Genetics and Genomics Commons^™

Motif-Vi Loop Acts As A Nucleotide Valve In The West Nile Virus Ns3 Helicase, Priti Roy, Zachary Walter, Lauren Berish, Holly Ramage, Martin Mccullagh

Department of Microbiology and Immunology Faculty Papers

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear …

Genetics Of Mdh In Humans, Adam Haberman, Celeste N. Peterson

Biology: Faculty Scholarship

Malate dehydrogenase (MDH) performs key roles in metabolism, but little is known about its function specifically in human health and disease. In this minireview, we describe the incomplete state of our knowledge of human MDH genetics. Humans have three MDH genes with a total of four validated isoforms. MDH1 and MDH2 are widely expressed, while MDH1B is only expressed in a small subset of tissues. Many mutations in MDH1 and MDH2 have been identified in patients, but only a few have been studied to determine what symptoms they cause. MDH1 has been associated with cancer and a neurodevelopmental disorder. MDH2 …

Rewiring The Sex-Determination Pathway During The Evolution Of Self-Fertility., Yongquan Shen, Shin-Yi Lin, Jonathan Harbin, Richa Amin, Allison Vassalotti, Joseph Romanowski, Emily Schmidt, Alexis Tierney, Ronald E Ellis

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Although evolution is driven by changes in how regulatory pathways control development, we know little about the molecular details underlying these transitions. The TRA-2 domain that mediates contact with TRA-1 is conserved in Caenorhabditis. By comparing the interaction of these proteins in two species, we identified a striking change in how sexual development is controlled. Identical mutations in this domain promote oogenesis in Caenorhabditis elegans but promote spermatogenesis in Caenorhabditis briggsae. Furthermore, the effects of these mutations involve the male-promoting gene fem-3 in C. elegans but are independent of fem-3 in C. briggsae. Finally, reciprocal mutations in these genes show …

Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum And Novel Clinical Observations In 241 Patients, Rachael C. Heath Jeffery, Jennifer A. Thompson, Johnny Lo, Enid S. Chelva, Sean Armstrong, Jose S. Pulido, Rebecca Procopio, Andrea L. Vincent, Lorenzo Bianco, Maurizio Battaglia Parodi, Lucia Ziccardi, Giulio Antonelli, Lucilla Barbano, João P. Marques, Sara Geada, Ana L. Carvalho, Wei C. Tang, Choi M. Chan, Camiel J. F. Boon, Jonathan Hensman, Ta-Ching Chen, Chien-Yu Lin, Pei-Lung Chen, Ajoy Vincent, Anupreet Tumber, Elise Heon, John R. Grigg, Robyn V. Jamieson, Elisa E. Cornish, Benjamin M. Nash, Shyamanga Borooah, Lauren N. Ayton, Alexis Ceecee Britten-Jones, Thomas L. Edwards, Jonathan B. Ruddle, Abhishek Sharma, Rowan G. Porter, Tina M. Lamey, Terri L. Mclaren, Samuel Mclenachan, Danial Roshandel, Fred K. Chen

Wills Eye Hospital Papers

PURPOSE: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene.

METHODS: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations.

RESULTS: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, …

Armc5 Controls The Degradation Of Most Pol Ii Subunits, And Armc5 Mutation Increases Neural Tube Defect Risks In Mice And Humans, Hongyu Luo, Linjiang Lao, Kit Sing Au, Hope Northrup, Xiao He, Diane Forget, Marie-Soleil Gauthier, Benoit Coulombe, Isabelle Bourdeau, Wei Shi, Lucia Gagliardi, Maria Candida Barisson Villares Fragoso, Junzheng Peng, Jiangping Wu

Student and Faculty Publications

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II).

RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized …

Fused In Sarcoma Regulates Glutamate Signaling And Oxidative Stress Response, Chiong-Hee Wong, Abu Rahat, Howard C Chang

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Mutations in fused in sarcoma (fust-1) are linked to ALS. However, how these ALS causative mutations alter physiological processes and lead to the onset of ALS remains largely unknown. By obtaining humanized fust-1 ALS mutations via CRISPR-CAS9, we generated a C. elegans ALS model. Homozygous fust-1 ALS mutant and fust-1 deletion animals are viable in C. elegans. This allows us to better characterize the molecular mechanisms of fust-1-dependent responses. We found FUST-1 plays a role in regulating superoxide dismutase, glutamate signaling, and oxidative stress. FUST-1 suppresses SOD-1 and VGLUT/EAT-4 in the nervous system. FUST-1 also regulates synaptic AMPA-type glutamate receptor …

