Biological Sciences Student Research Showcase 2010

Molecular Biology Abstracts

POSTER #21:

Selective Targeting of Head and Neck Squamous  Cancer Stem Cells Using Salinomycin

Katherine Blair
Dr. Martin Haas

Selective Targeting of Head and Neck Cancer Stem Cells Using Salinomycin  Authors: Katherine J. Blair2, Weg M. Ongkeko MD PhD1, Martin Haas PhD2 1 Division of Head and Neck Surgery, Department of Surgery, University of California, San Diego 2 Moores Cancer Center, University of California, San Diego  The cancer stem cell hypothesis states that within a solid tumor, there exists a heterogeneous population of cells. One of these populations displays the ability to self-renew and differentiate; properties tightly associated with stem cells. It is this population that is implicated in metastasis and chemoresistance: two of the greatest challenges in preventing recurrence after treatment. Current therapies target non-stem cell populations, thus leaving the cancer stem cell, allowing for regeneration of the tumor. A recent study implicated the livestock antibiotic salinomycin as a drug that could specifically kill cancer stem cells in breast cancer. The present study looks at the effects of salinomycin on cancer stem cells of head and neck squamous cell carcinomas (HNSCC).   This study used a putative cancer stem cell culture that was derived from a primary HNSCC tumor. These cells express CD44, a cancer stem cell marker, and BMI-1, a proto-oncogene. Cells were exposed to salinomycin in concentrations ranging from 0.5 – 8 μM. Flow cytometry showed a directly proportional, dose-dependent increase in cell death after four days’ exposure to salinomycin. Sphere-like aggregate formation in serum free media is used as measure of self-renewal capacity of cells, a characteristic property of stem cells. Cells treated with salinomycin produced a dose dependent decrease in number of spheres formed as well as size of spheres formed, indicating a decrease in “stem-ness”. Studies were also performed on mRNA expression via qRT-PCR. As expected, CD44 and BMI-1 mRNA expression decreased as salinomycin concentrations increased. Induction of genes associated with the epithelial-to-mesenchymal (EMT) transition has have been demonstrated to confer a stem cell phenotype to cancer cells. As such, it was hypothesized that as stem cell markers and stem cell properites decreased, so would too the expression of EMT genes. Interestingly, this was not exclusively the case. The mRNA transcripts of Twist indicated a decrease in EMT, while E-cadherin and Snail indicated an increase in EMT. To our knowledge, this is the first study that demonstrates a discord in the relationship between EMT expression and “stemness”, making salinomycin an even more interesting compound to study.  This study shows that salinomycin is able to decrease the proportion of CD44 and BMI-1 positive cells in a cancer stem cell culture. Whether this occurs by selectively killing the cancer stem cells or by inducing differentiation has yet to be determined. Either way, it is a promising potential treatment of HNSCC. An in vivo model using real-time imaging of fluorescent cancer stem cells will be initiated shortly to study the effects of salinomycin in vivo, including its effects on tumor initiation, size, metastasis, and survival.

POSTER #22:

Characterization of let-7 related microRNA primary transcripts and expression patterns in C. elegans

Victoria Burton
Dr. Amy Pasquinelli

microRNAs (miRNAs) are a class of small noncoding RNAs that regulate many important biological processes like developmental timing, cell differentiation and cell proliferation. miRNAs are encoded in the genome and transcribed by RNA polymerase II into multi-kilobase long primary transcripts that are capped and polyadenylated. The primary miRNA transcripts are cleaved by two RNase III enzymes, Drosha and Dicer, into 70 nt precursor, and 22 nt mature miRNAs respectively. Mature miRNAs imperfectly base pair with target mRNAs causing translational repression and mRNA decay. Originally identified in the nematode Caenorhabditis elegans, the let-7 miRNA is highly conserved in its sequence and temporal expression. Misregulation of let-7 can cause tumorigenesis in humans and lethality in C. elegans. miRNAs are categorized into “families” based on 5’ end sequence similarity and the potential to regulate common targets. A triple mutant worm of the let-7 sisters, mir-241, mir-48, and mir-84, displays growth defects showing that let-7 sisters regulate development. However, little is known about the identity of let-7 sister primary transcripts and how their maturation is regulated. I have used the Rapid Amplification of cDNA Ends technique (RACE) to map the 5’ and 3’ ends of the full-length primary transcripts of the let-7 sister miRNAs. I have also characterized their expression patterns by Northern Blotting analysis over a time course covering the embryonic, larval and adult stages. So far, my RACE results show that mir-241 and mir-48, which are separated by 1.7 kb, have the same 5’ and 3’ ends. This corroborates my Northern analysis, which reveals both similar size and parallel expression patterns of the mir-241 and mir-48 primary transcripts.  Altogether, my results suggest that mir-241 and mir-48 share the same primary transcript and thus may be commonly regulated. These results will further our understanding about the regulation of these important miRNAs and their role in development.

