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Molecular Genetic Characterization of Xiphophorus MOLECULAR GENETIC CHARACTERIZATION OF XIPHOPHORUS MACULATUS JP 163 B SKIN UPON EXPOSURE TO VARYING WAVELENGTHS OF LIGHT by Jordan Chang, B.S. A thesis submitted to the Graduate Council of Texas State University in partial fulfillment of the requirements for the degree of Master of Science with a Major in Biochemistry May 2016 Committee Members: Ronald Walter, Committee Chair Karen Lewis Steven Whitten COPYRIGHT by Jordan Chang 2016 FAIR USE AND AUTHOR’S PERMISSION STATEMENT Fair Use This work is protected by the Copyright Laws of the United States (Public Law 94-553, section 107). Consistent with fair use as defined in the Copyright Laws, brief quotations from this material are allowed with proper acknowledgment. Use of this material for financial gain without the author’s express written permission is not allowed. Duplication Permission As the copyright holder of this work I, Jordan Chang, authorize duplication of this work, in whole or in part, for educational or scholarly purposes only. ACKNOWLEDGEMENTS This work could not have been accomplished without the support and guidance I have received from my family, mentors, and colleagues. Although it would be impossible to name all who have helped, I would like to thank a few people in particular that have guided me through this journey. Thank you Dr. Ron Walter for the guidance and support these past 2 years. The mentorship that you have provided has helped me grow as a student and as a scientist. I am truly grateful for all the opportunities and lessons you have given me. I will bring with me all that I have learned and continue down my path to becoming a scientist. I hope that one day I can show you that the time and support you have shown me were well spent. I would also like to thank the various current and former members of the Molecular Biosciences Research Group. In particular, I would like to thank Dr. Yuan Lu, Will and Mikki Boswell and Esther Lee for the help in and outside the lab; and graduate students Kaela Caballero and Angelica Riojas for their support and fellowship during my time here. To the Xiphophorus Genetic Stock Center Staff, thank you Markita Savage, Natalie Taylor, and Cristina Mendoza for the work you do. Without the assistance of everyone here, I would not have been able to accomplish all that I have. iv Thank you to my committee members Drs. Karen Lewis and Steve Whitten for the academic input and mentorship during my time here. Your help has been most appreciated and invaluable. Lastly, to my parents, family, and friends, thank you for your continuous love and patience. I could not have made it this far without your love and encouragement. Thank you. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ..................................................................................... iv LIST OF TABLES ................................................................................................. xi LIST OF FIGURES ............................................................................................. xiii CHAPTERS .......................................................................................................... 1 I. INTRODUCTION .................................................................................... 1 Melanoma Models in Xiphophorus .................................................. 2 Classic Gordon-Kosswig Cross ............................................ 4 Inducible Melanoma Cross ................................................... 5 UV and Visible Light Properties....................................................... 7 Effects of Ultraviolet Light ..................................................... 7 DNA Damage and Photorepair of UV-Induced Lesions ........ 9 Visible Light Spectrum ........................................................ 10 UV vs. Fluorescent Light Effects on Gene Expression .................. 12 UV Induced Gene Expression ............................................ 12 Fluorescent Light Induced Gene Expression ...................... 12 Artificial Light Spectra ......................................................... 13 II. METHODS AND MATERIALS ............................................................. 17 Research Animals ......................................................................... 17 vi Specific Wavelength Exposure ..................................................... 17 RNA Isolation ................................................................................ 20 RNA Sequencing ........................................................................... 21 Computational Analysis ................................................................. 24 NanoString .................................................................................... 27 Quantitative Real Time PCR ......................................................... 29 III. GENETIC RESPONSE OF MALE XIPHOPHORUS SKIN TO VARYING WAVELENGTHS OF LIGHT ........................................ 32 Introduction ................................................................................... 32 Results .......................................................................................... 34 Determination of the Effects of Water on Light ................... 34 Distribution of Differentially Expressed Genes in Response to Varying Wavebands ............................................. 36 Circadian Gene Differential Expression and RNA-Seq Validations ............................................................... 37 Shared Genetic Response for 350-400 and 500-550 nm ... 41 Genetic Response in 350-400 and 500-550 nm Regions ... 43 Discussion ..................................................................................... 59 DE Gene Induction at 350-400 nm and 500-550 nm Waveband Regions ................................................. 59 Shared Genetic Response at Both 350-400 nm and 500-550 nm Regions ............................................................. 60 Modulation of Cell Cycle Regulators, atm and atr, at Both 350-400 nm and 500-550 nm Regions .................... 62 vii Genetic Response after Exposure to 350-400 nm Light ..... 63 Genetic Response after Exposure to 500-550 nm Light ..... 65 Conclusions .................................................................................. 68 IV. GENETIC RESPONSE OF FEMALE XIPHOPHORUS SKIN TO VARYING WAVELENGTHS OF LIGHT ........................................ 73 Introduction ................................................................................... 73 Results .......................................................................................... 74 Distribution of Differentially Expressed Genes in Females to Varying Wavebands ................................................. 74 Circadian Gene Expression in Females Over Six Waveband Regions ................................................................... 75 Shared Response between 300-350 and 400-450 nm Waveband Regions ................................................. 76 Genetic Response in the 300-350 and 400-450 nm Waveband Regions ................................................. 82 Discussion ..................................................................................... 96 DE Gene Distribution across 300-600 nm Waveband Regions ................................................................... 96 Shared Response between 300-350 and 400-450 nm Waveband Regions ................................................. 97 Modulation of Regulators Affected at 300-350 and 400-450 nm Regions ............................................................. 98 Genetic Response after Exposure to 300-350 nm Light ... 100 Genetic Response after Exposure to 400-450 nm Light ... 102 viii Conclusions ................................................................................ 104 V. COMPARISON OF SEX SPECIFIC GENETIC RESPONSES OF XIPHOPHORUS SKIN TO VARYING WAVELENGTHS OF LIGHT ......................................................................................... 106 Introduction ................................................................................. 106 Results ........................................................................................ 107 Differential Modulation of Genes between Male and Female Xiphophorus .......................................................... 107 Comparison of DE Genes between Each 50 nm Region from Male and Female Exposures ................................. 108 Shared DE Genes between Male and Female Xiphophorus at 450-500 nm ....................................................... 109 Shared DE Genes between Male and Female Xiphophorus at 500-550 nm ....................................................... 111 Shared DE Genes between Male and Female Xiphophorus at 550-600 nm ....................................................... 113 Discussion ................................................................................... 115 Comparison of DE Genes between Male and Female Light Exposures .............................................................. 115 Response of Shared Genes in Male and Female Xiphophorus at 450-500 nm .................................. 117 Response of Shared Genes in Male and Female Xiphophorus at 500-550 nm .................................. 118 Response of Shared Genes in Male and Female Xiphophorus at 550-600 nm .................................. 119 Conclusions ................................................................................ 120 ix REFERENCES ................................................................................................
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