Mad1 protein is required to localize Mad1 to the nuclear pore complex, but is not required for the checkpoint arrest. We found previously that if Nup60 is deleted, the spindle pole bodies do not grow in cells arrested at the checkpoint. Mad1 mutants containing only the C-terminus of the protein will be constructed. I will test if the ability of Mad1 to bind to Nup60 is required for spindle pole body growth. Spindle pole body size will be measured by fluorescence microscopy after arresting the cells in metaphase. I hypothesize that although Mad1 interaction with Nup60 is not required for the cell cycle arrest, it is required to signal spindle pole body growth. If true, then deletion of the Nup60 binding domain of Mad1 should block spindle pole body growth. Discovering what interactions are vital in these cells will give us a deeper understanding of how regulation of the cell cycle is maintained.

Duchenne Muscular Dystrophy (DMD) patients have mutated dystrophin genes and suffer from muscular degeneration, which leads to cardiac, respiratory and general skeletal muscle disabilities. Potential treatment of DMD patients could involve gene therapy in which Adeno-Associated Viruses (AAV) deliver functional dystrophin cDNA ligated to muscle specific regulatory cassettes that express dystrophin exclusively in striated muscle tissues. The Hauschka lab has been designing regulatory cassettes (e.g. MHCK7) that contain control regions from genes that are highly active in skeletal muscles such as the muscle Creatine Kinase (MCK) and α-Myosin Heavy Chain (α-MHC) genes. Due to the limited size of DNA that can fit into AAV capsids, the cassettes need to be as compact as possible. My goal is to test whether adding a small intronic enhancer (SIE) from the MCK gene, that is known to have high activity in skeletal muscle in vitro, will improve transcriptional activity in cardiac and skeletal muscle cultures compared to MHCK7. The SIE was isolated and cloned immediately 5’ of MHCK7. After sequencing to confirm the SIE orientation, we carried out a pilot study in which 5’-SIE-MHCK7 was transiently transfected into skeletal muscle cultures. 5’-SIE-MHCK7 activity, as measured by the reporter gene firefly luciferase, was several fold higher than MHCK7. This suggests that the addition of the SIE to two known muscle specific enhancers increases transcriptional activity in skeletal muscle cells. Future studies will focus on confirming this result, and comparing 5’-SIE-MHCK7 activity to aMHCK7-SIE cassette in which the SIE is located 3’ of the luciferase reporter, so as to mimic its normal location in the MCK gene. The 5’-SIE-MHCK7 cassette will also be tested in cardiomyocyte cultures, and smaller cassette versions will be designed that eliminates additional non-conserved DNA sequences from the SIE and MHCK7 components.