POSTER #23:

Structure-function study of protein-protein interactions between GIRK3 and the SNX27 PDZ domain

Christopher Childers
Dr. Paul Slesinger

Protein-protein interactions are fundamental to almost every biological process within the human body.  These interactions are vital to the transmission of information, the aggregation of protein complexes, and the modification of cellular components.  Understanding this complex array of interactions can provide insight into the mechanism of human disease and can provide the basis for therapeutic techniques.  The aim of the research is to find the amino acid residues responsible for protein-protein interaction between G protein coupled inwardly rectifying potassium channels (GIRK3) and the PDZ domain of sorting nexin 27 (SNX27). GIRK channels regulate neuronal excitability and are involved in the generation of slow inhibitory postsynaptic potentials in the brain; they are also involved in a variety of human diseases, including drug addiction. GIRK3 contains a class I C-terminal PDZ binding motif (-SKV).  SNX27 is a cytoplasmic protein which is upregulated in rodents sensitized to psychostimulants such as cocaine and methamphetamine.  SNX27 has a PDZ binding domain which has been shown to bind the PDZ binding motif of GIRK3 channels.  Interestingly, SNX27 does not bind the class I C-terminal PDZ binding motif (-SEI) of IRK1, a closely related inwardly-rectifying potassium channel.  Using site-directed mutagenesis and in vitro overlay assays we investigated the mutations required to induce binding of SNX27 to IRK1.  We found that electrostatic interactions between the SNX27 PDZ domain and the -5’ end of IRK1 determine the binding and strength of interaction.  Our results provide new information on the complex interaction between PDZ domains and class I PDZ motifs.  Furthermore, because of SNX27’s role in drug addiction, developing a potent and specific inhibitor of SNX27 PDZ domain could have possible use for therapy of drug addiction.

POSTER #24:

Analysis of Conditions for In Vitro Transcription with Human Factors

Yanhua Chi
Dr. James Kadonaga

In vitro transcription is an important method for the analysis of mammalian gene expression. However, the optimal conditions for in vitro transcription by the mammalian RNA polymerase II system have not been thoroughly investigated. A number of different conditions for transcription with human (HeLa) cell extracts such as dithiothreitol (DTT), spermadine, spermine, magnesium ion, and competitor DNA of various concentrations were examined. Of particular interest, it was found that 5mM DTT is the optimal concentration for transcription.

POSTER #25:

Pancreatic Shp2 Tyrosine Phosphatase regulates beta-cell mass in mice

Di Fang
Dr. GenSheng Feng

Shp2 is a tyrosine phosphatase encoded by PTPN11 and is widely expressed in various types of organs. It has been reported that the neuronal deletion of Shp2 can lead to disruption of energy homeostasis, obesity, and diabetes. Previous studies have also supported the hypothesis that Shp2 plays a very important role in coordinating glucose regulation by upregulating the expression of Pdx-1 that promotes pancreatic beta-cell maturation and insulin production. However, the role which Shp2 plays in the regulation of beta-cell mass is largely unknown. Evidences exist that the beta cell mass is capable to increase to compensate for increased metabolic stress, such as under high-fat diet or during pregnancy. In our experiment we used pancreas-specific Shp2 knockout mice to examine the function of Shp2 in the compensatory regeneration of beta-cell in vivo and in vitro, under conditions of high-fat diets induced Type II diabetes, and treatments of Streptozotocin, a structural analogue to glucose that specifically destructs the pancreatic beta- cells and can induce Type I-like diabetes.