Identification And Characterization Of Two Novel Kcnh2 Mutations Contributing To Long Qt Syndrome, Anthony Owusu-Mensah, Jacqueline Treat, Joyce Bernardi, Ryan Pfeiffer, Robert Goodrow, Bright Tsevi, Victoria Lam, Michel Audette, Jonathan M. Cordeiro, Makarand Deo

Electrical & Computer Engineering Faculty Publications

We identified two different inherited mutations in KCNH2 gene, or human ether-a-go-go related gene (hERG), which are linked to Long QT Syndrome. The first mutation was in a 1-day-old infant, whereas the second was in a 14-year-old girl. The two KCNH2 mutations were transiently transfected into either human embryonic kidney (HEK) cells or human induced pluripotent stem-cell derived cardiomyocytes. We performed associated multiscale computer simulations to elucidate the arrhythmogenic potentials of the KCNH2 mutations. Genetic screening of the first and second index patients revealed a heterozygous missense mutation in KCNH2, resulting in an amino acid change (P632L) in the …

Rab1a Haploinsufficiency Phenocopies The 2p14-P15 Microdeletion And Is Associated With Impaired Neuronal Differentiation, Jonathan J Rios, Yang Li, Nandina Paria, Ryan J Bohlender, Chad Huff, Jill A Rosenfeld, Pengfei Liu, Weimin Bi, Kentaro Haga, Mitsunori Fukuda, Shayal Vashisth, Kiran Kaur, Maria H Chahrour, Michael B Bober, Angela L Duker, Farah A Ladha, Neil A Hanchard, Kristhen Atala, Anas M Khanshour, Linsley Smith, Carol A Wise, Mauricio R Delgado

Student and Faculty Publications

Hereditary spastic parapareses (HSPs) are clinically heterogeneous motor neuron diseases with variable age of onset and severity. Although variants in dozens of genes are implicated in HSPs, much of the genetic basis for pediatric-onset HSP remains unexplained. Here, we re-analyzed clinical exome-sequencing data from siblings with HSP of unknown genetic etiology and identified an inherited nonsense mutation (c.523C>T [p.Arg175Ter]) in the highly conserved RAB1A. The mutation is predicted to produce a truncated protein with an intact RAB GTPase domain but without two C-terminal cysteine residues required for proper subcellular protein localization. Additional RAB1A mutations, including two frameshift mutations and …

Pls3 Missense Variants Affecting The Actin-Binding Domains Cause X-Linked Congenital Diaphragmatic Hernia And Body-Wall Defects, Florence Petit, Mauro Longoni, Julie Wells, Richard S Maser, Eric L Bogenschutz, Matthew J Dysart, Hannah T M Contreras, Frederic Frénois, Barbara R Pober, Robin D Clark, Philip F Giampietro, Hilger H Ropers, Hao Hu, Maria Loscertales, Richard Wagner, Xingbin Ai, Harrison Brand, Anne-Sophie Jourdain, Marie-Ange Delrue, Brigitte Gilbert-Dussardier, Louise Devisme, Boris Keren, David J Mcculley, Lu Qiao, Rebecca Hernan, Julia Wynn, Tiana M Scott, Daniel G Calame, Zeynep Coban-Akdemir, Patricia Hernandez, Andres Hernandez-Garcia, Hagith Yonath, James R Lupski, Yufeng Shen, Wendy K Chung, Daryl A Scott, Carol J Bult, Patricia K Donahoe, Frances A High

Student and Faculty Publications

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical …

A Rare Metastatic Mesenteric Malignant Pecoma With Tsc2 Mutation Treated With Palliative Surgical Resection And Nab-Sirolimus: A Case Report, Luke Meredith, Timothy Chao, Avinoam Nevler, Atrayee Basu Mallick, Rajan Singla, Peter Mccue, Wilbur Bowne, Wei Jiang, Md, Phd

Kimmel Cancer Center Faculty Papers

BACKGROUND: Malignant perivascular epithelioid cell tumors (PEComas) are exceedingly rare malignant mesenchymal neoplasms with characteristic morphological and immunohistochemical (IHC) patterns. However, some malignant PEComas are poorly differentiated with atypical histopathological features, making a definitive diagnosis difficult. PEComas are most commonly found in females and often show either TSC1 or TSC2 alterations, which result in the activation of the mTOR pathway, or TFE3 fusions. Given these molecular characteristics, mTOR inhibitors have recently been approved by the FDA in the treatment of malignant PEComas, particularly in those with TSC1/2 alterations. Therefore, molecular analyses may be helpful for both the diagnostic workup of …

