ISOLATION AND CHARACTERIZATION OF VIRUS-LIKE PARTICLES FROM LILIACEAE __________________ A University Thesis Presented to the Faculty of California State University, East Bay __________________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology __________________ By Anna-Louise Doss December, 2013 ! ! "! ! Abstract Transposable elements are mobile genetic elements that have the ability to replicate and relocate to different positions in the genome of a cell. These elements are ubiquitous in the genomes of all eukaryotic organisms and are credited with being the source of the vast variation in genome size among related organisms, such as angiosperms, which can differ in genome size by as much as 1000-fold. Retroelements in particular are very predominant in angiosperms and have been shown to account for anywhere from 30% to 90% of the genome. The massive genomes that are common in the Liliaceae family (mean C value of 50 pg) are extreme examples of retroelement proliferation. The Family Liliaceae provides a good model system for studying the mechanisms utilized by transposable elements to proliferate and evade host defenses, and for studying the impact transposable elements have on the evolution of genomes. However, due to their large genome size and the vast prevalence of repetitive DNA, members of the Liliaceae have not yet been fully sequenced nor have their transposable elements been fully characterized. In addition, there has been no attempt to isolate and characterize active retroelements from Liliaceae species in the form of virus-like particles. This thesis research will utilize and modify existing virus-like particle isolation techniques for retroelement isolation and characterization using barley as a model plant. Application of these techniques for the characterization of virus-like particles from Liliaceae species will allow us to gain insight into what elements are actively transposing in the Liliaceae and the extent of their activity. ! ""! Acknowledgments This work was carried out between summer 2011 and summer 2013 at California State University, East Bay under the supervision of Dr. Chris Baysdorfer as a requirement for the completion of a Masters of Science degree in Biology. My deepest appreciation goes to Dr. Baysdorfer for providing me the opportunity to be a member of his research lab and for the support, knowledge, patience, and inspiration he supplied during the course of this project. I am also sincerely grateful for the support of my friend and lab partner, Ghezal Saffi, who will continue this research project and master the next generation sequencing. ! """! Table of Contents Abstract........................................................................................................................ii Acknowledgements.....................................................................................................iv List of Tables..............................................................................................................vi List of Figures............................................................................................................vii Introduction................................................................................................................1 Experimental Objectives............................................................................................9 Methods and Procedures..........................................................................................10 Specific Aim 1: Develop and test an ultracentrifugation technique to isolate virus- like particles............................................................................................10 Specific Aim 2: Use the Product-Enhanced Reverse Transcriptase assay to test for the presence of virus-like particles.......................................................12 Specific Aim 3: Isolate and quantify total RNA from the virus-like particle Isolates………………………..........................................................................14 Specific Aim 4: Characterize purified RNA from virus-like particle samples using reverse transcription PCR techniques and sequencing by capillary electrophoresis...................................................................................15 Results and Discussion............................................................................................21 Ultracentrifugation...........................................................................................21 Product Enhanced Reverse Transcriptase Assay.............................................22 RNA Purification and Quantification..............................................................32 Reverse Transcription Reactions and PCR for Copia and Gypsy Reverse Transcriptase Gene..........................................................................................35 Sequencing and Analysis of Copia Reverse Transcriptase PCR Products...........................................................................................................45 Conclusions and Future Research.........................................................................49 References Cited......................................................................................................51 ! "#! List of Tables Table 1: List of all plant samples isolated by ultracentrifugation and the respective dates of the isolations............................................................................................21 Table 2: #"$%&'$!()#!*+,*$,-%&-.+,/!.,!0'1µ2!3%+4!567!0%$0&%&-.+,/!&,8!02&,-!! 2$&3!-.0/.................................................................................................................33! ! "! List of Figures Figure 1: Transposable elements...................................................................................................2 Figure 2: Structure of retroelements..............................................................................................3 Figure 3: Diploid angiosperm genomes........................................................................................6 Figure 4: Gel results of PERT assay on barley VLP isolates from 11/22/11..............................23 Figure 5: Gel results of PERT assay on Lilium VLP isolates from 01/17/12..............................25 Figure 6: Gel results of PERT assay on Lilium VLP isolates from 01/31/12..............................26 Figure 7: Gel results of PERT assay on Scoliopus VLP isolates from 04/13/12........................28 Figure 8: Gel results of PERT assay on Prosartes VLP isolates from 04/20/12........................28 Figure 9: Gel results of PERT assay on Calochortus VLP isolates from 04/30/12....................29 Figure 10: Gel results of PERT assay on barley VLP isolates from 12/10/12..............................31 Figure 11: Gel results from 07/20/12 of cDNA synthesis and PCR performed using Gypsy Friesen primers, RNA from barley VLP isolates and barley leaf samples.................36 Figure 12: Gel results from 07/24/12 of a negative control PCR reaction performed using RT-1 and RT-2 primers without cDNA template................................................................36 Figure 13: Gel results from 08/16/12 of cDNA synthesis and PCR performed using barley ubiquitin primers and RNA from barley VLP preparations and barley leaf samples....................................................................................................38 ! 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Figure 14: Gel results from 07/18/13 of cDNA synthesis and PCR performed using Liliaceae elongation factor-! primers, RNA from Liliaceae VLP isolates, and Liliaceae leaf samples...........................................................................................39 Figure 15: Gel results from 07/18/13 of cDNA synthesis and PCR performed with 50 cycles using Copia reverse transcriptase gene primers and RNA from barley and Liliales VLP preparations and leaf samples..............................................................................42 Figure 16: Gel results from 07/24/13 of cDNA synthesis and PCR performed with 35 cycles using Copia reverse transcriptase gene primers and RNA from barley and Liliales VLP isolates and RNA from leaf samples...................................................................44 Figure 17: A sample of the sequence obtained from Copia RT-PCR products using Smilax VLP RNA, displayed using 4Peaks software. ............................................................45 ! "##! ! "! Introduction It was Dr. Barbara McClintock who “accidentally” discovered the presence of what were first termed “controlling elements” while performing cytogenetic analysis of Zea mays about sixty years ago (McClintock 183). These “controlling elements” were referred to as such due to their ability to affect the phenotypic traits of the maize plants on which her research was focused. These “controlling elements” and McClintock’s proposal that they played a considerable (but yet-to-be explored) role in the evolution of genomes were the source of much skepticism in the scientific community for many years (Biemont 1089). These “controlling elements” proved to be one variety of what are now know as a diverse family of transposable elements - mobile genetic elements that have the ability to relocate to different positions in the genome of a cell. Transposable elements can be broadly divided into two groups: DNA transposons and retrotransposons. Retrotransposons use an RNA intermediate that is reverse transcribed