«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»
Phosphorylation status of a protein is important for activation of signaling pathways and affects many aspects of gene expression. The transcription factor myocyte enhancer factor 2 (MEF2) is known to have a complex phosphorylation pattern in vitro. This protein plays a vital role in activation of genes responsible for patterning of the sarcomeric muscle and is one of the components required for myogenesis. Although the phosphorylation status of MEF2 is thought to be a crucial determinant of its tissue specific function, there is little known about how this protein is modified in vivo. To better understand the phosphorylation pattern in vivo, we used tandem affinity purification along with western blotting, cell culture, and mass spectrometry to analyze MEF2 protein extracts from Drosophila melanogaster embryos. Initial work identified a modified residue at serine 98, with other potential sites of phosphorylation within close proximity.
Further investigation will include determination if phosphorylation of this residue is necessary for MEF2 function in vivo, generation of an S98 phosphomimetic mutant and analysis of its function in tissue culture, work to assess the effects of activating various signaling pathways upon MEF2 function, and comparison of phosphorylation patterns from adult tissues. By studying such patterns, we hope to gain insight into the factors that influence MEF2 regulation and ultimately affect the control of gene expression in muscle cells.
DIFFERENT SUBCELLULAR LOCALIZATION OF A MUTANT FORM OF ANKRD11 IS ASSOCIATED WITH KBGSYNDROME Ralph Valentin, Lei Cao, Clemer Abad, Katherina Walz.
University of Miami Miller School of Medicine, Miami, FL.
KBG syndrome is a rare, autosomal-dominant, and heterogeneous disorder. It is characterized by macrodontia of upper central incisors, learning disabilities, developmental delay, seizures, short stature, and skeletal and craniofacial abnormalities. Recently, it was reported several ankyrin repeat domain 11 protein (ANKRD11) mutations are responsible for KBG syndrome. We have characterized a mouse model with a spontaneous Ankrd11 missense mutation p.E2502K in the C-terminal. It recapitulated several phenotypic findings presented in human patients.
Primary culture of heterozygous mouse fibroblast revealed an accumulation of Ankrd11- mutated protein in the nucleolus while in the wild type, Ankrd11 protein localized in other regions of the nucleus. Most human mutations produce C-terminal truncated forms of ANKRD11. Thus, we expressed truncated forms of ANKRD11 in N2A cell lines to see if they have mislocalization. We found distinct localization of the proteins and higher expression when compared to wild type. This suggests a lack of proper proteolysis for the mutant proteins. Moreover, the missense mutation in the mouse is close to a putative D-box destruction domain which acts as a signal for the proteasome. This could explain the accumulation of the mutant protein in the nucleolus. Overall, our findings suggest mislocalization of Ankrd11 mutants in the nucleus is related to KBG syndrome. Furthermore, we are in the process of studying if this mutation affects the neuronal development in the mutant mouse which will explain the learning disability phenotype found in patients.
IDENTIFYING REGULATORY ELEMENTS IN THE TPNC41C PROMOTER RESPONSIBLE FOR MUSCLESPECIFIC EXPRESSION IN DROSOPHILA MELANOGASTERSara Maes, Maria Chechenova.
University of New Mexico, Albuquerque, NM.
Troponin C is one of three regulatory proteins that comprise the troponin complex, which plays an integral role in the execution of proper muscle contraction for all muscle types. In Drosophila melanogaster, 5 genes coding Troponin
100 UNDERGRADUATE POSTER ABSTRACTS
Biological Sciences C have been identified, and their expression is tissue-specific. The Troponin C at 41C gene (TpnC41C) encodes a protein that is expressed mostly in the thoraces of adult Drosophila. Particularly, this gene is expressed in tergal depressor of the trochanter muscles (TDT) or jump muscles, which are responsible for the motions associated with jumping prior to flight takeoff. In this work, we are focused on identification of the regulatory elements in the promoter of the TpnC41C gene that direct its tissue-specific expression. We developed 2 reporter plasmids with mutant forms of the TpnC41C promoter placed upstream of the LacZ gene. Flies containing genomic insertion of these plasmids were examined for expression of the reporter protein β-galactosidase. The results of this experiment will indicate whether the mutated regions of the TpnC41C promoter contain regulatory sequences that are essential for specific expression of the gene in jump muscles. These findings could potentially identify specific binding sequences for transcription factors that regulate gene expression in TDT muscles. Understanding the mechanisms of gene expression in model organisms such as Drosophila melanogaster provides us with knowledge of evolutionary-conserved regulatory pathways of muscle formation in all animals, including mammals, and could be applied towards the development of new medical approaches for treating muscle diseases.
CHARACTERIZING THE EFFECTS THAT TIN2 MUTATIONS HAVE ON TELOMERASE ACTIVITY:
AMPLIFICATION AND SEQUENCE ANALYSIS OF ENDOGENOUS AND ENGINEERED TIN2Sara Balla, Amanda Frank, Lifeng Xu.
University of California, Davis, Davis, CA.
Patients with dyskeratosis congenita (DC) are at high risk for developing bone marrow failure and multiple types of cancer. All DC patients have extremely short telomeres, regions of repetitive sequence at eukaryotic chromosome ends. The telomeres of highly proliferative cells are elongated by telomerase. Telomerase access to the telomeres is regulated by the telomere-binding shelterin protein complex. Mutations within the subunits of telomerase have been found in some patients with DC. Recently, however, heterozygous mutations within the shelterin component TIN2 have been discovered in DC patients that do not have telomerase mutations. It is unclear why the mutations within TIN2 lead to short telomeres in DC patients. We hypothesize that the TIN2 mutations affect telomerase function resulting in short telomeres. Previously, it was determined that the TIN2 mutations do not function in a dominant negative manner. To test if the TIN2 mutations are haploinsufficient, human cell lines with heterozygous TIN2 mutations were created by gene editing. Polymerase chain reaction (PCR) was performed to amplify both the endogenous TIN2 allele and the edited mutant or wild-type TIN2 allele. The TIN2 sequence was then analyzed to verify that there were no additional mutations. Thus far, the sequences for six wild-type and two mutant clones have been confirmed with no other mutations than the one we engineered. After verifying the TIN2 sequence, we plan to compare telomere length and telomerase activity of the mutant and wild-type clones to gain insight into the mechanism by which TIN2 mutations lead to short telomeres.
THE IMPACT OF HEAT STRESS ON POLLEN TUBE GROWTH IN TOMATODavid Barrera1, Alexander Markes1, Emma Corcoran1, Jenzel Espares1, Sandra Kimokoti1, Eric Chen1, Alexander Blum1, Austin Draycott1, Steven Michael1, Alison DeLong1, Ravishankar Palanivelu2, Mark Johnson1.
Brown University, Providence, RI, 2School of Plant Sciences, University of Arizona, Tucson, AZ.
1 In the summer of 2012, many farmers tended cornfields that failed to produce a crop because of extremely high temperatures during the 2 week period when the plants reproduce. Corn kernels and other critical seed crops including rice and wheat are produced when a pollen tube grows through floral tissue to deliver sperm to female gametes. If fertilization fails, so does the harvest. Fertilization in plants is thought to be highly temperature sensitive but little is known about how the pollen tube responds to increases in temperature. We are using tomato as a model system to understand the impact of high temperature on pollen tube function and gene expression. Our first goal is to define optimal pollen tube growth temperatures in multiple, genetically distinct tomato varieties including a series of heat-tolerant tomatoes known to naturally produce fruit at high temperatures. Our experiments suggest that tomato pollen tubes can tolerate mild temperature increases but pollen tube growth fails above a critical threshold. We are also analyzing the expression of critical, heat-responsive genes during tomato pollen-tube growth to determine whether heat tolerant varieties have a different response. Our long-term goals include defining the pollen tube’s response to high temperature using genomics and defining individual genes that confer thermal tolerance to tomato pollen-tube growth.
CRISPR/CAS9-MEDIATED GENE TARGETING IN MAMMALIAN FIBROBLASTSLuis Cedeno-Rosario1, Shuo-Ting Yen2, Jenny Deng3, Richard Behringer3.
