Using Complex Selection Dynamics to Reveal The

Total Page:16

File Type:pdf, Size:1020Kb

Using Complex Selection Dynamics to Reveal The USING COMPLEX SELECTION DYNAMICS TO REVEAL THE FITNESS LANDSCAPE AND CROSS EVOLUTIONARY VALLEYS by Barrett Steinberg A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland June 2015 Abstract Nature repurposes proteins via evolutionary processes. During evolution, the fitness landscapes of proteins change dynamically. Selection for new functionality leaves the protein susceptible to genetic drift in the absence of selective pressure for the former function. Drift is considered to be a driver of evolution, and functional tradeoffs are common during selection. We measured the effect on ampicillin resistance of ~12,500 unique mutants of alleles of TEM-1 β-lactamase along an adaptive path in the evolution of cefotaxime resistance. This series of shifting protein fitness landscapes provides a systematic, quantitative description of genetic drift and pairwise/tertiary intragenic epistasis involving adaptive mutations. Our study provides insight into the relationships between mutation, protein structure, protein stability, epistasis, and drift and reveals the tradeoffs inherent in the evolution of new functions. We further use principles of ruggedness and dynamic change in fitness landscapes to develop and evaluate novel directed evolution strategies using complex selection dynamics. Interestingly, the strategy that included negative selection relative to the original landscape yielded more highly active variants of β-lactamase than the other four selection strategies. We reconstructed evolutionary pathways leading to this highly active allele, confirmed the presence of a fitness valley, and found an initially deleterious mutation that serves as an epistatic bridge to cross this fitness valley. The ability of negative selection and changing environments to provide access to novel fitness peaks has important implications for applied directed evolution as well as the natural evolutionary mechanisms, particularly of antibiotic resistance. ii We finally applied principles of the influence of selection environment to a clinically relevant system by comparing the evolutionary pathways of cells evolving competitively and continuously to single antibiotics, a cocktail of two antibiotics, or alternating cycles of the two antibiotics. We find evidence for distinct evolutionary pathways between antibiotic strategies. Specifically, we suggest that cocktail strategies appear to select for “specialists” of varying activity while cycling more stringently selects for “generalists.” We hypothesize that this result is due to separately emerging populations to evolve in distinct niches during cocktail therapies. Our results have direct relevance to informing clinical antibiotic regimens. Advisor: Dr. Marc Ostermeier, Professor of Chemical and Biomolecular Engineering, Johns Hopkins University Readers: Dr. Michael Betenbaugh, Professor of Chemical and Biomolecular Engineering, Johns Hopkins University Dr. Robert Schleif, Professor of Biology, Johns Hopkins University iii Acknowledgments First and foremost, I would like to thank Kathleen January for her boundless support. I would not be here without both sharing help with her during harder times and joy with her at each small victory. I am relieved and ecstatic to finally see our greater plans begin to work out together. I also thank my parents and family for providing the building blocks towards my education. I couldn’t have finished without the support of members of the Ostermeier lab. Specifically, I couldn’t ask for a better coworker and friend than Nirav Shelat. Nirav, thank you for the science, pool, and whiskey. I’ve shared great camaraderie with Courtney Gonzalez, Nathan Nicholes, and Tina Xiong. Martin Kang, great job and best of luck. Outside the lab, both Dillon Nye and Joey Priola have shown to be excellent friends. I hope all of these connections are lasting, and wish you all the best of luck. I must directly thank Dr. Horacio Frydman for introducing me to proper scientific method and instilling the first true appreciation of science into me. Of course, I also thank Dr. Ostermeier for the freedom and critical feedback he has provided. He has given me the opportunity to try countless extra experiments, most of which have failed, but a few of which have succeeded. I have this freedom to thank for honing my scientific creativity, and these acknowledgements to thank for dealing with the many failures. I thank my committee members for providing feedback and support on this work. Finally, I cannot begin to acknowledge the great musicians, novelists, and scientists that have helped me. As Thomas Pynchon has helpfully summarized, “Life's single lesson: that there is more accident to it than a man can ever admit to in a lifetime and stay sane.” iv Table of contents Abstract .............................................................................................................................. ii Acknowledgments ............................................................................................................ iv Table of contents ............................................................................................................... v List of Tables .................................................................................................................. viii List of Figures ................................................................................................................... ix Chapter 1: Introduction and background ...................................................................... 1 The fitness landscape ........................................................................................ 