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Genetic Profiling of Mycoplasma Hyopneumoniae Melissa L Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2005 Genetic profiling of Mycoplasma hyopneumoniae Melissa L. Madsen Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons, Molecular Biology Commons, and the Veterinary Medicine Commons Recommended Citation Madsen, Melissa L., "Genetic profiling of Mycoplasma hyopneumoniae " (2005). Retrospective Theses and Dissertations. 1793. https://lib.dr.iastate.edu/rtd/1793 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. NOTE TO USERS This reproduction is the best copy available. ® UMI Genetic profiling of Mycoplasma hyopneumoniae by Melissa L. Madsen A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular and Developmental Biology Program of Study Committee: F. Chris Minion, Major Professor Daniel S. Nettleton Gregory J. Phillips Eileen L. Thacker Eve Wurtele Iowa State University Ames, Iowa 2005 UMI Number: 3200480 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3200480 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Melissa L. Madsen has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. For the Major Program iii TABLE OF CONTENTS LIST OF FIGURES vii LIST OF TABLES viii ABSTRACT ix CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 1 Literature Review 2 Mycoplasmas 2 Mycoplasma hyopneumoniae 4 Disease characteristics 4 Detection 5 Vaccines 6 Virulence mechanisms 8 Microarrays 9 History 9 Types of arrays 10 Informatics 14 Applications 17 Genomic comparisons (within/without species) 17 Single nucleotide polymorphism detection 18 Gene expression 19 Pathogens 19 Food safety 20 Mycoplasmas 21 Summary with Goals and Hypothesis 22 References 23 CHAPTER 2. TRANSCRIPTIONAL PROFILING OF MYCOPLASMA HYOPNEUMONIAE DURING HEAT SHOCK USING MICROARRAYS Abstract 37 Introduction 38 Materials and Methods 39 Mycoplasma strains and culture conditions 39 Microarray construction 39 RNA isolation 43 Random primer set for M. hyopneumoniae 43 Target generation and hybridization 44 Image acquisition and data analysis 44 IV Statistical analysis 45 Validation of microarray data 45 Results 46 Primer design and array construction 46 Heat shock studies 47 Validation of microarray data 53 Discussion 54 Acknowledgements 57 References 57 CHAPTER 3. TRANSCRIPTIONAL PROFILING OF MYCOPLASMA HYOPNEUMONIAE DURING IRON DEPLETION USING MICROARRAYS Abstract 61 Introduction 62 Materials and Methods 63 Mycoplasma strains and culture conditions 63 Microarray 64 Experimental design 64 RNA isolation 64 Target generation and hybridization 64 Data acquisition and normalization 65 Data analysis 65 Confirmation of microarray data 66 Results 67 Iron depletion studies 67 Microarray studies 67 Validation of microarray data 69 Discussion 72 Acknowledgements 75 References 75 CHAPTER 4. GENE EXPRESSION DIFFERENCES BETWEEN MYCOPLASMA HYOPNEUONIAE VIRULENT STRAIN 232 AND A VIRULENT STRAIN J Abstract 79 Introduction 79 Materials and Methods 81 Mycoplasma strains and culture conditions 81 Microarray 81 Experimental design 81 RNA isolation 82 Target generation and hybridization 82 Image acquisition and normalization 83 Data analysis 83 V Results 84 Microarray studies 84 Discussion 86 Acknowledgements 87 References 88 CHAPTER 5. TRANSCRIPTIONAL VARIATION BETWEEN HIGH AND LOW ADHERENT VARIANTS OF MYCOPLASMA HYOPNEUMONIAE Abstract 91 Introduction 91 Materials and Methods 93 Mycoplasma strains and culture conditions 93 Microarray 93 Experimental design 94 RNA isolation 94 Target generation and hybridization 94 Image acquisition and normalization 95 Data analysis 95 Results 96 Microarray studies 96 Discussion 98 Acknowledgements 99 References 102 CHAPTER 6. GENETIC ANALYSIS OF FIELD ISOLATES BY MICROARRAY Abstract 107 Introduction 107 Materials and Methods 109 Mycoplasma strains and culture conditions 109 Microarray 109 Experimental design 109 DNA isolation 110 TempliPhi™ reactions 110 Nebulization 110 Target generation and hybridization 111 Data acquisition and normalization 111 Data analysis 111 Results 112 TempliPhi™ reactions 112 Microarray studies 113 Discussion 116 Acknowledgements 117 References 117 VI CHAPTER 7. GENERAL CONCLUSIONS 121 General Discussion 121 Recommendations for Future Research 123 References 128 APPENDIX A. CIRCULAR REPRESENTATION OF THE MYCOPLASMA HYOPNEUMONIAE GENOME STRUCTURE 130 APPENDIX B. PRIMERS USED IN THE MYCOPLASMA HYOPNEUMONIAE MICROARRAY 132 APPENDIX C. PRINTING FILE FOR BIOROBOTICS MICROGRID PRINTING ROBOT 146 APPENDIX D. HEXAMER OLIGONUCLEOTIDE SEQUENCES USED FOR GENERATING CDNA FOR LABELING EXPERIMENTS 151 APPENDIX E. OPEN READING FAMES MISSING FROM THE MYCOPLASMA HYOPNEUMONIAE MICROARRAY 153 ACKNOWLEDGMENTS 156 vii LIST OF FIGURES Figure 2.1. Flow chart for PCR primer design 41 Figure 2.2. Example of agarose gel of purified PCR products 46 Figure 2.3. Determination of cross-linking energy 47 Figure 2.4. Volcano plot of transcriptional differences in M. hyopneumoniae during heat shock conditions 48 Figure 2.5. Semi-quantitative RT-PCR 53 Figure 3.1. Effect of 2,2'-dipyridyl on growth of M. pulmonis and M. gallisepticum 68 Figure 3.2. Volcano plot of transcriptional differences in M. hyopneumoniae during low iron growth conditions 69 Figure 3.3. Semi-quantitative RT-PCR 71 Figure 4.1. Volcano plot of transcriptional differences in M. hyopneumoniae strains 232 and J 84 Figure 5.1. Volcano plot of transcriptional differences in M. hyopneumoniae high-adherent variant 91-3 and low-adherent variant 60-3 98 Figure 6.1. Analysis of TempliPhi™ amplified and nebulized mycoplasma chromosomal DNA 113 Figure 6.2. Comparison of mean log signal intensity values of genomic vs TempliPhi™ amplified chromosomal DNA 114 Figure 7.1. Steps for microarray construction and utilization. 122 vin LIST OF TABLES Table 2.1. Initial Primer3 criteria for designing primers for the M. hyopneumoniae ORFs s. 42 Table 2.2. Heat shock up-regulated genes in M. hyopneumoniae 49 Table 2.3. Heat shock down-regulated genes in M. hyopneumoniae 51 Table 2.4. RT-PCR primers 53 Table 3.1. Differentially expressed genes of M. hyopneumoniae during growth under iron limiting conditions 70 Table 3.2. RT-PCR primer sequences. 71 Table 4.1. Transcriptional differences between M. hyopneumoniae strains 232 and J/KO.01 85 Table 5.1. Variation in signal intensities between variants 60-3 and 91-3 at ^<0.05 97 Table 6.1. Genetic variation of M. hyopneumoniae field isolates compared to strain 232 withp<0.05 115 Table. 7.1. Genes with significant (p<0.05) transcript values in both heat- shocked and iron-depleted studies 125 Table 7.2. Putative housekeeping genes 127 ix ABSTRACT A microarray was constructed and applied to transcriptional profiling and genetic variation studies of Mycoplasma hyopneumoniae. The genome sequence enabled the construction of the microarray to allow a global approach to understanding fundamental processes in M. hyopneumoniae. These studies focused on whether M. hyopneumoniae regulates its genes under different environmental conditions and if genetic changes can be correlated with virulence. The microarray consisted of 632 open reading frames represented by polymerase chain reaction products and were used in a two-color experimental design. Data were analyzed using a mixed linear statistical model. Unique features implemented in these studies included the printing of two complete arrays per substrate, reducing the slide to slide variation; the scanning of each dye channel of each array at different laser power settings to increase the dynamic range of expression measurement; and the use of a unique set of hexamer primers to generate fluorescently labeled targets for microarray analysis. The first series of studies focused on transcriptional profiling during heat shock and iron deprivation, two environmental changes that M. hyopneumoniae encounters on the respiratory epithelial surface during disease. Key genes responsive to these stresses were identified, and interestingly, fifty-three were regulated in common to the two conditions at p<0.05. Since adherence to swine cilia is a prerequisite for colonization and disease, the next series of studies involved the comparison of an adherent,
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