University of Puerto Rico at Humacao, Humacao, PR, 2Baylor College of Medicine, Houston, TX, 3The University of 1 Texas MD Anderson Cancer Center, Houston, TX.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems are adaptive immune systems which are found in bacteria and archea. These systems provide acquired immunity against bacteriophages by targeting nucleic acid in a sequence specific manner. Cas9 encodes an RNA-guided nuclease and together with an engineered RNA with 20 bases that match a target gene, will result in small deletions. Genome editing by CRISPR has been shown effective in many model organisms such as mouse, but little has been done in other mammalian species. The purpose of our study is to open up new genome editing mutagenesis methods in species that are not currently amenable to targeted gene manipulation. We hypothesize that the CRISPR/Cas9 system can induce a targeted mutation in a specific locus in fibroblasts derived from various mammalian species. Here, we coexpressed Cas9 and a chimeric guide RNA with which to target the HPRT locus in cultured mammalian cells from diverse species. Oligonucleotides were designed based on the target-site sequence and inserted into the CRISPR/Cas9 plasmid. The plasmid was introduced into the appropriate mammalian cells and HPRT-deficient cells were selected in medium using 6-thioguanine. Our studies will determine the feasibility of genome editing in diverse mammalian species.
IDENTIFYING DIFFERENTIALLY EXPRESSED GENES IN SLEEP-DEPRIVED MICEGabriel Vela, Susan McClatchy, Gary Churchil.
Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME.
Sleep is an incredibly complex behavior that remains a largely unexplained mystery. Sleep renders organisms vulnerable for extended periods of time, yet evidence suggests that sleep is evolutionarily conserved across species from worms to animals and thus must have an important function. It is clear that sleep is under genetic control, but the mechanisms behind this control are still the subject of inquiry. Understanding these mechanisms is increasingly important as sleep has been linked to diseases such as obesity or metabolic syndrome. We endeavor to find genes that will explain the genetic mechanisms underlying sleep and its function. We will identify genes with significant expression differences in the hypothalamus and cerebral cortex of sleep-deprived and control mice, which of these genes are upregulated or downregulated, and which share biological processes or functions. Preliminary studies show that a large amount of genes have significant expression differences in both the hypothalamus and the cerebral cortex. Identifying these genes will further our understanding of sleep and its relation to brain plasticity, metabolism, and biosynthesis. Understanding these relationships will lead to better treatments for sleep disorders and other sleeprelated diseases.
CHARACTERIZATION OF A FIELD ISOLATE OF TOMATO YELLOW LEAF CURL VIRUS - CLONINGXavier De Luna1, Biology, Garry Sunter2.
University of Texas at San Antonio, San Antonio, TX, 2University of San Antonio, San Antonio, TX.
1 Tomato yellow leaf curl virus (TYLCV) is a geminivirus and a major tomato pathogen, causing extensive crop loss.
The main controlling agent is the use of insecticides to limit the whitefly vector. Tomatoes are one of the leading fresh and processed vegetable crops worldwide, demonstrating the extreme importance for understanding mechanisms of TYLCV pathogenesis. This project involves the characterization of a strain of TYLCV that was shown to be present in plants showing symptoms of an infection in regions near San Antonio, Texas. The goals of the project are to clone and sequence the TYLCV strain and then perform infectivity studies in different hosts, including tomato. Rolling circle amplification was performed on DNA samples isolated from plants exhibiting symptoms typical of a TYLCV infection.
Following restriction of the RCA products, a unit-length genomic fragment of the viral genome was cloned into a pUC-based cloning vector. The entire genome was sequenced and was confirmed to be TYLCV. Infectious clones are being constructed that comprise tandem dimers of the TYLCV genome. We propose to analyze viral transcripts produced during infection and perform mutagenesis to determine the function of the 6 genes present in the viral genome. The replication kinetics of TYLCV will be determined using a protoplast system. The outcome of these experiments will increase our knowledge concerning transcription and replication in TYLCV and form the basis for the
TELOMERE LENGTHS IN INDIVIDUALS WITH IDIOPATHIC PULMONARY FIBROSISJoel Begay, Mary Beth Scholand, Roger Wolff, Richard Cawthon, Hilary Coon, Spencer Whipple, Patrick Carey.
University of Utah, Salt Lake City, UT.