1 Epistasis and the “tape of life” ......................................................................... 6 Genetic drift ..................................................................................................... 10 Directed Evolution .......................................................................................... 11 β-lactamase as a model gene in directed evolution ....................................... 14 Directed evolution informed by landscape ruggedness ............................... 19 Chapter 2: Shifting fitness and epistatic landscapes reflect tradeoffs along an evolutionary pathway ..................................................................................................... 21 Introduction ..................................................................................................... 21 Methods ............................................................................................................ 26 Mutagenesis .................................................................................................. 26 Selection ........................................................................................................ 26 Deep sequencing ........................................................................................... 26 Data analysis ................................................................................................. 27 v Results and Discussion .................................................................................... 32 Fitness landscapes along an adaptive pathway ............................................. 32 Structural map of epistasis ............................................................................ 40 Sign epistasis along an adaptive pathway ..................................................... 44 Reciprocal sign epistasis and ruggedness along an adaptive pathway ......... 46 Conclusions ................................................................................................... 47 Chapter 3: Environmental changes bridge fitness valleys .......................................... 49 Introduction ..................................................................................................... 49 Materials and Methods ................................................................................... 55 Plasmid conditions ........................................................................................ 55 Mutagenesis .................................................................................................. 55 Selection ........................................................................................................ 55 Deep sequencing and sequence analysis ....................................................... 56 Phylogenetic analysis .................................................................................... 57 Selection-weighted attractive graphing (SWAG) ......................................... 57 Calculation of epistasis ................................................................................. 57 Minimum inhibitory concentration testing ................................................... 58 Results and Discussion .................................................................................... 58 Chapter 4: Evaluation of antibiotic cycling and cocktail therapy on evolution of antibiotic resistance ........................................................................................................ 83 Introduction ..................................................................................................... 83 Methods ............................................................................................................ 86 Results .............................................................................................................
Recommended publications
  • The Global Population of SARS-Cov-2 Is Composed of Six
    www.nature.com/scientificreports OPEN The global population of SARS‑CoV‑2 is composed of six major subtypes Ivair José Morais1, Richard Costa Polveiro2, Gabriel Medeiros Souza3, Daniel Inserra Bortolin3, Flávio Tetsuo Sassaki4 & Alison Talis Martins Lima3* The World Health Organization characterized COVID‑19 as a pandemic in March 2020, the second pandemic of the twenty‑frst century. Expanding virus populations, such as that of SARS‑CoV‑2, accumulate a number of narrowly shared polymorphisms, imposing a confounding efect on traditional clustering methods. In this context, approaches that reduce the complexity of the sequence space occupied by the SARS‑CoV‑2 population are necessary for robust clustering. Here, we propose subdividing the global SARS‑CoV‑2 population into six well‑defned subtypes and 10 poorly represented genotypes named tentative subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, nsp12, nsp13 and nsp14) cistrons and structural (spike and nucleocapsid) and accessory (ORF8) genes. The six subtypes and the additional genotypes showed amino acid replacements that might have phenotypic implications. Notably, three mutations (one of them in the Spike protein) were responsible for the geographical segregation of subtypes. We hypothesize that the virus subtypes detected in this study are records of the early stages of SARS‑ CoV‑2 diversifcation that were randomly sampled to compose the virus populations around the world. The genetic structure determined for the SARS‑CoV‑2 population provides substantial guidelines for maximizing the efectiveness of trials for testing candidate vaccines or drugs. In December 2019, a local pneumonia outbreak of initially unknown aetiology was detected in Wuhan (Hubei, China) and quickly determined to be caused by a novel coronavirus1, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)2, with the disease referred to as COVID-193.
