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Evolution of the Mysrs Element in the Genome of Rodent Species Megan M

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Evolution of the Mysrs Element in the Genome of Rodent Species Megan M Eastern Michigan University DigitalCommons@EMU Master's Theses, and Doctoral Dissertations, and Master's Theses and Doctoral Dissertations Graduate Capstone Projects 2016 Evolution of the mysRS element in the genome of rodent species Megan M. Scott Follow this and additional works at: http://commons.emich.edu/theses Part of the Biology Commons Recommended Citation Scott, Megan M., "Evolution of the mysRS element in the genome of rodent species" (2016). Master's Theses and Doctoral Dissertations. 786. http://commons.emich.edu/theses/786 This Open Access Thesis is brought to you for free and open access by the Master's Theses, and Doctoral Dissertations, and Graduate Capstone Projects at DigitalCommons@EMU. It has been accepted for inclusion in Master's Theses and Doctoral Dissertations by an authorized administrator of DigitalCommons@EMU. For more information, please contact [email protected]. Evolution of the mysRS Element in the Genome of Rodent Species in the Cricetidae Family by Megan M. Scott Thesis Submitted to the Department of Biology Eastern Michigan University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biology Thesis Committee: David Kass, Ph.D, Chair Katherine Greenwald, Ph.D Lynne Shetron-Rama, Ph.D July 13, 2016 Ypsilanti, Michigan Acknowledgments I would like to thank the Eastern Michigan University Symposium Undergraduate Research Fellow Program for aid in funding for this project. I would also like to thank my committee members Dr. Katherine Greenwald, Dr. Lynne Shetron-Rama, and Dr. David Kass for all of their guidance. And finally I’d like to thank my family and friends for all of their support and encouragement. I couldn’t have done this without all of you! ii Abstract Retrotransposons are a type of transposable element in which DNA copies are produced via an RNA intermediate and integrate into new chromosomal sites continually contributing to the dynamics of genomes. While sequencing the mys element among the Cricetidae family of rodents, we identified a previously uncharacterized element in the genome of the Mexican volcano mouse that we refer to as mys-related sequence (mysRS). This investigation provides a unique opportunity to contrast the evolution of the two retrotransposon families apparently restricted to a specific taxonomic group of rodents. By analyzing the genomes of various cricetid rodents, our results provide evidence that the mysRS element originated in either an ancestor of the Neotominae subfamily or the Reithrodontomini tribe of rodents, which diverged roughly nine million years ago, and predates the appearance of the mys element. iii Table of Contents Acknowledgments………………………………………………………………………………………………..ii Abstract ........................................................................................................................................................ iii List of Tables ............................................................................................................................................. vii List of Figures ........................................................................................................................................... viii Introduction .................................................................................................................................................. 1 Methods ....................................................................................................................................................... 10 Isolation of DNA from Rodent Tissues ...................................................................................... 11 Generation of intra-mysRS Elements by Polymerase Chain Reactions (PCR) .......... 11 Analysis of intra-mysRS PCR Products by Gel Electrophoresis ...................................... 12 Generation of intra-mysRS PCR Libraries ................................................................................ 12 Selection of Clones Containing the mysRS Insert .................................................................. 13 Verification of mysRS Presence by Restriction Enzyme Digest ...................................... 13 SeQuencing and Analysis of the mysRS Element.. ................................................................. 13 Quantification of the mysRS Element Using Dot Blot Analysis ....................................... 14 Hybridization of the Dot Blot Filter Used for mysRS Quantification Analysis .......... 15 High-Efficiency Thermal Asymmetric Interlaced (hi-TAIL) PCR ................................... 16 hi-TAIL PCR Pre-Amplification ..................................................................................................... 18 Primary-Tail PCR Amplification ................................................................................................... 19 Secondary Tail PCR Amplification ............................................................................................... 20 Results .......................................................................................................................................................... 21 Amplification of the mysRS Element within Analyzed Species ....................................... 21 Analysis of the mysRS element and the Construction of a mysRS Element Master iv Sequence ................................................................................................................................................. 26 Dot Blot Analysis Estimating the Quantity of mysRS in Rodent Genomes ................. 32 Searching the Peromyscus Whole Genome Using mysRS to Extend the Element and Design hi-TAIL PCR Primer Sets .................................................................................................. 33 hi-TAIL PCR to Further Depict a Full-length mysRS Element .......................................... 42 hi-TAIL PCR SeQuence Analysis using MacVector Software………………………………44 Discussion ................................................................................................................................................... 47 References .................................................................................................................................................. 54 v List of Tables Table Page 1 Forward and Reverse mysRS Primers used to determine the presence/absence of the mysRS element in the genome of rodent species………………………………………………………………………...11 2 Dot Blot Filter Layout……………………………………………………………………………....14 3 Groups of primer seQuences and linkers used for hi-TAIL PCR…………………………………………………………………………………………….18 vi List of Figures Figure Page 1 Breakdown of Eukaryotic Transposable elements………………………………...1 2 Process of retrotransposon replication using an RNA intermediate……………………………………………………………………………………...5 3 Full length mys element……………………………………………………………........................7 4 Phylogenetic tree depicting the Cricitidae family of rodents which display the mysRS element…………………………………………………..8 5 Partial sequence of cloned intra-mys PCR results obtained from seQuencing Neotomodon alstoni and Peromyscus maniculatus………………………………………………………………………9 6 hi-TAIL PCR utilizes element specific primers…………………………………………..17 7 Analysis for the presence of the mysRS element among various rodent genera by intra-mysRS PCR using primers mysRS 40F and mysRS 430R……………………………………………………………………..22 8 Analysis for the presence of the mysRS element by amplification with primers mysRS 60F and mysRS 430R…………..……………….23 9 Analysis of the various rodent genera for the mysRS element by intra-mysRS PCR using primers mysRS 40F and mysRS 375R……………………………………………………………………………………...24 10 Analysis of Peromyscus species for the presence of the mysRS element using primers mysRS 40F and mysRS 375R……………………….25 vii 11 Phylogenetic tree displaying the potential point of origin for the mysRS element……………………………………….……………………….26 12 Analysis of cloned mysRS element among various rodent species using EcoRI……………………………………………….……………………...27 13 Analysis of cloned mysRS element among various rodent species using EcoRI……………………………………………………………………....27 14 Analysis of cloned mysRS elements from Peromyscus species via restriction enzyme digest using NcoRI-HF and NdeI…………………………………………………………………………………………………28 15 Analysis of cloned mysRS elements from Peromyscus species via restriction enzyme digest using NcoRI-HF and NdeI………………………………………………………………………………………………....28 16 Similarity scores of N. alstoni, generated via MacVector seQuencing software, from shotgun cloned PCR amplified products using mysRS primers 40F-375R…………………………………………………29 17 Similarity scores of O. leucogaster, generated via MacVector seQuencing software, from shotgun cloned PCR amplified products using mysRS primers 40F-375R…………………………………………………29 18 Similarity scores of R. fulvescens, generated via MacVector seQuencing software, from shotgun cloned PCR amplified products using mysRS primers 40F-375R…………………………………………………30 viii 19 Similarity scores of P. leucopus, generated via MacVector seQuencing software, from shotgun cloned PCR amplified products using mysRS primers 40F-375R………………………………………………....30 20 Similarity scores of species consensus seQuences generated via MacVector seQuencing software, from shotgun cloned PCR amplified products using mysRS primers 40F-375R………………………………………………………………….31 21 Displays the relationship between the different clones in comparison to the mysTR and mys elements………………...……………………...……31 22 Estimation of mysRS copy numbers using dot blot analysis using the lowest stringency (42
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