POSTER #26:

Toscana Virus Nucleoprotein Structure Determination Employing Amide Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS)

Henry Guan
Dr. Virgil Woods

Though Phlebotomine sandfly fever viruses first emerged in Europe, their diseases are spreading to affect humans around the world. The combination of these viruses, which include the Naples virus, Sicilian virus, and Toscana virus can lead to pappataci fever, headaches, myalgia and other illnesses. In particular, the Toscana virus alone is sufficient to cause serious illnesses such as meningitis and meningoencephalitis. Recent studies suggest that the viral family Bunyaviridae, which includes the Toscana virus, may be used as biological weapons in the future. In order to prevent a future epidemic, many pharmaceutical companies anticipate making antibodies to screen for those infected with the Toscana virus, and also raise immunity for the general public. To create effective antibodies against the Toscana virus, we need to identify antibody binding sites on its nucleoprotein (NP). Additionally there is no crystal structure of Toscana virus NP. Hydrogen/Deuterium Mass Spectrometry (DXMS) can help identify ligand binding sites as well as highly disordered, highly solvent accessible regions that are preventing proteins from crystallizing. Our objective is to use DXMS to identify those highly disordered regions in hopes of creating a crystallizable construct of the Toscana Virus NP for future applications.

POSTER #27:

Structural and biophysical characterization of Lassavirus matrix protein Z using Hydrogen/Deuterium Exchange Mass Spectrometry

Patrick Hancock
Dr. Virgil Woods

The Lassa virus is the causative agent of the deadly hemorrhagic Lassa fever which is endemic in Western Africa. The Lassa Virus Z protein is an abundant matrix protein which is closely associated with the viral membrane. Evidence suggests that this protein is the primary factor involving the initiation of viral particle release from infected cells.  Crystal structures of viral proteins offer tremendous insights into mechanism of infections and viral assembly. However, no viable crystalline structure of this protein has been achieved. The goal of this study was to identify the regions of the Lassa Z matrix protein which rapidly exchange amide hydrogens with the surrounding solvent. These regions of instability serve as an obstacle to protein crystallization. By using deuterium exchange mass spectroscopy (DXMS) such regions were identified and our data can now serve as a guide to the crystallization of the Z protein. This, in turn, can be used for 3-D structure determination and further vital Lassa virus analysis.

POSTER #28:

The Role of G-Protein Coupled Receptors in Regulating Myofibroblast Transformation

Steven He
Dr. Paul Insel

Cardiac fibrosis is a condition characterized by excessive extracellular matrix formation and scar tissue deposition after myocardial trauma. The excessive accumulation of collagen in the myocardium may lead to cardiac stiffness and impairment in contractile function, eventually causing heart failure. While cardiac fibrosis has long been considered an irreversible process, recent evidence has suggested otherwise. Specific G-Protein Coupled Receptors (GPCRs) may be key contributors to the propagation of the signal for the transformation of nesting fibroblasts into profibrogenic myofibroblasts by agents such as transforming growth factor β-1 ( TGFβ-1 ), angiotensin II (Ang II), and endothelin. Freshly isolated rat cardiac fibroblast cells were cultured, passaged twice and treated using 10 ng/mL of TGFβ-1 , followed by microarray profiling for GPCR expression.  Normalized results indicate that expression of adenosine A2A receptors increased 8-fold, A2B receptor increased <2-fold while A3 receptors decreased 4-fold. Treatment of transformed myofibroblasts using 1 µM 5'-N-ethylcarboxamidoadenosine (NECA), an agonist for Adenosine A2B receptor decreased profibrotic markers PAI-I by 30-fold , αSMA by 4-fold, CCN1 by 8-fold and CCN2 by 28-fold. Therefore, the increase in Adenosine A2A and A2B receptor abundance in transformed myofibroblasts combined with the receptors’ ability to increase cAMP levels, which have anti-fibrotic effects, make Adenosine receptors a potential drug target to inhibiting the fibrotic phenotype and reversing cardiac fibrosis.

POSTER #29:

Knockout and Overexpression Studies of Human mitoNEET in Synechococcus elongatus

Christina Homer
Dr. Mel Okamura

MitoNEET is a human protein of unknown function which contains two redox-active 2Fe-2S clusters. MitoNEET is thought to be an important protein in disease and has been linked to diabetes. Loss of another member of this protein family, Miner1, causes Wolfram’s Syndrome II, a neurodegenerative disorder, and Miner1 has been implicated in the proper regulation of autophagy in eukaryotic cells.  In an attempt to understand the function of mitoNEET and Miner1, we studied the only homolog (gene 1697) of these 2Fe-2S proteins in the cyanobacteria Synechococcus elongatus by knocking down the gene encoding the protein.  These knockdown strains were grown in a variety of stress conditions, including low light and nutrient deprivation conditions such as low iron, low nitrogen, no nutrients, and diluted media. The knockdown strains grew slower in the diluted and low iron media. Additionally, the knockdown strain lost its phycocyanin peak, a typical response to nitrogen stress, faster than the native in both no nitrogen and the no-nutrient conditions. This suggests a role for the mitoNEET homolog in regulating the cell’s response to nutrient deprivation. The faster degradation of the phycocyanin peak suggests that the homolog could be regulating how quickly non-essential proteins are broken down to generate amino acids for essential functions. Further studies are being conducted with a Synchecoccus strain overexpressing the homolog to confirm these results.