    [Show full text]
  • Soil Origin and Plant Genotype Structure Distinct Microbiome Compartments in the Model Legume Medicago Truncatula Shawn P
    Brown et al. Microbiome (2020) 8:139 https://doi.org/10.1186/s40168-020-00915-9 RESEARCH Open Access Soil origin and plant genotype structure distinct microbiome compartments in the model legume Medicago truncatula Shawn P. Brown1,3,4†, Michael A. Grillo1,5†, Justin C. Podowski1,6 and Katy D. Heath1,2* Abstract Background: Understanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities. Results: Bacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment × soil origin and compartment × plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless, all compartments were dominated by Ensifer, the genus of rhizobia that forms root nodule symbiosis with M. truncatula, and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere.
    [Show full text]
  • Lower Endoscopic Delivery of Freeze-Dried Intestinal Microbiota Results in More Rapid and Efficient Engraftment Than Oral Admini
    www.nature.com/scientificreports OPEN Lower endoscopic delivery of freeze‑dried intestinal microbiota results in more rapid and efcient engraftment than oral administration Christopher Staley1,2, Hossam Halaweish1,2, Carolyn Graiziger3, Matthew J. Hamilton2, Amanda J. Kabage3, Alison L. Galdys4, Byron P. Vaughn3, Kornpong Vantanasiri3, Raj Suryanarayanan5, Michael J. Sadowsky2,6,7 & Alexander Khoruts2,3* Fecal microbiota transplantation (FMT) is a highly efective treatment for recurrent Clostridioides difcile infection (rCDI). However, standardization of FMT products is essential for its broad implementation into clinical practice. We have developed an oral preparation of freeze‑dried, encapsulated microbiota, which is ~ 80% clinically efective, but results in delayed engraftment of donor bacteria relative to administration via colonoscopy. Our objective was to measure the engraftment potential of freeze‑dried microbiota without the complexity of variables associated with oral administration. We compared engraftment of identical preparations and doses of freeze‑dried microbiota following colonoscopic (9 patients) versus oral administration (18 patients). Microbiota were characterized by sequencing of the 16S rRNA gene, and engraftment was determined using the SourceTracker algorithm. Oligotyping analysis was done to provide high‑resolution patterns of microbiota engraftment. Colonoscopic FMT was associated with greater levels of donor engraftment within days following the procedure (ANOVA P = 0.035) and specifc increases in the relative
    [Show full text]
  • Article Reference
    Article HLA-DR polymorphism in a Senegalese Mandenka population : DNA oligotyping and population genetics of DRB1 specificities TIERCY, Jean-Marie, et al. Abstract HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of.884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a [...] Reference TIERCY, Jean-Marie, et al. HLA-DR polymorphism in a Senegalese Mandenka population : DNA oligotyping and population genetics of DRB1 specificities. American Journal of Human Genetics, 1992, vol. 51, no. 3, p. 592-608 PMID : 1496990 Available at: http://archive-ouverte.unige.ch/unige:14357 Disclaimer: layout of this document may differ from the published version. 1 / 1 Am. J.
    [Show full text]
  • Oligotyping Analysis of the Human Oral Microbiome PNAS PLUS
    Oligotyping analysis of the human oral microbiome PNAS PLUS A. Murat Erena, Gary G. Borisyb,1, Susan M. Husec, and Jessica L. Mark Welcha,1 aJosephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543; bDepartment of Microbiology, The Forsyth Institute, Cambridge, MA 02142; and cDepartment of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912 Contributed by Gary G. Borisy, May 27, 2014 (sent for review March 19, 2014; reviewed by Jack Gilbert, Jacques Ravel, and Jed A. Fuhrman) The Human Microbiome Project provided a census of bacterial Large-scale application of rRNA gene sequencing to the anal- populations in healthy individuals, but an understanding of the ysis of the human oral microbiota began with Aas et al. (2), who biomedical significance of this census has been hindered by limited sampled nine oral sites in five healthy individuals to assess dif- taxonomic resolution. A high-resolution method termed oligotyping ferences among individuals as well as among oral sites, whereas overcomes this limitation by evaluating individual nucleotide posi- Bik et al. (3) assessed the level of interindividual variation with tions using Shannon entropy to identify the most information-rich deeper sequencing of a pooled sample from each of 10 individuals. nucleotide positions, which then define oligotypes. We have applied The use of Sanger sequencing in these studies allowed species- this method to comprehensively analyze the oral microbiome. Using level taxon assignment but limited the depth of sequencing and Human Microbiome Project 16S rRNA gene sequence data for the thus limited our understanding of the degree to which taxa oc- curred consistently across individuals.