Comparative Risks Of Initial Aortic Events Associated With Genetic Thoracic Aortic Disease, Ellen S Regalado, Shaine A Morris, Alan C Braverman, Ellen M Hostetler, Julie De Backer, Ruosha Li, Reed E Pyeritz, Anji T Yetman, Elena Cervi, Sherene Shalhub, Richmond Jeremy, Scott Lemaire, Maral Ouzounian, Arturo Evangelista, Catherine Boileau, Guillaume Jondeau, Dianna M Milewicz

Student and Faculty Publications

BACKGROUND: Pathogenic variants in 11 genes predispose individuals to heritable thoracic aortic disease (HTAD), but limited data are available to stratify the risk for aortic events associated with these genes.

OBJECTIVES: This study sought to compare the risk of first aortic event, specifically thoracic aortic aneurysm surgery or an aortic dissection, among 7 HTAD genes and variant types within each gene.

METHODS: A retrospective cohort of probands and relatives with rare variants in 7 genes for HTAD (n = 1,028) was assessed for the risk of first aortic events based on the gene altered, pathogenic variant type, sex, proband status, …

Determinants Of Virus Variation, Evolution, And Host Adaptation, Katherine Latourrette, Hernan Garcia-Ruiz

Nebraska Center for Virology: Faculty Publications

Virus evolution is the change in the genetic structure of a viral population over time and results in the emergence of new viral variants, strains, and species with novel biological properties, including adaptation to new hosts. There are host, vector, environmental, and viral factors that contribute to virus evolution. To achieve or fine tune compatibility and successfully establish infection, viruses adapt to a particular host species or to a group of species. However, some viruses are better able to adapt to diverse hosts, vectors, and environments. Viruses generate genetic diversity through mutation, reassortment, and recombination. Plant viruses are exposed to …

Rnase Κ Promotes Robust Pirna Production By Generating 2',3'-Cyclic Phosphate-Containing Precursors, Megumi Shigematsu, Takuya Kawamura, Keisuke Morichika, Natsuko Izumi, Takashi Kiuchi, Shozo Honda, Venetia Pliatsika, Ryuma Matsubara, Isidore Rigoutsos, Susumu Katsuma, Yukihide Tomari, Yohei Kirino

Computational Medicine Center Faculty Papers

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated …

Frontotemporal Dementia Nonsense Mutation Of Progranulin Rescued By Aminoglycosides, Lisha Kuang, Kei Hashimoto, Eric J. Huang, Matthew S. Gentry, Haining Zhu

Molecular and Cellular Biochemistry Faculty Publications

Frontotemporal dementia (FTD) is an early onset dementia and is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5-26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its function under physiological and pathological conditions remains to be defined. Many FTD-causing nonsense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy.

Aminoglycosides are a class of antibiotics that possess a less known function …

Als Mutations Of Fus Suppress Protein Translation And Disrupt The Regulation Of Nonsense-Mediated Decay, Marisa Kamelgarn, Jing Chen, Lisha Kuang, Huan Jin, Edward J. Kasarskis, Haining Zhu

Toxicology and Cancer Biology Faculty Publications

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the fused in sarcoma (FUS) gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid–liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed …

The Zinc Transporter Zipt-7.1 Regulates Sperm Activation In Nematodes, Yanmei Zhao, Chieh-Hsiang Tan, Amber Krauchunas, Andrea Scharf, Nicholas Dietrich, Kurt Warnhoff, Zhiheng Yuan, Marina Druzhinina, Sam Guoping Gu, Long Miao, Andrew Singson, Ronald E Ellis, Kerry Kornfeld

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating …

Genotype-Phenotype Study In Patients With Valosin-Containing Protein Mutations Associated With Multisystem Proteinopathy, Ebaa Al-Obeidi, Sejad Al-Tahan, Abhilasha Surampalli, Namita Goyal, Annabel K. Wang, Andreas Hermann, Molly Omizo, Charles D. Smith, Tahseen Mozaffar, Virginia Kimonis

Neurology Faculty Publications

Mutations in valosin‐containing protein (VCP), an ATPase involved in protein degradation and autophagy, cause VCP disease, a progressive autosomal dominant adult onset multisystem proteinopathy. The goal of this study is to examine if phenotypic differences in this disorder could be explained by the specific gene mutations. We therefore studied 231 individuals (118 males and 113 females) from 36 families carrying 15 different VCP mutations. We analyzed the correlation between the different mutations and prevalence, age of onset and severity of myopathy, Paget's disease of bone (PDB), and frontotemporal dementia (FTD), and other comorbidities. Myopathy, PDB and FTD was present in …