POSTER #30:

Circadian Regulation of the Mouse Kiss1r gene in GnRH neurons

Lara Kose
Dr. Pamela Mellon

Background  Kisspeptin 1 receptor (Kiss1r), also known as G-protein coupled receptor 54 (GPR54) plays an integral role in maintaining the proper function of the reproductive system as well as in preventing the migration of metastic cancer cells.  Kiss1r functions by binding of the endogenous ligand kisspeptin (KP), a product of the Kiss1 gene, and signaling intracellularly.    The Kiss1/Kiss1r system was first identified in cancer biology. Strong data exist to indicate that Kiss1 is a melanoma metastasis suppressor in melanoma, bladder cancer, gastric carcinoma, thyroid cancer, endometrial cancer, and epithelial ovarian cancer to name a few.  For instance, one study that focused on analyzing ovarian cancer cell lines reported that an overexpression of Kiss1 inhibited cell migration and reduced the formation of metastic colonies (Makri 2008). However, the details of the mechanisms involved are still under investigation to ultimately target the Kiss1/Kiss1r system for therapeutic purposes.  Studies have also shown that the binding of the KP to Kiss1r in the hypothalamic-pituitary-gonadal axis plays a pivotal role in the initiation of puberty, ovulation, estrous cyclicity, and pregnancy. In a previous study, it has been shown that mutations in Kiss1r are associated with lack of puberty onset and hypogonadotropic hypogonadism.  Continuous expression of Kiss1 and Kiss1r mRNAs have been detected in male and female rat hypothalamus through postnatal development. During puberty, maximal expression was detected (Navarro 2004).        In previous studies, Kiss1r knockout mice have been used to study the role of Kiss1r in ovulation and the estrous cycle. Some studies suggest specificity to the regulation of KP and Kiss1r expression. For instance, KP-Kiss1r genes are expressed in only a few select tissues including the hypothalamus, pituitary, gonads, and placenta. Their expression also increases dramatically during pregnancy and then returns to initial levels after pregnancy. However, the mechanisms involved in the regulation of Kiss1r expression are not fully understood and few studies have been done to assess them.  Introduction   It is important to understand the molecular processes involved in maintaining the specific and tightly controlled transcriptional regulation of Kiss1r. From previous studies, it has been shown that GT1-7 cells display a circadian pattern of kisspeptin sensitivity(Zhao 2009). The master biological clock that functions to regulate 24-hour oscillations of circadian rhythm is regulated by clock genes in the suprachiasmatic nucleus (SCN). The main autoregulatory transcription-translation feedback loop is regulated by Clock and Bmal, which interact to form heterodimers and are known to regulate the circadian rhythm of GnRH secretion.   In rodents, the LH surge is restricted to a specific window of circadian time during proestrus. In previous studies, a neural signaling from the SCN was found to directly act on GnRH neurons to trigger GnRH secretion. SCN also projects to the anteroventral periventricular nucleus (AVPV), a region in which KP is highly expressed and has been linked to the initiation of the LH surge (Zhao 2009).  These studies suggest that Kiss1r may be the link between circadian KP signaling and GnRH secretion. Since GnRH neurons show a circadian pattern of KP sensitivity, it is important to investigate the regulation of the Kiss1 receptor. The purpose of our studies is to investigate the circadian regulation of the Kiss1r gene in GnRH neurons.

POSTER #31:

Utilization of Radiolabeled Transcript to Perform Nuclease Assays to Reveal Detail About Function of Ire-1 in RNA Splicing

Sari Lahham
Dr. Maho Niwa

Ire1 is an integral transmembrane protein involved in the Unfolded Protein Response (UPR) performed by the endoplasmic reticulum upon sensation of proteins that have either remained unfolded or have been folded improperly. A critical part of this response pathway is the Ire1 led catalysis of splicing of Hac-1 mRNA. This stereotyped splicing acts as a powerful marker to compare the activity levels of Ire1 when exposed to altering conditions. A multitude of diseases, ranging from cancers such as melanoma to other disorders such as diabetes have been linked to improper functioning of the UPR. Various novel drugs have been tested to see what kind of inhibition, if any, is exhibited onto Ire-1 during the UPR in order to help determine the normal functioning of the transmembrane protein itself. In the steps of drug development, it is critical to understand the biochemistry of ligand-receptor interaction before further research can be done.

POSTER #32:

The Effect of Schlafens on Notch Signaling

Elaine Lin
Dr. Michael David

Interferons play a crucial role in antiviral immunity and have been implicated in T-cell development. Interferons upregulate interferon stimulated genes, which include the novel Schlafen gene family. Because Schlafens are differentially regulated during thymocyte development, they may have a regulatory role in T-cell development. A Blast search of conserved short sequence elements within the Schlafen protein family revealed some homology with Numb, a known inhibitor of the Notch signaling pathway that is crucial in early T-cell development. This project aims to elucidate the possibility that Schlafens may hold similar regulatory functions as Numb in T-cell development by modulating the Notch-Numb pathway. We demonstrate that certain Schlafens enhance Notch signaling. It is hypothesized that Schlafens are expressed in the thymus to inhibit Numb in order to increase Notch signaling.

POSTER #33:

Bioinformatic Analysis of Bacterial Mercury Ion Transporters

Timothy Mok
Dr. Milton Saier

Currently there are five types of mercury protein transporters in bacteria that have been discovered: MerC, MerE, MerF, MerH, and MerT. Their general function is to mediate ion uptake across the bacterial membrane into the cell. Although they are present in prokaryotes of different phyla, and although they currently comprised of distinct families, my goal is to utilize bioinformatics and the Super Family Principle to determine if they are related by common descent. After using programs such as GAP and SSearch to establish homology, I will align and analyze their amino acid sequences to find conserved motifs. This will allow us to discern sequences that support the conclusion of homology, thus establishing that the proteins belong to a single superfamily. The significance of the conclusion will allow us to better predict structures, function, and mechanisms of the proteins.

POSTER #34:

THE ROLE OF TRPC AND Na+/Ca2+ EXCHANGER IN MEDIATING TGFβ-INDUCED PANCREATIC CANCER CELL MOTILITY

Tiffany Ornelas
Dr. Jimmy Chow

Pancreatic cancers show disrupted TGFβ-SMAD suppressive signaling, which unmasks the non-SMAD pathways to predominate and drive metastasis. It was reported that TGFβ induces increases in cytoplasmic free Ca2+ in pancreatic cancer cells, but how TGFβ mobilizes calcium is not known. Transient receptor potential (TRP) ion channels and Na+/Ca2+ exchanger (NCX) are plasma membrane proteins that play prominent roles in the control of cellular Ca2+ homeostasis, but little is known regarding their roles in cancer cells. We hypothesize that TGFβ induces cytoplasmic free Ca2+ and cell motility is due to functional coupling and activation of the TRPC and NCX1.  Therefore, we examined the role of TRPC and NCX1 in TGFβ-induced cell motility using a combination of biochemical, functional, and pharmacological studies.  We found that TGFβ triggers both intracellular Ca2+ release and extracellular Ca2+ entry via functional coupling of TRPCs and NCX1 in pancreatic cancer cells. In addition, our data indicated that either treatment of KB-R7943 (a selective NCX inhibitor) or transfection with siRNA against NCX1 reverses TGFβ-induced pancreatic cancer cell motility. The data suggest that TRPCs and NCX1 may be potential therapeutic targets for pancreatic cancer.

POSTER #35:

Encapsidating mCherry into bacteriophage Hong Kong 97 immature and mature capsid

Kristina Pedersen
Dr. Jack Johnson

Hong Kong 97 is a phage in which virus like particles (VLPs) are expressed and assembled in E. Coli. It incorporates ~60 copies of serine protease to remove the N-terminal domain (delta domain) that drives capsid assembly, followed by autoproteolysis to mature in vitro, forming a stable particle with a catanated ring structure. We employed this VLP as a transport capsule for therapeutic agents into targeted cells. For our experiments, we engineered a fusion protein that contained mCherry and the protease. This fusion protein has moderate affinity towards the capsomers and was encapsidated into the capsid. Our results showed that ~60 copies of mCherry were inside of particles and that the protease was active and removed the delta domain. However, the protease did not cleave itself free from mCherry. Therefore, we are currently revising of the fusion protein in order to optimize the activity of the protease. HK97 VLP’s incorporating therapeutic agents are being developed as the next generation tissue specific drug delivery system.

POSTER #36:

Expression and characterization of Plasmepsin X, a Plasmodium falciparum aspartic protease

Kenneth Pettersen
Dr. Joseph Vinetz

Malaria is among the most devastating infectious diseases affecting developing countries. Plasmodium, the parasite that causes malaria, must invade multiple tissue types in its vertebrate and invertebrate hosts. Numerous proteins secreted by Plasmodium are vital for its survival and successful propagation throughout its complex life cycle. Plasmepsin X (PM X) is an aspartic protease believed to play an essential role in the invasiveness of Plasmodium falciparum, the species responsible for the most lethal form of human malaria. The objective of this project is to express active, recombinant protease and to characterize its function. Recombinant PM X was successfully cloned and expressed in E. coli as insoluble protein. Solubilization was achieved under denaturing conditions but failed to generate active protease when refolded. PM X was then cloned and expressed in a cell-free wheat germ expression system, which produced soluble PM X. Attempts to induce autocatalysis under acidic conditions were unsuccessful. A construct containing an enterokinase cleavage site was subsequently cloned and expressed to better facilitate cleavage of the prodomain in vitro. GST purification and cleavage of the protease by enterokinase was successful with optimal conditions for proteolytic activity currently under investigation. Substrate analysis and screening for molecular inhibitors will also be performed. Expression of active, recombinant PM X will facilitate a better biochemical understanding of its role in malaria transmission and could lead to a novel anti-malarial drug.

POSTER #37:

Development of an ORF isolation protocol by expression of genomic clones in Nicotiana

Tiffany Su
Dr. Steve Kay

Transcription factors (TFs) are core to the regulation of gene expression during diverse processes, including developmental programs and responses to the environment.  A genome-wide search identified more than 2000 TFs in the Arabidopsis genome.  Our lab initiated the construction of a library of TFs to be used as a genomic tool to help dissect gene regulatory networks in the plant model Arabidopsis thaliana.  The ultimate objective is to generate a complete library of all TFs to facilitate both in vivo and in vitro experiments. Initially, traditional cloning techniques were used to isolate many of these TF genes from cDNA pools.  Despite numerous efforts, over 500 TFs have yet to be cloned.  The goal of this project was to develop a transient expression system in tobacco leaves to ectopically express properly spliced genomic amplicons of the remaining TFs.  This protocol bypasses the problems associated with low or tissue specific expression that can hinder gene isolation. The gene segment is cloned and transferred into a plasmid for ectopic expression in plants under a constitutive promoter.  The construct is transformed into Agrobacterium tumefaciens and the gene of interest is transformed into the tobacco leaves by infiltration.  The gene is transiently expressed, and introns are subsequently removed by the tobacco plant’s own splicing machinery.  The infiltrated leaves are cut, total RNA is extracted, and cDNA is synthesized.  From this cDNA, the TF is amplified, cloned, and included in the library. DNA sequencing of the clones is used to determine if the genes obtained are spliced correctly.  Using this protocol, the remaining TFs can now be cloned and incorporated into a complete TF library, which will be a resource made available to the plant science community to use in experiments to gain understanding of their functional roles in planta.

POSTER #38:

Structural Study of Deadly Viral Glycoprotein through Deuterium Exchange Mass Spectrometry

Brian Wong
Dr. Virgil Woods

The Zaire Ebolavirus strain causes severe, life-threatening hemorrhagic fevers and has one of the highest fatality rates of any viral pathogen that affects humans. Of the virally-encoded proteins, the Glycoprotein 1 subunit (GP1) of Ebolavirus found on the surface of the virion is critically responsible for attaching the Ebolavirus to host cells. Crystal structures of viral GPs offer tremendous insights into infection mechanisms, pathogenesis, viral assembly and immune recognition; however, no structure of any Ebolavirus GP exists to date. The goal of this study is to determine the structural characteristics of Ebolavirus GP1 by using Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS), in an effort to crystallize the protein. DXMS takes advantage of the observed ability of amide hydrogens on proteins to exchange with hydrogens in the surrounding solvent at a rate dependent on protein structure and solvent accessibility. By substituting solvent hydrogens with the isotope deuterium, the extra mass incorporated can be detected and the exchange rate localized to specific regions of the protein. Highly disordered, solvent accessible loops having no functional role have been known to prevent protein crystallization.  DXMS can identify such loops and help create functional GP1 constructs that are crystsallizable. Additionally, DXMS can and will be used to map antibody epitopes in order to design immunogens that boost effective antiviral immune response.

POSTER #39:

Characterization of two enhancers upstream of the Gonadotropin-releasing hormone (GnRH) gene

Kathleen Yip
Dr. Pamela Mellon

Gonadotropin-releasing hormone (GnRH) is a neuropeptide responsible for proper reproductive function, and its gene expression is limited to about 800 differentiated neurons in the hypothalamus of the mouse brain. The mechanism by which this gene is expressed only in these neurons is not clear. Although the GnRH enhancer-1 (GnRH-E1;  1863/ 1571) and promoter (GnRH-P; -173/+1) target the GnRH neurons exclusively, the two elements are not sufficient for complete expression, and target only 50% of GnRH neurons in the adult mouse. This suggests that there are additional regulatory elements for complete GnRH gene expression. Previously, two additional evolutionarily conserved far-upstream regions were determined to be involved in the regulation of GnRH gene expression. These regions are -3135/-2980 (GnRH-E2) and -4199/-3895 (GnRH-E3). We further characterized these regions of the GnRH gene to determine specific areas critical for activity using luciferase reporter plasmids transfected into the GT1-7 cell line, which is a model of the mature GnRH neuron. While GnRH-E2 displayed redundancy within itself, the activity of GnRH-E3 was narrowed down to the proximal end at  3952/-3895. Mutation of a conserved AT-rich site in the GnRH-E3 critical region shown to bind to Oct-1, Msx1 and Dlx2 resulted in decreased expression. Overexpression of Dlx2 increased expression of GnRH-E3 indicating that Dlx2 is a transcriptional activator of GnRH-E3. Understanding GnRH expression may lead to understanding hypogonadotropic hypogonadism and Kallmann’s Syndrome, which are forms of infertility and are the result of defects in the production of GnRH or development of the terminally differentiated GnRH neurons.

POSTER #40:

Proline-23-Histidine (P23H) Mutation in Rhodopsin Protein

Mark Yu
Dr. Jonathan Lin

The goal of this research to determine the molecular mechanism that leads to the expression of P23H mutation in Rhodopsin Protein   Methodologies used in this research would be Cloning mutated Rhodopsin protein into appropriate vectors for transfection into mammalian cell, testing expression of P23H by Western blot, and testing  expression of genes that indicate expression of P23H, studying mice with this mutation   Experiments planned for this research include Polymerase Chain Reaction (PCR), subcloning, bacterial Transformation, Deoxyribonucleic Acid (DNA) isolation/purification, sequencing, cell culture, transfection, Western Blotting, Ribonucleic Acid (RNA) Isolation/Purification, cDNA Synthesis, qPCR

POSTER #41:

A field guide to the cluster A1 and E phage isolated by the UCSD phage hunters

Roshmi Bhattacharya, Priya Chakrabarti, Aaron Kappe, Victoria Selzer, Alexandra Stanley, Oleg Stens, Yi Shuan Wu, Anne Lamsa
Dr. Kit Pogliano and Dr. Joe Pogliano

POSTER #42:

A field guide to the cluster B1 and F1 phage isolated by BIMM171 students

Jeremy Chang, Mahathee Chetlapalli, David Horstman, Michael Pham, Andryus Planutis, Kyle Szeto, Cliff Wu, Anne Lamsa Dr. Kit Pogliano and Dr. Joe Pogliano

POSTER #43:

The unidentified phage of the UCSD phage genomics course 2009-2010

Kristina Chun, Abby Conroy, Payal Desi, Hubert Jenq, Brittany Khong, Catherine Kuo, Joseph Steward, Anne Lamsa
Dr. Kit Pogliano and Dr.Joe Pogliano

POSTER #44:

Analysis of the Dot Product Genome

Andrew Grainger, Zac Hann, Lawrence Ku, Sean Lund, Amy Nguyen, Tasha Thompson, Lisa Zeng, Anne Lamsa
Dr. Kit Pogliano and Dr. Joe Pogliano