    [Show full text]
  • Diverse, Rare Microbial Taxa Responded to the Deepwater Horizon Deep-Sea Hydrocarbon Plume
    The ISME Journal (2016) 10, 400–415 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 OPEN www.nature.com/ismej ORIGINAL ARTICLE Diverse, rare microbial taxa responded to the Deepwater Horizon deep-sea hydrocarbon plume Sara Kleindienst1,4, Sharon Grim2,5, Mitchell Sogin2, Annalisa Bracco3, Melitza Crespo-Medina1,6 and Samantha B Joye1 1Department of Marine Sciences, University of Georgia, Athens, GA, USA; 2Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA, USA and 3School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA, USA The Deepwater Horizon (DWH) oil well blowout generated an enormous plume of dispersed hydrocarbons that substantially altered the Gulf of Mexico’s deep-sea microbial community. A significant enrichment of distinct microbial populations was observed, yet, little is known about the abundance and richness of specific microbial ecotypes involved in gas, oil and dispersant biodegradation in the wake of oil spills. Here, we document a previously unrecognized diversity of closely related taxa affiliating with Cycloclasticus, Colwellia and Oceanospirillaceae and describe their spatio-temporal distribution in the Gulf’s deepwater, in close proximity to the discharge site and at increasing distance from it, before, during and after the discharge. A highly sensitive, computational method (oligotyping) applied to a data set generated from 454-tag pyrosequencing of bacterial 16S ribosomal RNA gene V4–V6 regions, enabled the detection of population dynamics at the sub-operational taxonomic unit level (0.2% sequence similarity). The biogeochemical signature of the deep-sea samples was assessed via total cell counts, concentrations of short-chain alkanes (C1–C5), nutrients, (colored) dissolved organic and inorganic carbon, as well as methane oxidation rates.
    [Show full text]
  • ORIGINAL ARTICLE Ubiquity and Quantitative Significance of Bacterioplankton Lineages Inhabiting the Oxygenated Hypolimnion of Deep Freshwater Lakes
    OPEN The ISME Journal (2017) 11, 2279–2293 www.nature.com/ismej ORIGINAL ARTICLE Ubiquity and quantitative significance of bacterioplankton lineages inhabiting the oxygenated hypolimnion of deep freshwater lakes Yusuke Okazaki1, Shohei Fujinaga1, Atsushi Tanaka2, Ayato Kohzu2, Hideo Oyagi3 and Shin-ichi Nakano1 1Center for Ecological Research, Kyoto University, Otsu, Japan; 2National Institute for Environmental Studies, Tsukuba, Japan and 3College of Humanities and Sciences, Nihon University, Tokyo, Japan The oxygenated hypolimnion accounts for a volumetrically significant part of the global freshwater systems. Previous studies have proposed the presence of hypolimnion-specific bacterioplankton lineages that are distinct from those inhabiting the epilimnion. To date, however, no consensus exists regarding their ubiquity and abundance, which is necessary to evaluate their ecological importance. The present study investigated the bacterioplankton community in the oxygenated hypolimnia of 10 deep freshwater lakes. Despite the broad geochemical characteristics of the lakes, 16S rRNA gene sequencing demonstrated that the communities in the oxygenated hypolimnia were distinct from those in the epilimnia and identified several predominant lineages inhabiting multiple lakes. Catalyzed reporter deposition fluorescence in situ hybridization revealed that abundant hypolimnion-specific lineages, CL500-11 (Chloroflexi), CL500-3, CL500-37, CL500-15 (Planctomy- cetes) and Marine Group I (Thaumarchaeota), together accounted for 1.5–32.9% of all bacterioplank- ton in the hypolimnion of the lakes. Furthermore, an analysis of single-nucleotide variation in the partial 16S rRNA gene sequence (oligotyping) suggested the presence of different sub-populations between lakes and water layers among the lineages occurring in the entire water layer (for example, acI-B1 and acI-A7).