Gain-Of-Function Experiments With Bacteriophage Lambda Uncover Residues Under Diversifying Selection In Nature, Rohan Maddamsetti, Daniel T. Johnson, Stephanie J. Spielman, Katherine L. Petrie, Debora S. Marks, Justin R. Meyer

Biological Sciences Faculty Publications

Viral gain-of-function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ’s J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative …

Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ω_ave. We found …

Zinc Transporters Ybtx And Znuabc Are Required For The Virulence Of Yersinia Pestis In Bubonic And Pneumonic Plague In Mice, Alexander G. Bobrov, Olga Kirillina, Marina Y. Fosso, Jacqueline D. Fetherston, M. Clarke Miller, Tiva T. Vancleave, Joseph A. Burlison, William K. Arnold, Matthew B. Lawrenz, Sylvie Garneau-Tsodikova, Robert D. Perry

Microbiology, Immunology, and Molecular Genetics Faculty Publications

A number of bacterial pathogens require the ZnuABC Zinc (Zn²⁺) transporter and/or a second Zn²⁺ transport system to overcome Zn²⁺ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn²⁺ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn²⁺ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic …

High Resolution Time-Course Mapping Of Early Transcriptomic, Molecular And Cellular Phenotypes In Huntington's Disease Cag Knock-In Mice Across Multiple Genetic Backgrounds., Seth A Ament, Jocelynn R Pearl, Andrea Grindeland, Jason St Claire, John C Earls, Marina Kovalenko, Tammy Gillis, Jayalakshmi Mysore, James F Gusella, Jong-Min Lee, Seung Kwak, David Howland, Min Young Lee, David Baxter, Kelsey Scherler, Kai Wang, Donald Geman, Jeffrey B Carroll, Marcy E Macdonald, George Carlson, Vanessa C Wheeler, Nathan D Price, Leroy Hood

Articles, Abstracts, and Reports

Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, …

Identification Of A Novel Gene On 10q22.1 Causing Autosomal Dominant Retinitis Pigmentosa (Adrp)., Stephen P Daiger, Lori S Sullivan, Sara J Bowne, Daniel C Koboldt, Susan H Blanton, Dianna K Wheaton, Cheryl E Avery, Elizabeth D Cadena, Robert K Koenekoop, Robert S Fulton, Richard K Wilson, George M Weinstock, Richard A Lewis, David G Birch

Faculty Publications

Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Recurrent Tissue-Specific Mtdna Mutations Are Common In Humans, David C. Samuels, Chun Li, Bingshan Li, Zhuo Song, Eric Torstenson, Hayley Boyd Clay, Antonis Rokas, Tricia A. Thornton-Wells, Jason H. Moore, Tia M. Hughes, Robert D. Hoffman, Jonathan L. Haines, Deborah G. Murdock, Douglas P. Mortlock, Scott M. Williams

Dartmouth Scholarship

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in …

Epigenetic Dominance Of Prion Conformers, Eri Saijo, Hae-Eun Kang, Jifeng Bian, Kristi G. Bowling, Shawn Browning, Sehun Kim, Nora Hunter, Glenn C. Telling

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, …

Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza

Dartmouth Scholarship

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes …

Accumulation Of Rhodopsin In Late Endosomes Triggers Photoreceptor Cell Degeneration, Yashodhan Chinchore, Amitavo Mitra, Patrick J. Dolph, Norbert Perrimon

Dartmouth Scholarship

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates …

H2-M3-Restricted Cd8+ T Cells Are Not Required For Mhc Class Ib-Restricted Immunity Against Listeria Monocytogenes, Sarah E. F. D'Orazio, Christine A. Shaw, Michael N. Starnbach

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Studies using major histocompatibility complex (MHC)-Ia–deficient mice have shown that MHC-Ib–restricted CD8⁺ T cells can clear infections caused by intracellular pathogens such as Listeria monocytogenes. M3-restricted CD8⁺ T cells, which recognize short hydrophobic N-formylated peptides, appear to comprise a substantial portion of the MHC-Ib–restricted T cell response in the mouse model of L. monocytogenes infection. In this study, we isolated formyltransferase (fmt) mutant strains of L. monocytogenes that lacked the ability to add formyl groups to nascent polypeptides. These fmt mutant Listeria strains did not produce antigens that could be recognized by M3-restricted T …

Estimation Of The Incidence Of A Rare Genetic Disease Through A Two-Tier Mutation Survey, R Chakraborty, M R Srinivasan, S Raskin

Student and Faculty Publications

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates …