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2015 Research Showcase
GAMB Abstracts
Abstract Title : Comparative Genomics of Mycobacteriophage Kersh
Abstract : In the Phage Genomics Research Initiative 2014-2015, UCSD freshmen isolated mycobacteriophages, viruses that infect host bacteria, from soil samples. We further characterized Kersh, a temperate phage that belongs to the F1 cluster. Upon sequencing using Illumina technology, we determined Kersh to be 60190 base pairs long. We analyzed Kersh using DNA Master, a program that determines putative genes, and manually adjusted some gene lengths to maximize coding potential and alignment with other known phage genes. Once Kersh?s genome was finalized, we utilized Phamerator, a tool for comparative bacteriophage genomics, to create a map that shows both nucleotide and protein similarities across multiple genomes. In comparing Kersh to multiple F1 phages, including Ibhubesi, Estave1, and Pacc40, we observed that Kersh displays a mosaic structure, which is indicative of horizontal genetic exchange. The analysis and annotation of Kersh?s genome expands our knowledge of mycobacteriophage and mycobacterium, which could lead to the discovery of therapeutic solutions to mycobacterial diseases. ADDITIONAL PRESENTERS: Areian Eghbali, Rishi Modi, Alison Fanton, and Lynley Fernandez
Abstract Title : The Characterization of Human Specific Genes, CHRFAM7A and TBC1D3, in Myeloid Cells
Abstract : Genes that have been evolutionarily conserved only in humans, known as human-specific genes, may be used as markers in detecting human specific responses to diseases. The function of human specific genes, such as CHRFAM7A and TBC1D3, remains largely unknown. Our lab is interested in defining human specific inflammation responses. The project presented here analyzes the expression and signaling of CHRFAM7A and TBC1D3 in myeloid cells to refine and validate a reverse transcriptase polymerase chain reaction (RT-PCR) assay with gel electrophoresis with cell lines as positive controls for application on humanized mice. Heterogeneity of CHRFAM7A and TBC1D3 expression was observed in myeloid cells collected from healthy donors and burn patients.
Abstract Title : Isolation and characterization of ER-derived pre-peroxisomal vesicles
Abstract : Previous studies suggest a role of the endoplasmic reticulum (ER) in de novo peroxisome biogenesis. Peroxisomes form by the heterotypic fusion of two specific pre-peroxisomal vesicles (ppVs) that bud from the ER, each carrying one subcomplex (RING and docking subcomplex) of the peroxisomal importomer, which is necessary for import competent peroxisomes. The exact composition and coat characteristics of ppVs still remain unknown. We blocked their heterotypic fusion (by deleting the PEX1 or PEX6 genes required for ppV fusion) to allow their enrichment and isolation, prior to their molecular characterization. Initial results with buoyant-density centrifugation suggest different buoyant characteristics for vesicular fractions containing RING and docking subcomplex proteins. Additionally, cells lacking Pex1p and Pex6p demonstrated slightly different peroxisomal membrane protein fractionation results in our comparative studies. Further analysis of density gradients, which still do not completely separate the ER, cytosolic and peroxisomal proteins, is required for reliable identification of the ppVs.
Abstract Title : Studying the Structure-Function Relationship of Syndecan-1 in Hepatic Uptake of Remnant Lipoproteins in Mice Using Adeno-associated Viral 2/8 Vectors
Abstract : The objective of this project is to examine how the structure of syndecan-1 affects its capacity to clear remnant lipoproteins in the liver in mice. The experimental design involves engineering Adeno-associated viral vectors containing the cDNA for syndecan-1. This is done by inserting cDNA encoding the full length of syndecan-1 into the vector, making mutations in the cDNA, and transfection studies in cultured hepatoma cells or by injection into syndecan-deficient mice. Lipoprotein binding and uptake will be measured in vitro and in cells and lipoprotein clearance will be measured in mice. Standard electrophoretic methods, PCR analysis, and DNA sequencing will be used for the cloning experiments. Lipoprotein uptake experiments are performed using optical methods and by measuring triglyceride levels.
Abstract Title : Predicting and Confirming Putative Functions of Genes in the Mycobacteriophage Kersh
Abstract : Earlier in the year we isolated mycobacteriophage Kersh in the lab by infecting host mycobacterium smegmatis and performing plaque purifications. To provide more data for practical applications of mycobacteriophage such as antibacterial therapeutics, this study aimed to predict and confirm the functions of the genes of Kersh. To accomplish this, we first annotated the sequenced genome of Kersh with DNA Master, a bioinformatics tool that utilizes several DNA analysis programs to predict protein-coding genes. After the protein coding genes were located, we used BLASTp and Phamerator to identify their potential functions and conserved domains. After putative functions were identified, the expression of some of the phage proteins was confirmed by using mass spectrometry to analyze a phage lysate. The expression of several putative structural genes was confirmed by the mass spectrometry results. ADDITIONAL PRESENTERS: Nathan Board, Jaidev Bapat, Taylor Harman, and Sean Guy
Abstract Title : Isolation and Purification of Mycobacteriophage from Soil
Abstract : In Fall of 2014, UCSD freshmen gathered soil samples from around the La Jolla and greater San Diego area in order to isolate and study mycobacteriophages. The soil samples were mixed with an enrichment culture containing Mycobacterium smegmatis in order to increase the phage yield. The enrichment culture was then filtered, the filtrate was streaked onto a lawn of Mycobacterium smegmatis and top agar, and the plates were incubated at 37℃. The phage plaques were then picked and re-streaked at least five times to ensure isolation of a single phage. Next, serial dilutions and spot titers were performed in order to determine phage concentrations. Phage lysates were created to increase the concentration, amplifying the phage so that enough genomic DNA could be obtained for sequencing. Electron microscopy imaging was performed in order to determine phage size and characteristics. Finally, DNA was digested using specific restriction enzymes, and gel electrophoresis was conducted to determine genome size. Of the twenty phages isolated, one phage, Kersh, was selected for sequencing. ADDITIONAL PRESENTERS: Chandra Couzens, Alyssa Hu, Marissa Parkhurst, and Amanda Tjitro
Advisor : DR. JING WANG
Abstract Title : Chemical Control of Gene Expression in Drosophila
Abstract : Spatiotemporal control of gene expression is a powerful technique for addressing gene function. The GAL4-UAS bipartite expression system is an important tool for spatial control of gene expression. Additionally, the GAL4/UAS system can be coupled with temperature to confer saptio-temporal control. However, variation in temperature alters physiology and behavior, particularly in Drosophila melanogaster. As an alternative, we have developed a chemical approach to control gene expression. Specifically, we have adopted a chemical method to regulate protein stability by fusing an Escherichia coli dihydrofolate reductase (ecDHFR)-derived destabilizing domain (DD) to any protein of interest (Iwamoto et al, 2010). This domain destabilizes the fusion protein and marks it for degradation by the proteasome. Trimethoprim (TMP), a small molecule, can bind to DD and prevent the degradation of the protein in a dose dependent, reversible manner. We have tested the applicability of this technology in Drosophila. We now aim to utilize this approach for spatio-temporal control of gene expression by fusing DD to GAL4. In this way, we hope to express any gene in a specified cell population in an inducible manner by feeding a small molecule to flies.
Abstract Title : Phage Hunting: A Survey of the Mycobacteriophages Isolated by UCSD Students
Abstract : The Phage Genomics Research Initiative is an innovative program at UC San Diego that began in 2007 as a part of the SEA-PHAGES program through HHMI (Howard Hughes Medical Institute). This educational opportunity introduced a nationwide genomics course available solely for college freshmen. The program focuses on the study of mycobacteriophage, which are viruses that infect mycobacteria such as M. tuberculosis. The analysis of their genomes and their proteins is a relatively new field of study that holds much potential for medical research, evolutionary studies, and more. As of 2015, UCSD freshmen have discovered 147 different phages, 6 of which have been sequenced and annotated. In this year?s course we isolated and sequenced the genome of a phage called Kersh, found by Joseph Chang in La Jolla, CA. This phage is a part of the F1 cluster and is a siphoviridae. Kersh, like every phage found during this program, uniquely adds to the body of knowledge about phage genomics and contributes to the greater understanding of phage and their specific traits.The Phage Genomics Research Initiative provides a foundation for aspiring microbiologists, phage enthusiasts, and scientific researchers alike. ADDITIONAL PRESENTERS: Madeleine Yu, Danielle Escueta, and Emelie Knight
Abstract Title : SIRT1 Gene Expression in Mouse Tail-Looping Models of Induced Disc Degeneration
Abstract : Although there are many determinants of degenerative scoliosis, the asymmetrical degeneration of the intervertebral disc (IVD) is thought to be a main contributor. Therefore, it may be clinically relevant to elucidate the pathology of disc degeneration and the role of genetics on the disc microenvironment. The present study observes the effect of SIRT1 expression in mice tail-looping models of induced disc degeneration. SIRT1 is a gene associated with DNA repair, transcriptional silencing, and regulation of apoptosis, and it has been identified to be abnormally expressed in IVDs of patients with degenerative disc disorders. MicroCT (mCT) imaging was performed on SIRT1 knockout and overexpression mice to visualize the progression of the scoliotic curve over a 4-week period. An analysis of disc height differentials indicated that disc height increased in SIRT1 knockout mice from week 2 to week 4. Additional research is necessary to determine the mechanism underlying these unexpected results.
Abstract Title : The role of varying RUNX1 3'UTR on RUNX1 expression
Abstract : Runt-related transcription factor 1 (RUNX1) is a transcription factor and a tumor suppressor gene that regulates differentiation of hematopoietic stem cells into mature blood cells. The three prime untranslated region (3'UTR) of mRNA contains regulatory regions that influence mRNA stability and translation. Various RUNX1 3'UTR variants have been reported , but little is known about functional differences between these variants. We aim to characterize how these RUNX1 3'UTR variants affect mRNA stability and protein expression. We cloned and analyzed the effect of various RUNX1 3'UTR fragments on protein expression through luciferase assay. We analyzed RNA-seq data to determine the RUNX1 3'UTR variants preferentially expressed by various human tissues and cell lines. We used database to predict RNA binding proteins sites on RUNX1 3'UTR sequence to explore how the length of RUNX1 3'UTR may regulate RUNX1 expression. This study will provide insights on RUNX1 expression regulation and direction for future research.
Abstract Title : The Inhibitory Role of ISG15 in Antigen Presentation Pathway
Abstract : Type I interferons implicate antigen presentation pathways. ISG15 is an interferon-simulated gene encoded protein, which is strongly upregulated by interferon α/β during pathogen infection and tumorigenesis. Using an antigen presentation T cell activation assay, we demonstrate that ISG15 treatment results in a significant decrease in CD8+ T cell activation with the GST-ovalbumin peptide fusion protein primed dendritic cells. Whereas ISG15 does not affect antigen presentation when the ovalbumin-peptide is used in the assay. Moreover, ISG15 does not affect the uptake of GST-ovalbumin peptide fusion protein into dendritic cells. These findings suggest ISG15 inhibits the antigen cross-presentation pathway ? perhaps playing a role in endosome mediated degradation. Based on these preliminary data, we aim to determine how ISG15 inhibits the antigen presentation pathway mechanistically. We will use the ovalbumin peptide reactive OT-1 and OT-2 mice to determine the physiological function of ISG15 in both MHC-I and MHC-II antigen presenting pathways
Abstract Title : The Roles of the Cell Adhesion Molecule Cadm4 in Zebrafish Cardiac Outflow Tract Development
Abstract : The cardiac outflow tract (OFT) is an important structure in all species of vertebrates. It is a small conduit that connects the cardiac chambers to the vascular network. Our previous study implicated that cadm4 functions to restrict the size of the OFT. Zebrafish embryos whose cadm4 function was knocked down by morpholinos exhibited an elongated OFT. However, the phenotype of zebrafish embryos with a cadm4 mutation is still unknown. In our study, we aim to find the function of cadm4 in OFT development in a genetic mutant background. To achieve this goal, we obtained a nonsense mutation in exon 2 of cadm4 from the Zebrafish Mutation Project. After incrossing the heterozygous cadm4 mutant fish to get homozygous mutant embryos, we observed cardiac morphology using a fluorescent reporter transgene. We found that not all homozygous mutant embryos had an elongated OFT. This suggests that the cadm4 mutation is not fully penetrant.