    [Show full text]
  • Downloaded from the Integrated Microbial Genomes and Microbiomes Database (IMG/M) (Chen Et Al., 2019C) and Refined (See Methods) and a MAG from Marcy Et Al
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.069278; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome Alon Shaiber1,2, Amy D. Willis3, Tom O. Delmont4, Simon Roux5, Lin-Xing Chen6, Abigail 5 C. Schmid7, Mahmoud Yousef8, Andrea R. Watson1,9, Karen Lolans1, Özcan C. Esen1, Sonny T. M. Lee10, Nora Downey11, Hilary G. Morrison11, Floyd E. Dewhirst12,13, Jessica L. Mark Welch11,*, A. Murat Eren1,2,9,11,* 1Department of Medicine, University of Chicago, Chicago, IL 60637, USA; 2Graduate Program in Biophysical Sciences, University of Chicago, Chicago, IL 60637, USA; 3Department of Biostatistics, 10 University of Washington, Seattle WA 98195, USA; 4Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France; 5Department of Energy Joint Genome Institute, Berkeley CA 94720, USA; 6Department of Earth and Planetary Sciences, University of California, Berkeley, CA 94720, USA; 7Undergraduate Student, Computational and Applied Mathematics, University of Chicago, Chicago, IL 60637, USA; 15 8Undergraduate Student, Computer Science, University of Chicago, Chicago, IL 60637, USA; 9Committee on Microbiology, University of Chicago, Chicago, IL 60637, USA; 10
    [Show full text]
  • Oligotyping Reveals Community Level Habitat Selection Within the Genus Vibrio
    Oligotyping reveals community level habitat selection within the genus Vibrio Victor Schmidt, Julie Reveillaud, Erik Zettler, Tracy Mincer, Leslie Murphy, Linda Amaral-Zettler To cite this version: Victor Schmidt, Julie Reveillaud, Erik Zettler, Tracy Mincer, Leslie Murphy, et al.. Oligotyping reveals community level habitat selection within the genus Vibrio. Frontiers in Microbiology, Frontiers Media, 2014, 5 (563), pp.1-14. 10.3389/fmicb.2014.00563. hal-01104930 HAL Id: hal-01104930 https://hal.univ-brest.fr/hal-01104930 Submitted on 19 Jan 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ORIGINAL RESEARCH ARTICLE published: 13 November 2014 MICROBIOLOGY doi: 10.3389/fmicb.2014.00563 Oligotyping reveals community level habitat selection within the genus Vibrio Victor T. Schmidt 1,2, Julie Reveillaud 1†, Erik Zettler 3, Tracy J. Mincer 4, Leslie Murphy 1 and Linda A. Amaral-Zettler 1,5* 1 Marine Biological Laboratory, Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Woods Hole, MA, USA 2 Department of Ecology and Evolutionary Biology, Brown University, Providence, RI, USA 3 Sea Education Association, Woods Hole, MA, USA 4 Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, USA 5 Department of Earth, Environmental and Planetary Sciences, Brown University, Providence, RI, USA Edited by: The genus Vibrio is a metabolically diverse group of facultative anaerobic bacteria, Rachel Susan Poretsky, University of common in aquatic environments and marine hosts.
    [Show full text]
  • Oligotyping Reveals Stronger Relationship of Organic Soil Bacterial
    University of New Hampshire University of New Hampshire Scholars' Repository New Hampshire Agricultural Experiment Station Publications New Hampshire Agricultural Experiment Station 2-16-2015 Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA Swathi A. Turlapati University of New Hampshire, Durham Rakesh Minocha United States Department of Agriculture Forest Service, [email protected] Stephanie Long United States Department of Agriculture Forest Service Jordan Ramsdell University of New Hampshire, Durham Subhash C. Minocha University of New Hampshire, Durham, [email protected] Follow this and additional works at: https://scholars.unh.edu/nhaes Recommended Citation Turlapati SA, Minocha R, Long S, Ramsdell J and Minocha SC (2015) Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA. Front. Microbiol. 6:49. https://dx.doi.org/ 10.3389/fmicb.2015.00049 This Article is brought to you for free and open access by the New Hampshire Agricultural Experiment Station at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in New Hampshire Agricultural Experiment Station Publications by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. ORIGINAL RESEARCH ARTICLE published: 16 February 2015 doi: 10.3389/fmicb.2015.00049 Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA Swathi A.
    [Show full text]
  • Oligotyping Bacterial Symbionts of Hindgut Protists Reveals Complex
    bioRxiv preprint doi: https://doi.org/10.1101/299255; this version posted April 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 Oligotyping Bacterial Symbionts of Hindgut Protists Reveals Complex 3 Population Structures and Transmission Trends 4 in the Termite Reticulitermes flavipes 5 6 Michael E. Stephens1 and Daniel J. Gage1 7 8 1Department of Molecular and Cell Biology, University of Connecticut, Storrs, USA 9 10 Running Title: Oligotyping the symbionts of hindgut protists. 11 12 13 Corresponding Author: 14 Daniel J. Gage 15 Email:[email protected] 16 Phone: 860-486-3092 17 Address: 91 North Eagleville Rd. 18 Unit 3125 Room 406 19 Storrs CT, 06269 20 21 22 1 bioRxiv preprint doi: https://doi.org/10.1101/299255; this version posted April 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 Author Contributions: 24 MES and DJG designed experiments. MES performed experiments and data analysis. MES and 25 DJG wrote the manuscript. 26 27 Conflict of interest 28 The authors declare no conflict of interest. 29 30 Source of Funding 31 This research was funded by the National Science Foundation (NSF) division of Emerging 32 Frontiers in Research and Innovation in Multicellular and Interkingdom Signaling.
    [Show full text]
  • Use of Monoclonal Antibodies to Detect Disease Associated HLA
    Annals of the Rheumatic Diseases 1997;56:135–139 135 Use of monoclonal antibodies to detect disease Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from associated HLA-DRB1 alleles and the shared epitope in rheumatoid arthritis Ian Wicks, GeoV McColl, Angela D’Amico, Loretta Dougherty, Brian Tait Abstract earlier and more aggressive therapeutic Objective—To use a panel of monoclonal intervention. Early stratification of patients antibodies (Mab) which recognise HLA who are at risk for severe disease is an class II alleles associated with rheumatoid important clinical issue for these reasons. arthritis for fluorescence activated cell Many studies have confirmed an association sorter (FACS) analysis of peripheral blood between rheumatoid arthritis and a restricted mononuclear cells (PBMNC) from pa- number of alleles at the highly polymorphic tients with early and established rheuma- HLA-DRB1 locus (primarily DRB1*0401, toid arthritis and to compare these results *0404, and *0101).23 Each of these alleles against DNA oligotyping of HLA class II shares sequence homology in the third molecules in the same patients. hypervariable region of the â chain, which is Methods—27 patients (18 from an early referred to as the shared epitope. In a study of arthritis clinic, nine with established 201 patients with rheumatoid arthritis, who rheumatoid arthritis) were studied using were unselected for disease severity, the both techniques. PBMNC were stained presence of a single copy of *0401, *0404, or with Mab which recognise the shared *0101 was associated with an odds ratio for epitope, the HLA-DRB1*04 molecule and rheumatoid arthritis of 3.5.4 Two identical its *0401, *0404 subtypes in the presence of copies of these alleles gave an odds ratio of 7.8 bound peptide.
    [Show full text]